Recombinant human GLP-1(rhGLP-1) alleviating renal tubulointestitial injury in diabetic STZ-induced rats.

PubMed

Yin, Weiqin; Xu, Shiqing; Wang, Zai; Liu, Honglin; Peng, Liang; Fang, Qing; Deng, Tingting; Zhang, Wenjian; Lou, Jinning

2018-01-01

GLP-1-based treatment improves glycemia through stimulation of insulin secretion and inhibition of glucagon secretion. Recently, more and more findings showed that GLP-1 could also protect kidney from diabetic nephropathy. Most of these studies focused on glomeruli, but the effect of GLP-1 on tubulointerstitial and tubule is not clear yet. In this study, we examined the renoprotective effect of recombinant human GLP-1 (rhGLP-1), and investigated the influence of GLP-1 on inflammation and tubulointerstitial injury using diabetic nephropathy rats model of STZ-induced. The results showed that rhGLP-1 reduced urinary albumin without influencing the body weight and food intake. rhGLP-1 could increased the serum C-peptide slightly but not lower fasting blood glucose significantly. In diabetic nephropathy rats, beside glomerular sclerosis, tubulointerstitial fibrosis was very serious. These lesions could be alleviated by rhGLP-1. rhGLP-1 decreased the expression of profibrotic factors collagen I, α-SMA, fibronectin, and inflammation factors MCP-1 and TNFα in tubular tissue and human proximal tubular cells (HK-2 cells). Furthermore, rhGLP-1 significantly inhibited the phosphorylation of NF-κB, MAPK in both diabetic tubular tissue and HK-2 cells. The inhibition of the expression of TNFα, MCP-1, collagen I and α-SMA in HK-2 cells by GLP-1 could be mimicked by blocking NF-κB or MAPK. These results indicate that rhGLP-1 exhibit renoprotective effect by alleviation of tubulointerstitial injury via inhibiting phosphorylation of MAPK and NF-κB. Therefore, rhGLP-1 may be a potential drug for treatment of diabetic nephropathy. Copyright © 2017 Elsevier Inc. All rights reserved.

Discovery of Potent and Selective Inhibitors for G9a-Like Protein (GLP) Lysine Methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Yan; Li, Fengling; Babault, Nicolas

G9a-like protein (GLP) and G9a are highly homologous protein lysine methyltransferases (PKMTs) sharing approximately 80% sequence identity in their catalytic domains. GLP and G9a form a heterodimer complex and catalyze mono- and dimethylation of histone H3 lysine 9 and nonhistone substrates. Although they are closely related, GLP and G9a possess distinct physiological and pathophysiological functions. Thus, GLP or G9a selective small-molecule inhibitors are useful tools to dissect their distinct biological functions. We previously reported potent and selective G9a/GLP dual inhibitors including UNC0638 and UNC0642. Here we report the discovery of potent and selective GLP inhibitors including 4 (MS0124) and 18more » (MS012), which are >30-fold and 140-fold selective for GLP over G9a and other methyltransferases, respectively. The cocrystal structures of GLP and G9a in the complex with either 4 or 18 displayed virtually identical binding modes and interactions, highlighting the challenges in structure-based design of selective inhibitors for either enzyme.« less

Brain GLP-1 and insulin sensitivity

USDA-ARS?s Scientific Manuscript database

Type 2 diabetes is often treated with a class of drugs referred to as glucagon-like peptide-1 (GLP-1) analogs. GLP-1 is a peptide secreted by the gut that acts through only one known receptor, the GLP-1 receptor. The primary function of GLP-1 is thought to be lowering of postprandial glucose levels....

Involvement of gut microbiota in association between GLP-1/GLP-1 receptor expression and gastrointestinal motility.

PubMed

Yang, Mo; Fukui, Hirokazu; Eda, Hirotsugu; Xu, Xin; Kitayama, Yoshitaka; Hara, Ken; Kodani, Mio; Tomita, Toshihiko; Oshima, Tadayuki; Watari, Jiro; Miwa, Hiroto

2017-04-01

The microbiota in the gut is known to play a pivotal role in host physiology by interacting with the immune and neuroendocrine systems in gastrointestinal (GI) tissues. Glucagon-like peptide 1 (GLP-1), a gut hormone, is involved in metabolism as well as GI motility. We examined how gut microbiota affects the link between GLP-1/GLP-1 receptor (GLP-1R) expression and motility of the GI tract. Germ-free (GF) mice (6 wk old) were orally administered a fecal bacterial suspension prepared from specific pathogen-free (SPF) mice, and then after fecal transplantation (FT) GI tissues were obtained from the GF mice at various time points. The expression of GLP-1 and its receptor was examined by immunohistochemistry, and gastrointestinal transit time (GITT) was measured by administration of carmine red solution. GLP-1 was expressed in endocrine cells in the colonic mucosa, and GLP-1R was expressed in myenteric neural cells throughout the GI wall. GLP-1R-positive cells throughout the GI wall were significantly fewer in GF mice with FT than in GF mice without gut microbiota reconstitution. GITT was significantly shorter in GF mice with FT than in control GF mice without FT and correlated with the number of GLP-1R-positive cells throughout the GI wall. GITT was significantly longer in GF control mice than in SPF mice. When those mice were treated with GLP-1 agonist extendin4, GITT was significantly longer in the GF mice. The gut microbiota may accelerate or at least modify GI motility while suppressing GLP-1R expression in myenteric neural cells throughout the GI tract. NEW & NOTEWORTHY The gut microbiota has been intensively studied, because it plays a pivotal role in various aspects of host physiology. On the other hand, glucagon-like peptide 1 (GLP-1) plays important roles in metabolism as well as gastrointestinal motility. In the present study, we have suggested that the gut microbiota accelerates gastrointestinal motility while suppressing the expression of GLP-1 receptor in

GAD-specific T cells are induced by GAD-alum treatment in Type-1 diabetes patients.

PubMed

Pihl, Mikael; Barcenilla, Hugo; Axelsson, Stina; Chéramy, Mikael; Åkerman, Linda; Johansson, Ingela; Ludvigsson, Johnny; Casas, Rosaura

2017-03-01

Administration of Glutamic Acid Decarboxylase (GAD) 65 formulated in aluminium hydroxide preserved insulin secretion in a phase II trial in recent onset Type 1 Diabetes. A subsequent European phase III trial was closed at 15months after failing to reach primary endpoint, but the majority of the Swedish patients completed the 21months follow-up. We studied the frequencies and phenotype of T cells, suppressive capacity of Tregs, GAD 65 -induced proliferation, and frequencies of T cells with a GAD 65 -specific TCR in Swedes participating in the trial. Stimulation with GAD 65 induced activated T cells and also cells with a suppressive phenotype. Activated GAD 65 -specific effector T cells were detected by tetramer staining while the frequency of GAD 65 -specific Treg was not affected by the treatment. Additional doses of GAD-alum increased frequencies of CD25 + CD127 + , but had no effect on CD25 hi CD127 lo . Our findings indicate that GAD-alum treatment primarily induced activated T cells. GAD 65 -specific cells were mainly of activated phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

Loss of dorsomedial hypothalamic GLP-1 signaling reduces BAT thermogenesis and increases adiposity.

PubMed

Lee, Shin J; Sanchez-Watts, Graciela; Krieger, Jean-Philippe; Pignalosa, Angelica; Norell, Puck N; Cortella, Alyssa; Pettersen, Klaus G; Vrdoljak, Dubravka; Hayes, Matthew R; Kanoski, Scott; Langhans, Wolfgang; Watts, Alan G

2018-05-01

Glucagon-like peptide-1 (GLP-1) neurons in the hindbrain densely innervate the dorsomedial hypothalamus (DMH), a nucleus strongly implicated in body weight regulation and the sympathetic control of brown adipose tissue (BAT) thermogenesis. Therefore, DMH GLP-1 receptors (GLP-1R) are well placed to regulate energy balance by controlling sympathetic outflow and BAT function. We investigate this possibility in adult male rats by using direct administration of GLP-1 (0.5 ug) into the DMH, knocking down DMH GLP-1R mRNA with viral-mediated RNA interference, and by examining the neurochemical phenotype of GLP-1R expressing cells in the DMH using in situ hybridization. GLP-1 administered into the DMH increased BAT thermogenesis and hepatic triglyceride (TG) mobilization. On the other hand, Glp1r knockdown (KD) in the DMH increased body weight gain and adiposity, with a concomitant reduction in energy expenditure (EE), BAT temperature, and uncoupling protein 1 (UCP1) expression. Moreover, DMH Glp1r KD induced hepatic steatosis, increased plasma TG, and elevated liver specific de-novo lipogenesis, effects that collectively contributed to insulin resistance. Interestingly, DMH Glp1r KD increased neuropeptide Y (NPY) mRNA expression in the DMH. GLP-1R mRNA in the DMH, however, was found in GABAergic not NPY neurons, consistent with a GLP-1R-dependent inhibition of NPY neurons that is mediated by local GABAergic neurons. Finally, DMH Glp1r KD attenuated the anorexigenic effects of the GLP-1R agonist exendin-4, highlighting an important role of DMH GLP-1R signaling in GLP-1-based therapies. Collectively, our data show that DMH GLP-1R signaling plays a key role for BAT thermogenesis and adiposity. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

A Targeted RNAi Screen Identifies Endocytic Trafficking Factors That Control GLP-1 Receptor Signaling in Pancreatic β-Cells.

PubMed

Buenaventura, Teresa; Kanda, Nisha; Douzenis, Phoebe C; Jones, Ben; Bloom, Stephen R; Chabosseau, Pauline; Corrêa, Ivan R; Bosco, Domenico; Piemonti, Lorenzo; Marchetti, Piero; Johnson, Paul R; Shapiro, A M James; Rutter, Guy A; Tomas, Alejandra

2018-03-01

The glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) is a key target for type 2 diabetes (T2D) treatment. Because endocytic trafficking of agonist-bound receptors is one of the most important routes for regulation of receptor signaling, a better understanding of this process may facilitate the development of new T2D therapeutic strategies. Here, we screened 29 proteins with known functions in G protein-coupled receptor trafficking for their role in GLP-1R potentiation of insulin secretion in pancreatic β-cells. We identify five (clathrin, dynamin1, AP2, sorting nexins [SNX] SNX27, and SNX1) that increase and four (huntingtin-interacting protein 1 [HIP1], HIP14, GASP-1, and Nedd4) that decrease insulin secretion from murine insulinoma MIN6B1 cells in response to the GLP-1 analog exendin-4. The roles of HIP1 and the endosomal SNX1 and SNX27 were further characterized in mouse and human β-cell lines and human islets. While HIP1 was required for the coupling of cell surface GLP-1R activation with clathrin-dependent endocytosis, the SNXs were found to control the balance between GLP-1R plasma membrane recycling and lysosomal degradation and, in doing so, determine the overall β-cell incretin responses. We thus identify key modulators of GLP-1R trafficking and signaling that might provide novel targets to enhance insulin secretion in T2D. © 2017 by the American Diabetes Association.

GLP-1 is both anxiogenic and antidepressant; divergent effects of acute and chronic GLP-1 on emotionality.

PubMed

Anderberg, Rozita H; Richard, Jennifer E; Hansson, Caroline; Nissbrandt, Hans; Bergquist, Filip; Skibicka, Karolina P

2016-03-01

Glucagon-like peptide 1 (GLP-1), produced in the intestine and hindbrain, is known for its glucoregulatory and appetite suppressing effects. GLP-1 agonists are in clinical use for treatment of type 2 diabetes and obesity. GLP-1, however, may also affect brain areas associated with emotionality regulation. Here we aimed to characterize acute and chronic impact of GLP-1 on anxiety and depression-like behavior. Rats were subjected to anxiety and depression behavior tests following acute or chronic intracerebroventricular or intra-dorsal raphe (DR) application of GLP-1 receptor agonists. Serotonin or serotonin-related genes were also measured in the amygdala, DR and the hippocampus. We demonstrate that both GLP-1 and its long lasting analog, Exendin-4, induce anxiety-like behavior in three rodent tests of this behavior: black and white box, elevated plus maze and open field test when acutely administered intraperitoneally, into the lateral ventricle, or directly into the DR. Acute central GLP-1 receptor stimulation also altered serotonin signaling in the amygdala. In contrast, chronic central administration of Exendin-4 did not alter anxiety-like behavior but significantly reduced depression-like behavior in the forced swim test. Importantly, this positive effect of Exendin-4 was not due to significant body weight loss and reduced food intake, since rats pair-fed to Exendin-4 rats did not show altered mood. Collectively we show a striking impact of central GLP-1 on emotionality and the amygdala serotonin signaling that is divergent under acute versus chronic GLP-1 activation conditions. We also find a novel role for the DR GLP-1 receptors in regulation of behavior. These results may have direct relevance to the clinic, and indicate that Exendin-4 may be especially useful for obese patients manifesting with comorbid depression. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

HIP1-ALK, a novel fusion protein identified in lung adenocarcinoma.

PubMed

Hong, Mineui; Kim, Ryong Nam; Song, Ji-Young; Choi, So-Jung; Oh, Ensel; Lira, Maruja E; Mao, Mao; Takeuchi, Kengo; Han, Joungho; Kim, Jhingook; Choi, Yoon-La

2014-03-01

The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non-small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3. In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)-ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK. Reverse-transcriptase polymerase chain reaction and immunohistochemical analysis were used to detect this fusion gene's transcript and protein expression, respectively. We had amplified the full-length cDNA sequence of this novel fusion gene by using 5'-rapid amplification of cDNA ends. The causative genomic translocation t(2;7)(p23;q11.23) for generating this novel fusion gene was verified by using genomic sequencing. The examined adenocarcinoma showed predominant acinar pattern, and ALK immunostaining was localized to the cytoplasm, with intense staining in the submembrane region. In break-apart, fluorescence in situ hybridization analysis for ALK, split of the 5' and 3' probe signals, and isolated 3' signals were observed. Reverse-transcriptase polymerase chain reaction revealed that the tumor harbored a novel fusion transcript in which exon 21 of HIP1 was fused to exon 20 of ALK in-frame. The novel fusion gene and its protein HIP1-ALK harboring epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains, which could play a role in carcinogenesis, could become diagnostic and therapeutic target of the lung adenocarcinoma and deserve a further study in the future.

Glucagon Like Peptide-1 (GLP-1) Modulates OVA-Induced Airway Inflammation and Mucus Secretion Involving a Protein Kinase A (PKA)-Dependent Nuclear Factor-κB (NF-κB) Signaling Pathway in Mice.

PubMed

Zhu, Tao; Wu, Xiao-Ling; Zhang, Wei; Xiao, Min

2015-08-26

Asthma is a common chronic pulmonary inflammatory disease, featured with mucus hyper-secretion in the airway. Recent studies found that glucagon like peptide-1 (GLP-1) analogs, including liraglutide and exenatide, possessed a potent anti-inflammatory property through a protein kinase A (PKA)-dependent signaling pathway. Therefore, the aim of current study was to investigate the value of GLP-1 analog therapy liraglutide in airway inflammation and mucus secretion in a murine model of ovalbumin (OVA)-induced asthma, and its underlying molecular mechanism. In our study, BALB/c mice were sensitized and challenged by OVA to induce chronic asthma. Pathological alterations, the number of cells and the content of inflammatory mediators in bronchoalveolar lavage fluid (BALF), and mucus secretion were observed and measured. In addition, the mRNA and protein expression of E-selectin and MUC5AC were analyzed by qPCR and Western blotting. Then, the phosphorylation of PKA and nuclear factor-κB (NF-κB) p65 were also measured by Western blotting. Further, NF-κB p65 DNA binding activity was detected by ELISA. OVA-induced airway inflammation, airway mucus hyper-secretion, the up-regulation of E-selectin and MUC5AC were remarkably inhibited by GLP-1 in mice (all p < 0.01). Then, we also found that OVA-reduced phosphorylation of PKA, and OVA-enhanced NF-κB p65 activation and NF-κB p65 DNA binding activity were markedly improved by GLP-1 (all p < 0.01). Furthermore, our data also figured out that these effects of GLP-1 were largely abrogated by the PKA inhibitor H-89 (all p < 0.01). Taken together, our results suggest that OVA-induced asthma were potently ameliorated by GLP-1 possibly through a PKA-dependent inactivation of NF-κB in mice, indicating that GLP-1 analogs may be considered an effective and safe drug for the potential treatment of asthma in the future.

PSCs and GLP-1R: occurrence in normal pancreas, acute/chronic pancreatitis and effect of their activation by a GLP-1R agonist

PubMed Central

Nakamura, Taichi; Ito, Tetsuhide; Uchida, Masahiko; Hijioka, Masayuki; Igarashi, Hisato; Oono, Takamasa; Kato, Masaki; Nakamura, Kazuhiko; Suzuki, Koichi; Jensen, Robert T.; Takayanagi, Ryoichi

2013-01-01

Background and Aims There is increasing concern about the development of pancreatitis in patients with diabetes mellitus who received long-term GLP-1 analog treatment. Its pathogenesis is unknown. The effects of GLP-1 agonists on pancreatic endocrine cells is well studied, however there is little information on effects on other pancreatic tissues that might be involved in inflammatory processes. Pancreatic stellate cells (PSCs) can play an important role in pancreatitis, secreting various inflammatory cytokines/chemokines, as well as collagen. In this study, we investigated GLP-1R occurrence in normal pancreas, acute/chronic pancreatitis, and the effects of GLP-1 analog on normal PSCs, their ability to stimulate inflammatory mediator secretion or proliferation. Methods GLP-1R expression/localization in normal pancreas and pancreatitis (acute/chronic) tissues were evaluated with histological/immunohistochemical analysis. PSCs were isolated from male Wistar rats. GLP1R expression and effects of GLP-1 analog on activated PSCs was examined with realtime PCR, MTS assays and Western Blotting. Results In normal pancreas, pancreatic β cells expressed GLP-1R, with only low expression in acinar cells, whereas in acute or chronic pancreatitis, acinar cells, ductal cells and activated PSCs expressed GLP-1R. With activation of normal PSCs, GLP-1R is markedly increased, as is multiple other incretin-related receptors. The GLP-1 analog, liraglutide, did not induce inflammatory genes expression in activated PSCs, but induced proliferation. Liraglutide activated multiple signaling cascades in PSCs, and the ERK pathway mediated the PSCs proliferation. Conclusions GLP-1Rs are expressed in normal pancreas and there is marked enhanced expression in acute/chronic pancreatitis. GLP-1-agonist induced cell proliferation of activated PSCs without increasing release of inflammatory mediators. These results suggest chronic treatment with GLP-1R agonists could lead to proliferation

Markers of β-Cell Failure Predict Poor Glycemic Response to GLP-1 Receptor Agonist Therapy in Type 2 Diabetes.

PubMed

Jones, Angus G; McDonald, Timothy J; Shields, Beverley M; Hill, Anita V; Hyde, Christopher J; Knight, Bridget A; Hattersley, Andrew T

2016-02-01

To assess whether clinical characteristics and simple biomarkers of β-cell failure are associated with individual variation in glycemic response to GLP-1 receptor agonist (GLP-1RA) therapy in patients with type 2 diabetes. We prospectively studied 620 participants with type 2 diabetes and HbA1c ≥58 mmol/mol (7.5%) commencing GLP-1RA therapy as part of their usual diabetes care and assessed response to therapy over 6 months. We assessed the association between baseline clinical measurements associated with β-cell failure and glycemic response (primary outcome HbA1c change 0-6 months) with change in weight (0-6 months) as a secondary outcome using linear regression and ANOVA with adjustment for baseline HbA1c and cotreatment change. Reduced glycemic response to GLP-1RAs was associated with longer duration of diabetes, insulin cotreatment, lower fasting C-peptide, lower postmeal urine C-peptide-to-creatinine ratio, and positive GAD or IA2 islet autoantibodies (P ≤ 0.01 for all). Participants with positive autoantibodies or severe insulin deficiency (fasting C-peptide ≤0.25 nmol/L) had markedly reduced glycemic response to GLP-1RA therapy (autoantibodies, mean HbA1c change -5.2 vs. -15.2 mmol/mol [-0.5 vs. -1.4%], P = 0.005; C-peptide <0.25 nmol/L, mean change -2.1 vs. -15.3 mmol/mol [-0.2 vs. -1.4%], P = 0.002). These markers were predominantly present in insulin-treated participants and were not associated with weight change. Clinical markers of low β-cell function are associated with reduced glycemic response to GLP-1RA therapy. C-peptide and islet autoantibodies represent potential biomarkers for the stratification of GLP-1RA therapy in insulin-treated diabetes. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

GLP-1 Treatment Improves Diabetic Retinopathy by Alleviating Autophagy through GLP-1R-ERK1/2-HDAC6 Signaling Pathway.

PubMed

Cai, Xiangsheng; Li, Jingjing; Wang, Mingzhu; She, Miaoqin; Tang, Yongming; Li, Jinlong; Li, Hongwei; Hui, Hongxiang

2017-01-01

Objective: Apoptosis and autophagy of retinal cells, which may be induced by oxidative stress, are tightly associated with the pathogenesis of diabetic retinopathy (DR). The autophagy induced by oxidative stress is considered as excessively stimulated autophagy, which accelerates the progression of DR. This study aims to investigate the protective effect of GLP-1 treatment on alleviating apoptosis and autophagy of retinal cells in type 2 diabetic rats and reveals its possible mechanism. Methods: Type 2 diabetic rats were induced by fed with high sugar, high fat diet and followed with streptozotocin injection. GLP-1 was applied to treat the diabetic rats for one week after the onset of diabetes. The expressions of oxidative stress-related enzymes, retinal GLP-1R, mitochondria-dependent apoptosis- related genes, autophagy markers, and autophagy-associated pathway genes were studied by Western blotting or immunohistochemistry analysis. Results: GLP-1treatment reduced the levels of NOX3 and SOD2 in DR. The expression of BCL2 was increased, while the levels of caspase3 and LC3B were reduced through GLP-1 treatment in DR . GLP-1 treatment restored the GLP-1R expression and decreased the levels of phosphorylated AKT and phosphorylated ERK1/2, which was accompanied with the reduction of the HDAC6 levels in DR. Conclusions: GLP-1 treatment can alleviate autophagy which may be induced by oxidative stress; this protective effect is likely through GLP-1R-ERK1/2-HDAC6 signaling pathway.

The clinical and immunological significance of GAD-specific autoantibody and T-cell responses in type 1 diabetes.

PubMed

Boettler, Tobias; Pagni, Philippe P; Jaffe, Rachel; Cheng, Yang; Zerhouni, Peter; von Herrath, Matthias

2013-08-01

Antigen-specific interventions are desirable approaches in Type 1 Diabetes (T1D) as they can alter islet-specific autoimmunity without systemic side effects. Glutamic acid decarboxylase of 65 kDa (GAD65) is a major autoantigen in type 1 diabetes (T1D) and GAD-specific autoimmunity is a common feature of T1D in humans but also in mouse models of the disease. In humans, administration of the GAD65 protein in an alum formulation has been shown to reduce C-peptide decline in recently diagnosed patients, however, these observations were not confirmed in subsequent phase II/III clinical trials. As GAD-based immune interventions in different formulations have successfully been employed to prevent the establishment of T1D in mouse models of T1D, we sought to analyze the efficacy of GAD-alum treatment and the effects on the GAD-specific immune response in two different mouse models of T1D. Consistent with the latest clinical trials, mice treated with GAD-alum were not protected from diabetes, although GAD-alum induced a GAD-specific Th2-deviated immune response in transgenic rat insulin promoter-glycoprotein (RIP-GP) mice. These observations underline the importance of a thorough, preclinical evaluation of potential drugs before the initiation of clinical trials. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Generalized Anxiety Disorder (GAD) and Comorbid Major Depression with GAD Are Characterized by Enhanced Nitro-oxidative Stress, Increased Lipid Peroxidation, and Lowered Lipid-Associated Antioxidant Defenses.

PubMed

Maes, Michael; Bonifacio, Kamila Landucci; Morelli, Nayara Rampazzo; Vargas, Heber Odebrecht; Moreira, Estefânia Gastaldello; St Stoyanov, Drozdstoy; Barbosa, Décio Sabbatini; Carvalho, André F; Nunes, Sandra Odebrecht Vargas

2018-05-07

Accumulating evidence shows that nitro-oxidative pathways play an important role in the pathophysiology of major depressive disorder (MDD) and bipolar disorder (BD) and maybe anxiety disorders. The current study aims to examine superoxide dismutase (SOD1), catalase, lipid hydroperoxides (LOOH), nitric oxide metabolites (NOx), advanced oxidation protein products (AOPP), malondialdehyde (MDA), glutathione (GSH), paraoxonase 1 (PON1), high-density lipoprotein cholesterol (HDL), and uric acid (UA) in participants with and without generalized anxiety disorder (GAD) co-occurring or not with BD, MDD, or tobacco use disorder. Z unit-weighted composite scores were computed as indices of nitro-oxidative stress driving lipid and protein oxidation. SOD1, LOOH, NOx, and uric acid were significantly higher and HDL and PON1 significantly lower in participants with GAD than in those without GAD. GAD was more adequately predicted by increased SOD + LOOH + NOx and lowered HDL + PON1 composite scores. Composite scores of nitro-oxidative stress coupled with aldehyde and AOPP production were significantly increased in participants with comorbid GAD + MDD as compared with all other study groups, namely MDD, GAD + BD, BD, GAD, and healthy controls. In conclusion, GAD is characterized by increased nitro-oxidative stress and lipid peroxidation and lowered lipid-associated antioxidant defenses, while increased uric acid levels in GAD may protect against aldehyde production and protein oxidation. This study suggests that increased nitro-oxidative stress and especially increased SOD1 activity, NO production, and lipid peroxidation as well as lowered HDL-cholesterol and PON1 activity could be novel drug targets for GAD especially when comorbid with MDD.

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

PubMed

Peisajovich, S G; Samuel, O; Shai, Y

2000-03-10

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. Copyright 2000 Academic Press.

Adverse Effects of GLP-1 Receptor Agonists

PubMed Central

Filippatos, Theodosios D.; Panagiotopoulou, Thalia V.; Elisaf, Moses S.

2014-01-01

Glucagon-like peptide-1 (GLP-1) receptor agonists are a class of injective anti-diabetic drugs that improve glycemic control and many other atherosclerosis-related parameters in patients with type 2 diabetes (T2D). However, the use of this relatively new class of drugs may be associated with certain adverse effects. Concerns have been expressed regarding the effects of these drugs on pancreatic and thyroid tissue, since animal studies and analyses of drug databases indicate an association of GLP-1 receptor agonists with pancreatitis, pancreatic cancer, and thyroid cancer. However, several meta-analyses failed to confirm a cause-effect relation between GLP-1 receptor agonists and the development of these adverse effects. One benefit of GLP-1 receptor agonists is that they do not cause hypoglycemia when combined with metformin or thiazolidinediones, but the dose of concomitant sulphonylurea or insulin may have to be decreased to reduce the risk of hypoglycemic episodes. On the other hand, several case reports have linked the use of these drugs, mainly exenatide, with the occurrence of acute kidney injury, primarily through hemodynamic derangement due to nausea, vomiting, and diarrhea. The most common symptoms associated with the use of GLP-1 receptor agonists are gastrointestinal symptoms, mainly nausea. Other common adverse effects include injection site reactions, headache, and nasopharyngitis, but these effects do not usually result in discontinuation of the drug. Current evidence shows that GLP-1 receptor agonists have no negative effects on the cardiovascular risk of patients with T2D. Thus, GLP-1 receptor agonists appear to have a favorable safety profile, but ongoing trials will further assess their cardiovascular effects. The aim of this review is to analyze critically the available data regarding adverse events of GLP-1 receptor agonists in different anatomic systems published in Pubmed and Scopus. Whenever possible, certain differences between GLP-1

IL-6-Type Cytokine Signaling in Adipocytes Induces Intestinal GLP-1 Secretion.

PubMed

Wueest, Stephan; Laesser, Céline I; Böni-Schnetzler, Marianne; Item, Flurin; Lucchini, Fabrizio C; Borsigova, Marcela; Müller, Werner; Donath, Marc Y; Konrad, Daniel

2018-01-01

We recently showed that interleukin (IL)-6-type cytokine signaling in adipocytes induces free fatty acid release from visceral adipocytes, thereby promoting obesity-induced hepatic insulin resistance and steatosis. In addition, IL-6-type cytokines may increase the release of leptin from adipocytes and by those means induce glucagon-like peptide 1 (GLP-1) secretion. We thus hypothesized that IL-6-type cytokine signaling in adipocytes may regulate insulin secretion. To this end, mice with adipocyte-specific knockout of gp130, the signal transducer protein of IL-6, were fed a high-fat diet for 12 weeks. Compared with control littermates, knockout mice showed impaired glucose tolerance and circulating leptin, GLP-1, and insulin levels were reduced. In line, leptin release from isolated adipocytes was reduced, and intestinal proprotein convertase subtilisin/kexin type 1 ( Pcsk1 ) expression, the gene encoding PC1/3, which controls GLP-1 production, was decreased in knockout mice. Importantly, treatment with the GLP-1 receptor antagonist exendin 9-39 abolished the observed difference in glucose tolerance between control and knockout mice. Ex vivo, supernatant collected from isolated adipocytes of gp130 knockout mice blunted Pcsk1 expression and GLP-1 release from GLUTag cells. In contrast, glucose- and GLP-1-stimulated insulin secretion was not affected in islets of knockout mice. In conclusion, adipocyte-specific IL-6 signaling induces intestinal GLP-1 release to enhance insulin secretion, thereby counteracting insulin resistance in obesity. © 2017 by the American Diabetes Association.

Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells

PubMed Central

Li, Jing; Keller, Mark P.; Hohmeier, Hans E.; Wang, Yong; Feng, Yue; Zhou, Heather H.; Shen, Xiaolan; Rabaglia, Mary; Soni, Mufaddal; Attie, Alan D.; Newgard, Christopher B.; Thornberry, Nancy A.; Howard, Andrew D.; Zhou, Yun-Ping

2015-01-01

Better understanding how glucagon-like peptide 1 (GLP-1) promotes pancreatic β-cell function and/or mass may uncover new treatment for type 2 diabetes. In this study, we investigated the potential involvement of microRNAs (miRNAs) in the effect of GLP-1 on glucose-stimulated insulin secretion. miRNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR. The role of miRNAs on insulin secretion was studied by transfecting INS-1 cells with either precursors or antisense inhibitors of miRNAs. Among the 250 miRNAs surveyed, miR-132 and miR-212 were significantly up-regulated by GLP-1 by greater than 2-fold in INS-1 832/3 cells, which were subsequently reproduced in freshly isolated rat, mouse, and human islets, as well as the islets from GLP-1 infusion in vivo in mice. The inductions of miR-132 and miR-212 by GLP-1 were correlated with cAMP production and were blocked by the protein kinase A inhibitor H-89 but not affected by the exchange protein activated by cAMP activator 8-pCPT-2′-O-Me-cAMP-AM. GLP-1 failed to increase miR-132 or miR-212 expression levels in the 832/13 line of INS-1 cells, which lacks robust cAMP and insulin responses to GLP-1 treatment. Overexpression of miR-132 or miR-212 significantly enhanced glucose-stimulated insulin secretion in both 832/3 and 832/13 cells, and restored insulin responses to GLP-1 in INS-1 832/13 cells. GLP-1 increases the expression of miRNAs 132 and 212 via a cAMP/protein kinase A-dependent pathway in pancreatic β-cells. Overexpression of miR-132 or miR-212 enhances glucose and GLP-1-stimulated insulin secretion. PMID:26218441

Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic β-Cells.

PubMed

Shang, Jin; Li, Jing; Keller, Mark P; Hohmeier, Hans E; Wang, Yong; Feng, Yue; Zhou, Heather H; Shen, Xiaolan; Rabaglia, Mary; Soni, Mufaddal; Attie, Alan D; Newgard, Christopher B; Thornberry, Nancy A; Howard, Andrew D; Zhou, Yun-Ping

2015-09-01

Better understanding how glucagon-like peptide 1 (GLP-1) promotes pancreatic β-cell function and/or mass may uncover new treatment for type 2 diabetes. In this study, we investigated the potential involvement of microRNAs (miRNAs) in the effect of GLP-1 on glucose-stimulated insulin secretion. miRNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR. The role of miRNAs on insulin secretion was studied by transfecting INS-1 cells with either precursors or antisense inhibitors of miRNAs. Among the 250 miRNAs surveyed, miR-132 and miR-212 were significantly up-regulated by GLP-1 by greater than 2-fold in INS-1 832/3 cells, which were subsequently reproduced in freshly isolated rat, mouse, and human islets, as well as the islets from GLP-1 infusion in vivo in mice. The inductions of miR-132 and miR-212 by GLP-1 were correlated with cAMP production and were blocked by the protein kinase A inhibitor H-89 but not affected by the exchange protein activated by cAMP activator 8-pCPT-2'-O-Me-cAMP-AM. GLP-1 failed to increase miR-132 or miR-212 expression levels in the 832/13 line of INS-1 cells, which lacks robust cAMP and insulin responses to GLP-1 treatment. Overexpression of miR-132 or miR-212 significantly enhanced glucose-stimulated insulin secretion in both 832/3 and 832/13 cells, and restored insulin responses to GLP-1 in INS-1 832/13 cells. GLP-1 increases the expression of miRNAs 132 and 212 via a cAMP/protein kinase A-dependent pathway in pancreatic β-cells. Overexpression of miR-132 or miR-212 enhances glucose and GLP-1-stimulated insulin secretion.

Ghrelin suppresses cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) in the intestine, and attenuates the anorectic effects of CCK, PYY and GLP-1 in goldfish (Carassius auratus).

PubMed

Blanco, Ayelén Melisa; Bertucci, Juan Ignacio; Valenciano, Ana Isabel; Delgado, María Jesús; Unniappan, Suraj

2017-07-01

Ghrelin is an important gut-derived hormone with an appetite stimulatory role, while most of the intestinal hormones, including cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1), are appetite-inhibitors. Whether these important peptides with opposing roles on food intake interact to regulate energy balance in fish is currently unknown. The aim of this study was to characterize the putative crosstalk between ghrelin and CCK, PYY and GLP-1 in goldfish (Carassius auratus). We first determined the localization of CCK, PYY and GLP-1 in relation to ghrelin and its main receptor GHS-R1a (growth hormone secretagogue 1a) in the goldfish intestine by immunohistochemistry. Colocalization of ghrelin/GHS-R1a and CCK/PYY/GLP-1 was found primarily in the luminal border of the intestinal mucosa. In an intestinal explant culture, a significant decrease in prepro-cck, prepro-pyy and proglucagon transcript levels was observed after 60min of incubation with ghrelin, which was abolished by preincubation with the GHS-R1a ghrelin receptor antagonist [D-Lys3]-GHRP-6 (except for proglucagon). The protein expression of PYY and GLP-1 was also downregulated by ghrelin. Finally, intraperitoneal co-administration of CCK, PYY or GLP-1 with ghrelin results in no modification of food intake in goldfish. Overall, results of the present study show for the first time in fish that ghrelin exerts repressive effects on enteric anorexigens. It is likely that these interactions mediate the stimulatory effects of ghrelin on feeding and metabolism in fish. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression, purification, and C-terminal site-specific PEGylation of cysteine-mutated glucagon-like peptide-1.

PubMed

Gao, Mingming; Tian, Hong; Ma, Chen; Gao, Xiangdong; Guo, Wei; Yao, Wenbing

2010-09-01

Glucagon-like peptide-1 (GLP-1) is attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical application is limited because of short biological half-life. This study was designed to produce a C-terminal site-specific PEGylated analog of cysteine-mutated GLP-1 (cGLP-1) to prolong its action. The gene of cGLP-1 was inserted into pET32a to construct a thioredoxinA fusion protein. After expression in BL21 (DE3) strain, the fusion protein was purified with Ni-affinity chromatography and then was PEGylated with methoxy-polyethylene glycol-maleimide (mPEG(10K)-MAL). The PEGylated fusion protein was purified with anion exchange chromatography and then was cleaved by enterokinase. The digested product was further purified with reverse-phase chromatography. Finally, 8.7 mg mPEG(10K)-cGLP-1 with a purity of up to 98% was obtained from the original 500 ml culture. The circular dichroism spectra indicated that mPEG(10K)-cGLP-1 maintained the secondary structure of native GLP-1. As compared with that of native GLP-1, the plasma glucose lowering activity of mPEG(10K)-cGLP-1 was significantly extended. These results suggest that our method will be useful in obtaining a large quantity of mPEG(10K)-cGLP-1 for further study and mPEG(10K)-cGLP-1 might find a role in the therapy of type 2 diabetes through C-terminal site-specific PEGylation.

Astrocytes Regulate GLP-1 Receptor-Mediated Effects on Energy Balance

PubMed Central

Reiner, David J.; Mietlicki-Baase, Elizabeth G.; McGrath, Lauren E.; Zimmer, Derek J.; Bence, Kendra K.; Sousa, Gregory L.; Konanur, Vaibhav R.; Krawczyk, Joanna; Burk, David H.; Kanoski, Scott E.; Hermann, Gerlinda E.; Rogers, Richard C.

2016-01-01

Astrocytes are well established modulators of extracellular glutamate, but their direct influence on energy balance-relevant behaviors is largely understudied. As the anorectic effects of glucagon-like peptide-1 receptor (GLP-1R) agonists are partly mediated by central modulation of glutamatergic signaling, we tested the hypothesis that astrocytic GLP-1R signaling regulates energy balance in rats. Central or peripheral administration of a fluorophore-labeled GLP-1R agonist, exendin-4, localizes within astrocytes and neurons in the nucleus tractus solitarius (NTS), a hindbrain nucleus critical for energy balance control. This effect is mediated by GLP-1R, as the uptake of systemically administered fluorophore-tagged exendin-4 was blocked by central pretreatment with the competitive GLP-1R antagonist exendin-(9–39). Ex vivo analyses show prolonged exendin-4-induced activation (live cell calcium signaling) of NTS astrocytes and neurons; these effects are also attenuated by exendin-(9–39), indicating mediation by the GLP-1R. In vitro analyses show that the application of GLP-1R agonists increases cAMP levels in astrocytes. Immunohistochemical analyses reveal that endogenous GLP-1 axons form close synaptic apposition with NTS astrocytes. Finally, pharmacological inhibition of NTS astrocytes attenuates the anorectic and body weight-suppressive effects of intra-NTS GLP-1R activation. Collectively, data demonstrate a role for NTS astrocytic GLP-1R signaling in energy balance control. SIGNIFICANCE STATEMENT Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce food intake and are approved by the Food and Drug Administration for the treatment of obesity, but the cellular mechanisms underlying the anorectic effects of GLP-1 require further investigation. Astrocytes represent a major cellular population in the CNS that regulates neurotransmission, yet the role of astrocytes in mediating energy balance is largely unstudied. The current data provide novel evidence that

Immunization against GAD Induces Antibody Binding to GAD-Independent Antigens and Brainstem GABAergic Neuronal Loss

PubMed Central

Chang, Thashi; Alexopoulos, Harry; Pettingill, Philippa; McMenamin, Mary; Deacon, Robert; Erdelyi, Ferenc; Szabó, Gabor; Buckley, Camilla J.; Vincent, Angela

2013-01-01

Stiff person syndrome (SPS) is a highly-disabling neurological disorder of the CNS characterized by progressive muscular rigidity and spasms. In approximately 60–80% of patients there are autoantibodies to glutamic acid decarboxylase (GAD), the enzyme that synthesizes gamma-amino butyric acid (GABA), the predominant inhibitory neurotransmitter of the CNS. Although GAD is intracellular, it is thought that autoimmunity to GAD65 may play a role in the development of SPS. To test this hypothesis, we immunized mice, that expressed enhanced green fluorescent protein (EGFP) under the GAD65 promoter, with either GAD65 (n = 13) or phosphate buffered saline (PBS) (n = 13). Immunization with GAD65 resulted in autoantibodies that immunoprecipitated GAD, bound to CNS tissue in a highly characteristic pattern, and surprisingly bound not only to GAD intracellularly but also to the surface of cerebellar neurons in culture. Moreover, immunization resulted in immunoglobulin diffusion into the brainstem, and a partial loss of GAD-EGFP expressing cells in the brainstem. Although immunization with GAD65 did not produce any behavioral abnormality in the mice, the induction of neuronal-surface antibodies and the trend towards loss of GABAergic neurons in the brainstem, supports a role for humoral autoimmunity in the pathogenesis of SPS and suggests that the mechanisms may involve spread to antigens expressed on the surface of these neurons. PMID:24058450

A high carbohydrate, but not fat or protein meal attenuates postprandial ghrelin, PYY and GLP-1 responses in Chinese men

PubMed Central

Parvaresh Rizi, Ehsan; Loh, Tze Ping; Baig, Sonia; Chhay, Vanna; Huang, Shiqi; Caleb Quek, Jonathan; Tai, E. Shyong; Toh, Sue-Anne

2018-01-01

It is known that the macronutrient content of a meal has different impacts on the postprandial satiety and appetite hormonal responses. Whether obesity interacts with such nutrient-dependent responses is not well characterized. We examined the postprandial appetite and satiety hormonal responses after a high-protein (HP), high-carbohydrate (HC), or high-fat (HF) mixed meal. This was a randomized cross-over study of 9 lean insulin-sensitive (mean±SEM HOMA-IR 0.83±0.10) and 9 obese insulin-resistant (HOMA-IR 4.34±0.41) young (age 21–40 years), normoglycaemic Chinese men. We measured fasting and postprandial plasma concentration of glucose, insulin, active glucagon-like peptide-1 (GLP-1), total peptide-YY (PYY), and acyl-ghrelin in response to HP, HF, or HC meals. Overall postprandial plasma insulin response was more robust in the lean compared to obese subjects. The postprandial GLP-1 response after HF or HP meal was higher than HC meal in both lean and obese subjects. In obese subjects, HF meal induced higher response in postprandial PYY compared to HC meal. HP and HF meals also suppressed ghrelin greater compared to HC meal in the obese than lean subjects. In conclusion, a high-protein or high-fat meal induces a more favorable postprandial satiety and appetite hormonal response than a high-carbohydrate meal in obese insulin-resistant subjects. PMID:29385178

Insulin chains as efficient fusion tags for prokaryotic expression of short peptides.

PubMed

Deng, Ligang; Xue, Xiaoying; Shen, Cangjie; Song, Xiaohan; Wang, Chunyang; Wang, Nan

2017-10-01

Insulin chains are usually expressed in Escherichia coli as fusion proteins with different tags, including various low molecular weight peptide tags. The objective of this study was to determine if insulin chains could facilitate the recombinant expression of other target proteins, with an emphasis on low molecular weight peptides. A series of short peptides were fused to mini-proinsulin, chain B or chain A, and induced for expression in Escherichia coli. All the tested peptides including glucagon-like peptide 1 (GLP-1), a C-terminal extended GLP-1, oxyntomodulin, enfuvirtide, linaclotide, and an unstructured artificial peptide were expressed with reasonable yields, identified by Tricine-SDS-PAGE and immunoblotting. All recombinant products were expressed in inclusion bodies. The effective accumulation of products was largely attributed to the insoluble expression induced by fusion with insulin chains, and was confirmed by the fusion expression of transthyretin. Insulin chains thus show promise as efficient fusion tags for mass production of heterologous peptides in prokaryotes. Copyright © 2017 Elsevier Inc. All rights reserved.

GLP-1 promotes mitochondrial metabolism in vascular smooth muscle cells by enhancing endoplasmic reticulum-mitochondria coupling.

PubMed

Morales, Pablo E; Torres, Gloria; Sotomayor-Flores, Cristian; Peña-Oyarzún, Daniel; Rivera-Mejías, Pablo; Paredes, Felipe; Chiong, Mario

2014-03-28

Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)-mitochondria communication, as it allows for a more efficient transfer of Ca(2+) into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER-mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3h of GLP-1 treatment, paralleled by increased Ca(2+) transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca(2+) increases in GLP-1 treated cells. Inhibiting both Ca(2+) release from the ER and Ca(2+) entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER-mitochondria communication in VSMC, resulting in higher mitochondrial activity. Copyright © 2014 Elsevier Inc. All rights reserved.

CORRELATION BETWEEN PRE AND POSTOPERATIVE LEVELS OF GLP-1/GLP-2 AND WEIGHT LOSS AFTER ROUX-EN-Y GASTRIC BYPASS: A PROSPECTIVE STUDY.

PubMed

Cazzo, Everton; Gestic, Martinho Antonio; Utrini, Murillo Pimentel; Pareja, José Carlos; Chaim, Elinton Adami; Geloneze, Bruno; Barreto, Maria Rita Lazzarini; Magro, Daniéla Oliveira

2016-01-01

The role of gut hormones in glucose homeostasis and weight loss achievement and maintenance after bariatric surgery appears to be a key point in the understanding of the beneficial effects observed following these procedures. To determine whether there is a correlation between the pre and postoperative levels of both GLP-1 and GLP-2 and the excess weight loss after Roux-en-Y gastric bypass (RYGB). An exploratory prospective study which enrolled 11 individuals who underwent RYGB and were followed-up for 12 months. GLP-1 and GLP-2 after standard meal tolerance test (MTT) were determined before and after surgery and then correlated with the percentage of excess loss (%EWL). GLP-2 AUC presented a significant postoperative increase (945.3±449.1 vs.1787.9±602.7; p=0.0037); GLP-1 AUC presented a non-significant trend towards increase after RYGB (709.6±320.4 vs. 1026.5±714.3; p=0.3808). Mean %EWL was 66.7±12.2%. There was not any significant correlation between both the pre and postoperative GLP-1 AUCs and GLP-2 AUCs and the %EWL achieved after one year. There was no significant correlation between the pre and postoperative levels of the areas under the GLP-1 and GLP-2 curves with the percentage of weight loss reached after one year. O papel de hormônios gastrointestinais sobre a homeostase glicêmica e a obtenção e manutenção da perda de peso após a cirurgia bariátrica parece ser elemento fundamental na compreensão dos benefícios observados após estes procedimentos. Determinar se há correlação entre os níveis pré e pós-operatórios de GLP-1 e GLP-2 com a perda do excesso de peso após o bypass gástrico em Y-de-Roux. Estudo prospectivo exploratório que envolveu 11 indivíduos submetidos ao bypass gástrico, acompanhados por 12 meses. Os níveis GLP-1 e GLP-2 após um teste de refeição padrão foram determinados antes e 12 meses após a operação e então foram correlacionados com o percentual de perda do excesso de peso. Houve aumento

The truncated metabolite GLP-2 (3-33) interacts with the GLP-2 receptor as a partial agonist.

PubMed

Thulesen, Jesper; Knudsen, Lotte Bjerre; Hartmann, Bolette; Hastrup, Sven; Kissow, Hannelouise; Jeppesen, Palle Bekker; Ørskov, Cathrine; Holst, Jens Juul; Poulsen, Steen Seier

2002-01-15

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

PubMed

Iuchi, S; Cole, S T; Lin, E C

1990-01-01

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.

Duodenal GLP-1 signaling regulates hepatic glucose production through a PKC-δ-dependent neurocircuitry

PubMed Central

Yang, Mengliu; Wang, Jinzhi; Wu, Shaobo; Yuan, Lei; Zhao, Xiaodong; Liu, Chaohong; Xie, Jing; Jia, Yanjun; Lai, Yerui; Zhao, Allan Zijian; Boden, Guenther; Li, Ling; Yang, Gangyi

2017-01-01

Intestinal glucagon-like peptide-1 (GLP-1) is a hormone that stimulates insulin secretion and acts as a neuropeptide to control glucose homeostasis, but little is known whether intestinal GLP-1 has any effect in the control of hepatic glucose production (HGP). Here we found that intraduodenal infusion of GLP-1 activated duodenal PKC-δ, lowered HGP and was accompanied by a decrease in hepatic expression of gluconeogenic enzymes and an increase in hepatic insulin signaling in rats. However, gut co-infusion of either the GLP-1 receptor antagonist Ex-9, or the PKC-δ inhibitor rottlerin with GLP-1, negated the ability of gut GLP-1 to lower HGP and to increase hepatic insulin signaling during clamps. The metabolic and molecular signal effects of duodenal GLP-1 were also negated by co-infusion with tetracaine, pharmacologic inhibition of N-methyl-d-aspartate receptors within the dorsalvagal complex, or hepatic vagotomy in rats. In summary, we identified a neural glucoregulatory function of gut GLP-1 signaling. PMID:28182013

The endocrine disrupting potential of monosodium glutamate (MSG) on secretion of the glucagon-like peptide-1 (GLP-1) gut hormone and GLP-1 receptor interaction.

PubMed

Shannon, Maeve; Green, Brian; Willars, Gary; Wilson, Jodie; Matthews, Natalie; Lamb, Joanna; Gillespie, Anna; Connolly, Lisa

2017-01-04

Monosodium glutamate (MSG) is a suspected obesogen with epidemiological evidence positively correlating consumption to increased body mass index and higher prevalence of metabolic syndrome. ELISA and high content analysis (HCA) were employed to examine the disruptive effects of MSG on the secretion of enteroendocrine hormone glucagon-like peptide-1 (GLP-1) and GLP-1 receptor (GLP-1R), respectively. Following 3h MSG exposure of the enteroendocrine pGIP/neo: STC-1 cell line model (500μg/ml) significantly increased GLP-1 secretion (1.8 fold; P≤0.001), however, 72h exposure (500μg/ml) caused a 1.8 fold decline (P≤0.05). Also, 3h MSG exposure (0.5-500μg/ml) did not induce any cytotoxicity (including multiple pre-lethal markers) but 72h exposure at 250-500μg/ml, decreased cell number (11.8-26.7%; P≤0.05), increased nuclear area (23.9-29.8%; P≤0.001) and decreased mitochondrial membrane potential (13-21.6%; P≤0.05). At 500μg/ml, MSG increased mitochondrial mass by 16.3% (P≤0.01). MSG did not agonise or antagonise internalisation of the GLP-1R expressed recombinantly in U2OS cells, following GLP-1 stimulation. In conclusion, 72h exposure of an enteroendocrine cell line at dietary levels of MSG, results in pre-lethal cytotoxicity and decline in GLP-1 secretion. These adverse events may play a role in the pathogenesis of obesity as outlined in the obesogen hypothesis by impairing GLP-1 secretion, related satiety responses and glucose-stimulated insulin release. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Elevated Serum GAD65 and GAD65-GADA Immune Complexes in Stiff Person Syndrome.

PubMed

Gu Urban, Gucci Jijuan; Friedman, Mikaela; Ren, Ping; Törn, Carina; Fex, Malin; Hampe, Christiane S; Lernmark, Åke; Landegren, Ulf; Kamali-Moghaddam, Masood

2015-06-16

Glutamic acid decarboxylase 65 (GAD65) and autoantibodies specific for GAD65 (GADA) are associated with autoimmune diseases including Stiff Person Syndrome (SPS) and Type 1 diabetes (T1D). GADA is recognized as a biomarker of value for clinical diagnosis and prognostication in these diseases. Nonetheless, it remains medically interesting to develop sensitive and specific assays to detect GAD65 preceding GADA emergence, and to monitor GADA-GAD65 immune complexes in blood samples. In the present study, we developed a highly sensitive proximity ligation assay to measure serum GAD65. This novel assay allowed detection of as little as 0.65 pg/ml GAD65. We were also able to detect immune complexes involving GAD65 and GADA. Both free GAD65 and GAD65-GADA levels were significantly higher in serum samples from SPS patients compared to healthy controls. The proximity ligation assays applied for detection of GAD65 and its immune complexes may thus enable improved diagnosis and better understanding of SPS.

Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens

PubMed Central

Valansi, Clari; Moi, David; Leikina, Evgenia; Matveev, Elena; Chernomordik, Leonid V.

2017-01-01

Cell–cell fusion is inherent to sexual reproduction. Loss of HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) proteins results in gamete fusion failure in diverse organisms, but their exact role is unclear. In this study, we show that Arabidopsis thaliana HAP2/GCS1 is sufficient to promote mammalian cell–cell fusion. Hemifusion and complete fusion depend on HAP2/GCS1 presence in both fusing cells. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus results in homotypic virus–cell fusion. We demonstrate that the Caenorhabditis elegans Epithelial Fusion Failure 1 (EFF-1) somatic cell fusogen can replace HAP2/GCS1 in one of the fusing membranes, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Structural modeling of the HAP2/GCS1 protein family predicts that they are homologous to EFF-1 and viral class II fusion proteins (e.g., Zika virus). We name this superfamily Fusexins: fusion proteins essential for sexual reproduction and exoplasmic merger of plasma membranes. We suggest a common origin and evolution of sexual reproduction, enveloped virus entry into cells, and somatic cell fusion. PMID:28137780

Prediction of thyroid C-cell carcinogenicity after chronic administration of GLP1-R agonists in rodents.

PubMed

van den Brink, Willem; Emerenciana, Annette; Bellanti, Francesco; Della Pasqua, Oscar; van der Laan, Jan Willem

2017-04-01

Increased incidence of C-cell carcinogenicity has been observed for glucagon-like-protein-1 receptor (GLP-1r) agonists in rodents. It is suggested that the duration of exposure is an indicator of carcinogenic potential in rodents of the different products on the market. Furthermore, the role of GLP-1-related mechanisms in the induction of C-cell carcinogenicity has gained increased attention by regulatory agencies. This study proposes an integrative pharmacokinetic/pharmacodynamic (PKPD) framework to identify explanatory factors and characterize differences in carcinogenic potential of the GLP-1r agonist products. PK models for four products (exenatide QW (once weekly), exenatide BID (twice daily), liraglutide and lixisenatide) were developed using nonlinear mixed effects modelling. Predicted exposure was subsequently linked to GLP-1r stimulation using in vitro GLP-1r potency data. A logistic regression model was then applied to exenatide QW and liraglutide data to assess the relationship between GLP-1r stimulation and thyroid C-cell hyperplasia incidence as pre-neoplastic predictor of a carcinogenic response. The model showed a significant association between predicted GLP-1r stimulation and C-cell hyperplasia after 2years of treatment. The predictive performance of the model was evaluated using lixisenatide, for which hyperplasia data were accurately described during the validation step. The use of a model-based approach provided insight into the relationship between C-cell hyperplasia and GLP-1r stimulation for all four products, which is not possible with traditional data analysis methods. It can be concluded that both pharmacokinetics (exposure) and pharmacodynamics (potency for GLP-1r) factors determine C-cell hyperplasia incidence in rodents. Our work highlights the pharmacological basis for GLP-1r agonist-induced C-cell carcinogenicity. The concept is promising for application to other drug classes. Copyright © 2017 Elsevier Inc. All rights reserved.

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

PubMed

Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

2002-11-01

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus.

PubMed

Ludvigsson, Johnny; Krisky, David; Casas, Rosaura; Battelino, Tadej; Castaño, Luis; Greening, James; Kordonouri, Olga; Otonkoski, Timo; Pozzilli, Paolo; Robert, Jean-Jacques; Veeze, Henk J; Palmer, Jerry; Samuelsson, Ulf; Elding Larsson, Helena; Åman, Jan; Kärdell, Gunilla; Neiderud Helsingborg, Jan; Lundström, Göran; Albinsson, Eva; Carlsson, Annelie; Nordvall, Maria; Fors, Hans; Arvidsson, Carl-Göran; Edvardson, Stig; Hanås, Ragnar; Larsson, Karin; Rathsman, Björn; Forsgren, Henrik; Desaix, Helena; Forsander, Gun; Nilsson, Nils-Östen; Åkesson, Carl-Göran; Keskinen, Päivi; Veijola, Riitta; Talvitie, Timo; Raile, Klemens; Kapellen, Thomas; Burger, Walter; Neu, Andreas; Engelsberger, Ilse; Heidtmann, Bettina; Bechtold, Suzanne; Leslie, David; Chiarelli, Francesco; Cicognani, Alesandro; Chiumello, Giuseppe; Cerutti, Franco; Zuccotti, Gian Vincenzo; Gomez Gila, Ana; Rica, Itxaso; Barrio, Raquel; Clemente, Maria; López Garcia, Maria José; Rodriguez, Mercedes; Gonzalez, Isabel; Lopez, Juan Pedro; Oyarzabal, Mirentxu; Reeser, H M; Nuboer, Roos; Stouthart, Pauline; Bratina, Natasa; Bratanic, Nina; de Kerdanet, Marc; Weill, Jacques; Ser, Nicole; Barat, Pascal; Bertrand, Anne Marie; Carel, Jean-Claude; Reynaud, Rachel; Coutant, Regis; Baron, Sabine

2012-02-02

The 65-kD isoform of glutamic acid decarboxylase (GAD65) is a major autoantigen in type 1 diabetes. We hypothesized that alum-formulated GAD65 (GAD-alum) can preserve beta-cell function in patients with recent-onset type 1 diabetes. We studied 334 patients, 10 to 20 years of age, with type 1 diabetes, fasting C-peptide levels of more than 0.3 ng per milliliter (0.1 nmol per liter), and detectable serum GAD65 autoantibodies. Within 3 months after diagnosis, patients were randomly assigned to receive one of three study treatments: four doses of GAD-alum, two doses of GAD-alum followed by two doses of placebo, or four doses of placebo. The primary outcome was the change in the stimulated serum C-peptide level (after a mixed-meal tolerance test) between the baseline visit and the 15-month visit. Secondary outcomes included the glycated hemoglobin level, mean daily insulin dose, rate of hypoglycemia, and fasting and maximum stimulated C-peptide levels. The stimulated C-peptide level declined to a similar degree in all study groups, and the primary outcome at 15 months did not differ significantly between the combined active-drug groups and the placebo group (P=0.10). The use of GAD-alum as compared with placebo did not affect the insulin dose, glycated hemoglobin level, or hypoglycemia rate. Adverse events were infrequent and mild in the three groups, with no significant differences. Treatment with GAD-alum did not significantly reduce the loss of stimulated C peptide or improve clinical outcomes over a 15-month period. (Funded by Diamyd Medical and the Swedish Child Diabetes Foundation; ClinicalTrials.gov number, NCT00723411.).

Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia.

PubMed

Hyde, Thomas M; Lipska, Barbara K; Ali, Towhid; Mathew, Shiny V; Law, Amanda J; Metitiri, Ochuko E; Straub, Richard E; Ye, Tianzhang; Colantuoni, Carlo; Herman, Mary M; Bigelow, Llewellyn B; Weinberger, Daniel R; Kleinman, Joel E

2011-07-27

GABA signaling molecules are critical for both human brain development and the pathophysiology of schizophrenia. We examined the expression of transcripts derived from three genes related to GABA signaling [GAD1 (GAD67 and GAD25), SLC12A2 (NKCC1), and SLC12A5 (KCC2)] in the prefrontal cortex (PFC) and hippocampal formation of a large cohort of nonpsychiatric control human brains (n = 240) across the lifespan (from fetal week 14 to 80 years) and in patients with schizophrenia (n = 30-31), using quantitative RT-PCR. We also examined whether a schizophrenia risk-associated promoter SNP in GAD1 (rs3749034) is related to expression of these transcripts. Our studies revealed that development and maturation of both the PFC and hippocampal formation are characterized by progressive switches in expression from GAD25 to GAD67 and from NKCC1 to KCC2. Previous studies have demonstrated that the former leads to GABA synthesis, and the latter leads to switching from excitatory to inhibitory neurotransmission. In the hippocampal formation, GAD25/GAD67 and NKCC1/KCC2 ratios are increased in patients with schizophrenia, reflecting a potentially immature GABA physiology. Remarkably, GAD25/GAD67 and NKCC1/KCC2 expression ratios are associated with rs3749034 genotype, with risk alleles again predicting a relatively less mature pattern. These findings suggest that abnormalities in GABA signaling critical to brain development contribute to genetic risk for schizophrenia.

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

PubMed

Robert, S; Van Huynegem, K; Gysemans, C; Mathieu, C; Rottiers, P; Steidler, L

2015-01-01

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Prediction of thyroid C-cell carcinogenicity after chronic administration of GLP1-R agonists in rodents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brink, Willem van den; Emerenciana, Annette

Increased incidence of C-cell carcinogenicity has been observed for glucagon-like-protein-1 receptor (GLP-1r) agonists in rodents. It is suggested that the duration of exposure is an indicator of carcinogenic potential in rodents of the different products on the market. Furthermore, the role of GLP-1-related mechanisms in the induction of C-cell carcinogenicity has gained increased attention by regulatory agencies. This study proposes an integrative pharmacokinetic/pharmacodynamic (PKPD) framework to identify explanatory factors and characterize differences in carcinogenic potential of the GLP-1r agonist products. PK models for four products (exenatide QW (once weekly), exenatide BID (twice daily), liraglutide and lixisenatide) were developed using nonlinearmore » mixed effects modelling. Predicted exposure was subsequently linked to GLP-1r stimulation using in vitro GLP-1r potency data. A logistic regression model was then applied to exenatide QW and liraglutide data to assess the relationship between GLP-1r stimulation and thyroid C-cell hyperplasia incidence as pre-neoplastic predictor of a carcinogenic response. The model showed a significant association between predicted GLP-1r stimulation and C-cell hyperplasia after 2 years of treatment. The predictive performance of the model was evaluated using lixisenatide, for which hyperplasia data were accurately described during the validation step. The use of a model-based approach provided insight into the relationship between C-cell hyperplasia and GLP-1r stimulation for all four products, which is not possible with traditional data analysis methods. It can be concluded that both pharmacokinetics (exposure) and pharmacodynamics (potency for GLP-1r) factors determine C-cell hyperplasia incidence in rodents. Our work highlights the pharmacological basis for GLP-1r agonist-induced C-cell carcinogenicity. The concept is promising for application to other drug classes. - Highlights: • An integrative PKPD model is

GLP-1 acts on habenular avoidance circuits to control nicotine intake.

PubMed

Tuesta, Luis M; Chen, Zuxin; Duncan, Alexander; Fowler, Christie D; Ishikawa, Masago; Lee, Brian R; Liu, Xin-An; Lu, Qun; Cameron, Michael; Hayes, Matthew R; Kamenecka, Theodore M; Pletcher, Matthew; Kenny, Paul J

2017-05-01

Tobacco smokers titrate their nicotine intake to avoid its noxious effects, sensitivity to which may influence vulnerability to tobacco dependence, yet mechanisms of nicotine avoidance are poorly understood. Here we show that nicotine activates glucagon-like peptide-1 (GLP-1) neurons in the nucleus tractus solitarius (NTS). The antidiabetic drugs sitagliptin and exenatide, which inhibit GLP-1 breakdown and stimulate GLP-1 receptors, respectively, decreased nicotine intake in mice. Chemogenetic activation of GLP-1 neurons in NTS similarly decreased nicotine intake. Conversely, Glp1r knockout mice consumed greater quantities of nicotine than wild-type mice. Using optogenetic stimulation, we show that GLP-1 excites medial habenular (MHb) projections to the interpeduncular nucleus (IPN). Activation of GLP-1 receptors in the MHb-IPN circuit abolished nicotine reward and decreased nicotine intake, whereas their knockdown or pharmacological blockade increased intake. GLP-1 neurons may therefore serve as 'satiety sensors' for nicotine that stimulate habenular systems to promote nicotine avoidance before its aversive effects are encountered.

Endogenous GLP-1 as a key self-defense molecule against lipotoxicity in pancreatic islets.

PubMed

Huang, Chenghu; Yuan, Li; Cao, Shuyi

2015-07-01

The number of pro-α cells is known to increase in response to β cell injury and these cells then generate glucagon-like peptide-1 (GLP-1), thus attenuating the development of diabetes. The aim of the present study was to further examine the role and the mechanisms responsible for intra-islet GLP-1 production as a self-protective response against lipotoxicity. The levels of the key enzyme, prohormone convertase 1/3 (PC1/3), as well as the synthesis and release of GLP-1 in models of lipotoxicity were measured. Furthermore, islet viability, apoptosis, oxidative stress and inflammation, as well as islet structure were assessed after altering GLP-1 receptor signaling. Both prolonged exposure to palmitate and a high-fat diet facilitated PC1/3 expression, as well as the synthesis and release of GLP-1 induced by β cell injury and the generation of pro-α cells. Prolonged exposure to palmitate increased reactive oxygen species (ROS) production, and the antioxidant, N-acetylcysteine (NAC), partially prevented the detrimental effects induced by palmitate on β cells, resulting in decreased GLP-1 levels. Furthermore, the inhibition of GLP-1 receptor (GLP-1R) signaling by treatment with exendin‑(9-39) further decreased cell viability, increased cell apoptosis and caused a stronger inhibition of the β cell-specific transcription factor, pancreatic duodenal homeobox 1 (PDX1). Moreover, treatment with the GLP-1R agonist, liraglutide, normalized islet structure and function, resulting in a decrease in cell death and in the amelioration of β cell marker expression. Importantly, liraglutide maintained the oxidative balance and decreased inflammatory factor and p65 expression. Overall, our data demonstrate that an increase in the number of pro-α cells and the activation of the intra-islet GLP-1 system comprise a self-defense mechanism for enhancing β cell survival to combat lipid overload, which is in part mediated by oxidative stress and inflammation.

GLP-1 Receptor Stimulation of the Lateral Parabrachial Nucleus Reduces Food Intake: Neuroanatomical, Electrophysiological, and Behavioral Evidence

PubMed Central

Richard, Jennifer E.; Farkas, Imre; Anesten, Fredrik; Anderberg, Rozita H.; Dickson, Suzanne L.; Gribble, Fiona M.; Reimann, Frank; Jansson, John-Olov; Liposits, Zsolt

2014-01-01

The parabrachial nucleus (PBN) is a key nucleus for the regulation of feeding behavior. Inhibitory inputs from the hypothalamus to the PBN play a crucial role in the normal maintenance of feeding behavior, because their loss leads to starvation. Viscerosensory stimuli result in neuronal activation of the PBN. However, the origin and neurochemical identity of the excitatory neuronal input to the PBN remain largely unexplored. Here, we hypothesize that hindbrain glucagon-like peptide 1 (GLP-1) neurons provide excitatory inputs to the PBN, activation of which may lead to a reduction in feeding behavior. Our data, obtained from mice expressing the yellow fluorescent protein in GLP-1-producing neurons, revealed that hindbrain GLP-1-producing neurons project to the lateral PBN (lPBN). Stimulation of lPBN GLP-1 receptors (GLP-1Rs) reduced the intake of chow and palatable food and decreased body weight in rats. It also activated lPBN neurons, reflected by an increase in the number of c-Fos-positive cells in this region. Further support for an excitatory role of GLP-1 in the PBN is provided by electrophysiological studies showing a remarkable increase in firing of lPBN neurons after Exendin-4 application. We show that within the PBN, GLP-1R activation increased gene expression of 2 energy balance regulating peptides, calcitonin gene-related peptide (CGRP) and IL-6. Moreover, nearly 70% of the lPBN GLP-1 fibers innervated lPBN CGRP neurons. Direct intra-lPBN CGRP application resulted in anorexia. Collectively, our molecular, anatomical, electrophysiological, pharmacological, and behavioral data provide evidence for a functional role of the GLP-1R for feeding control in the PBN. PMID:25116706

Rate of Homologous Desensitization and Internalization of the GLP-1 Receptor.

PubMed

Shaaban, Ghina; Oriowo, Mabayoje; Al-Sabah, Suleiman

2016-12-26

The glucagon-like peptide-1 receptor (GLP-1R) is an important target in the treatment of type 2 diabetes mellitus. The aim of this study was to compare the rate of agonist stimulated desensitization and internalization of GLP-1R. To this end, an N-terminally myc-tagged GLP-1R was stably expressed in HEK-293 cells. Homologous desensitization was assessed by measuring the cAMP response to agonist stimulation following pre-incubation with agonist for up to 120 min. Receptor internalization was monitored using an indirect ELISA-based method and confocal microscopy. Pre-incubation with GLP-1 resulted in a time-dependent loss of response to a second stimulation. Washing cells following pre-incubation failed to bring cAMP levels back to basal. Taking this into account, two desensitization rates were calculated: "apparent" (t 1/2 = 19.27 min) and "net" (t 1/2 = 2.99 min). Incubation of cells with GLP-1 also resulted in a time-dependent loss of receptor cell surface expression (t 1/2 = 2.05 min). Rapid agonist-stimulated internalization of GLP-1R was confirmed using confocal microscopy. Stimulation of GLP-1R with GLP-1 results in rapid desensitization and internalization of the receptor. Interestingly, the rate of "net" desensitization closely matches the rate of internalization. Our results suggest that agonist-bound GLP-1R continues to generate cAMP after it has been internalized.

FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing's sarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Liu, E-mail: lyang@u.washington.edu; Medical Research Service, VA Puget Sound Health Care System, Seattle, WA 98108; Hu, Hsien-Ming

2010-11-05

Research highlights: {yields} Inducible and reversible siRNA knockdown of an oncogenic fusion protein such as EWS-Fli1 is feasible and more advantageous than other siRNA methods. {yields} The tumor suppressor gene FOXO1 is a new EWS-Fli1 target. {yields} While trans-activators are known for the FOXO1 gene, there has been no report on negative regulators of FOXO1 transcription. {yields} This study provides first evidence that the EWS-Fli1 oncogenic fusion protein can function as a transcriptional repressor of the FOXO1 gene. -- Abstract: Ewing's family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusionmore » protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing's cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing's sarcoma cells. Here we show that FOXO1 expression in Ewing's cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing's family tumors.« less

Validation of the Generalized Anxiety Disorder-7 (GAD-7) and GAD-2 in patients with migraine.

PubMed

Seo, Jong-Geun; Park, Sung-Pa

2015-01-01

Psychiatric problems have been commonly reported in patients with migraine. This study investigated the reliability and validity of the Generalized Anxiety Disorder-7 (GAD-7) and Generalized Anxiety Disorder-2 (GAD-2) in patients with migraine. Subjects were recruited from a headache clinic and a neuropsychologist examined their GAD using the Mini International Neuropsychiatric Interview-Plus Version 5.0.0 (MINI). Subjects completed several instruments, including the GAD-7, the Beck Anxiety Inventory (BAI), the Migraine Disability Assessment Scale (MIDAS), the Headache Impact Test-6 (HIT-6), and the Migraine-Specific Quality of Life (MSQoL). Among 146 participants, 32 patients (21.9 %) had GAD as determined by the MINI. Cronbach's α for the GAD-7 and GAD-2 were 0.915 and 0.820, respectively. At a cutoff score of 5, the GAD-7 had a sensitivity of 78.1 %, a specificity of 74.6 %, a positive predictive value (PPV) of 46.3 %, and a negative predictive value (NPV) of 92.4 %. At a cutoff score of 1, the GAD-2 had a sensitivity of 84.4 %, a specificity of 72.8 %, a PPV of 46.6 %, and a NPV of 94.3 %. The scores of the GAD-7 and GAD-2 well correlated with the BAI score, the MIDAS score, the HIT-6 score, and the MSQoL score. The GAD-7 and GAD-2 are both reliable and valid screening instruments for GAD in patients with migraine.

EGO-1, a Putative RNA-Directed RNA Polymerase, Promotes Germline Proliferation in Parallel With GLP-1/Notch Signaling and Regulates the Spatial Organization of Nuclear Pore Complexes and Germline P Granules in Caenorhabditis elegans

PubMed Central

Vought, Valarie E.; Ohmachi, Mitsue; Lee, Min-Ho; Maine, Eleanor M.

2005-01-01

Caenorhabditis elegans EGO-1, a putative cellular RNA-directed RNA polymerase, promotes several aspects of germline development, including proliferation, meiosis, and gametogenesis, and ensures a robust response to RNA interference. In C. elegans, GLP-1/Notch signaling from the somatic gonad maintains a population of proliferating germ cells, while entry of germ cells into meiosis is triggered by the GLD-1 and GLD-2 pathways. GLP-1 signaling prevents germ cells from entering meiosis by inhibiting GLD-1 and GLD-2 activity. We originally identified the ego-1 gene on the basis of a genetic interaction with glp-1. Here, we investigate the role of ego-1 in germline proliferation. Our data indicate that EGO-1 does not positively regulate GLP-1 protein levels or GLP-1 signaling activity. Moreover, GLP-1 signaling does not positively regulate EGO-1 activity. EGO-1 does not inhibit expression of GLD-1 protein in the distal germline. Instead, EGO-1 acts in parallel with GLP-1 signaling to influence the proliferation vs. meiosis fate choice. Moreover, EGO-1 and GLD-1 act in parallel to ensure germline health. Finally, the size and distribution of nuclear pore complexes and perinuclear P granules are altered in the absence of EGO-1, effects that disrupt germ cell biology per se and probably limit germline growth. PMID:15911573

The amyloid fold of Gad m 1 epitopes governs IgE binding

PubMed Central

Sánchez, Rosa; Martínez, Javier; Castro, Ana; Pedrosa, María; Quirce, Santiago; Rodríguez-Pérez, Rosa; Gasset, María

2016-01-01

Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. β-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish β-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy. PMID:27597317

Hepatic functions of GLP-1 and its based drugs: current disputes and perspectives.

PubMed

Jin, Tianru; Weng, Jianping

2016-09-01

GLP-1 and its based drugs possess extrapancreatic metabolic functions, including that in the liver. These direct hepatic metabolic functions explain their therapeutic efficiency for subjects with insulin resistance. The direct hepatic functions could be mediated by previously assumed "degradation" products of GLP-1 without involving canonic GLP-1R. Although GLP-1 analogs were created as therapeutic incretins, extrapancreatic functions of these drugs, as well as native GLP-1, have been broadly recognized. Among them, the hepatic functions are particularly important. Postprandial GLP-1 release contributes to insulin secretion, which represses hepatic glucose production. This indirect effect of GLP-1 is known as the gut-pancreas-liver axis. Great efforts have been made to determine whether GLP-1 and its analogs possess direct metabolic effects on the liver, as the determination of the existence of direct hepatic effects may advance the therapeutic theory and clinical practice on subjects with insulin resistance. Furthermore, recent investigations on the metabolic beneficial effects of previously assumed "degradation" products of GLP-1 in the liver and elsewhere, including GLP-128-36 and GLP-132-36, have drawn intensive attention. Such investigations may further improve the development and the usage of GLP-1-based drugs. Here, we have reviewed the current advancement and the existing controversies on the exploration of direct hepatic functions of GLP-1 and presented our perspectives that the direct hepatic metabolic effects of GLP-1 could be a GLP-1 receptor-independent event involving Wnt signaling pathway activation. Copyright © 2016 the American Physiological Society.

The autonomic nervous system and cardiac GLP-1 receptors control heart rate in mice.

PubMed

Baggio, Laurie L; Ussher, John R; McLean, Brent A; Cao, Xiemin; Kabir, M Golam; Mulvihill, Erin E; Mighiu, Alexandra S; Zhang, Hangjun; Ludwig, Andreas; Seeley, Randy J; Heximer, Scott P; Drucker, Daniel J

2017-11-01

Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine cells and exerts a broad number of metabolic actions through activation of a single GLP-1 receptor (GLP-1R). The cardiovascular actions of GLP-1 have garnered increasing attention as GLP-1R agonists are used to treat human subjects with diabetes and obesity that may be at increased risk for development of heart disease. Here we studied mechanisms linking GLP-1R activation to control of heart rate (HR) in mice. The actions of GLP-1R agonists were examined on the control of HR in wild type mice (WT) and in mice with cardiomyocyte-selective disruption of the GLP-1R (Glp1r CM-/- ). Complimentary studies examined the effects of GLP-1R agonists in mice co-administered propranolol or atropine. The direct effects of GLP-1R agonism on HR and ventricular developed pressure were examined in isolated perfused mouse hearts ex vivo, and atrial depolarization was quantified in mouse hearts following direct application of liraglutide to perfused atrial preparations ex vivo. Doses of liraglutide and lixisenatide that were equipotent for acute glucose control rapidly increased HR in WT and Glp1r CM-/- mice in vivo. The actions of liraglutide to increase HR were more sustained relative to lixisenatide, and diminished in Glp1r CM-/- mice. The acute chronotropic actions of GLP-1R agonists were attenuated by propranolol but not atropine. Neither native GLP-1 nor lixisenatide increased HR or developed pressure in perfused hearts ex vivo. Moreover, liraglutide had no direct effect on sinoatrial node firing rate in mouse atrial preparations ex vivo. Despite co-localization of HCN4 and GLP-1R in primate hearts, HCN4-directed Cre expression did not attenuate levels of Glp1r mRNA transcripts, but did reduce atrial Gcgr expression in the mouse heart. GLP-1R agonists increase HR through multiple mechanisms, including regulation of autonomic nervous system function, and activation of the atrial GLP-1R. Surprisingly, the

Crystal structure of the C-terminal SH3 domain of the adaptor protein GADS in complex with SLP-76 motif peptide reveals a unique SH3-SH3 interaction.

PubMed

Dimasi, Nazzareno

2007-01-01

The Grb2-like adaptor protein GADS is essential for tyrosine kinase-dependent signaling in T lymphocytes. Following T cell receptor ligation, GADS interacts through its C-terminal SH3 domain with the adaptors SLP-76 and LAT, to form a multiprotein signaling complex that is crucial for T cell activation. To understand the structural basis for the selective recognition of GADS by SLP-76, herein is reported the crystal structure at 1.54 Angstrom of the C-terminal SH3 domain of GADS bound to the SLP-76 motif 233-PSIDRSTKP-241, which represents the minimal binding site. In addition to the unique structural features adopted by the bound SLP-76 peptide, the complex structure reveals a unique SH3-SH3 interaction. This homophilic interaction, which is observed in presence of the SLP-76 peptide and is present in solution, extends our understanding of the molecular mechanisms that could be employed by modular proteins to increase their signaling transduction specificity.

Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro

To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two typesmore » of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.« less

GLP-1 secretion is stimulated by 1,10-phenanthroline via colocalized T2R5 signal transduction in human enteroendocrine L cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jiyoung; Kim, Ki-Suk; Kim, Kang-Hoon

Glucagon-like peptide-1 (GLP-1) hormone is known to regulate blood glucose by an insulinotropic effect and increases proliferation as and also prevents apoptosis of pancreatic β cells. We know that GLP-1 is secreted by nutrients such as fatty acids and sweet compounds but also bitter compounds via stimulation of G-protein coupled receptors (GPCRs) in the gut. Among these, bitter compounds are multiply-contained in phytochemicals or artificial materials and perceived as ligands of various bitter taste receptors. We hypothesized that GLP-1 hormone is secreted through stimulation of a single bitter taste receptor by 1,10-phenanthroline which is known agonist of taste receptor typemore » 2 member 5 (T2R5). To prove this hypothesis, we used the representatively well-known 1,10-phenanthroline as ligand of single receptor and evaluated the existence of T2R5 by double-labeling immunofluorescence and then 1,10-phenanthroline is able to secrete GLP-1 hormone through stimulation of T2R5 in human enteroendocrine cells. Consequently, we verify that GLP-1 hormone is colocalized with T2R5 in the human duodenum and ileum tissue and is secreted by 1,10-phenanthroline via T2R5 signal transduction in differentiated human enteroendocrine L cells. - Highlights: • Taste receptor type 2 member 5 (T2R5) is colocalized with GLP-1 hormone in human enteroendocrine cells. • GLP-1 secretion is stimulated by 1,10-phenanthroline via stimulation of T2R5. • Inhibition of the bitter taste pathway reduce GLP-1 secretion.« less

Functional importance of GLP-1 receptor species and expression levels in cell lines.

PubMed

Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

2012-04-10

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

PubMed

Ferry, Laure; Fournier, Alexandra; Tsusaka, Takeshi; Adelmant, Guillaume; Shimazu, Tadahiro; Matano, Shohei; Kirsh, Olivier; Amouroux, Rachel; Dohmae, Naoshi; Suzuki, Takehiro; Filion, Guillaume J; Deng, Wen; de Dieuleveult, Maud; Fritsch, Lauriane; Kudithipudi, Srikanth; Jeltsch, Albert; Leonhardt, Heinrich; Hajkova, Petra; Marto, Jarrod A; Arita, Kyohei; Shinkai, Yoichi; Defossez, Pierre-Antoine

2017-08-17

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

Hindbrain GLP-1 receptor mediation of cisplatin-induced anorexia and nausea.

PubMed

De Jonghe, Bart C; Holland, Ruby A; Olivos, Diana R; Rupprecht, Laura E; Kanoski, Scott E; Hayes, Matthew R

2016-01-01

While chemotherapy-induced nausea and vomiting are clinically controlled in the acute (<24 h) phase following treatment, the anorexia, nausea, fatigue, and other illness-type behaviors during the delayed phase (>24 h) of chemotherapy are largely uncontrolled. As the hindbrain glucagon-like peptide-1 (GLP-1) system contributes to energy balance and mediates aversive and stressful stimuli, here we examine the hypothesis that hindbrain GLP-1 signaling mediates aspects of chemotherapy-induced nausea and reductions in feeding behavior in rats. Specifically, hindbrain GLP-1 receptor (GLP-1R) blockade, via 4th intracerebroventricular (ICV) exendin-(9-39) injections, attenuates the anorexia, body weight reduction, and pica (nausea-induced ingestion of kaolin clay) elicited by cisplatin chemotherapy during the delayed phase (48 h) of chemotherapy-induced nausea. Additionally, the present data provide evidence that the central GLP-1-producing preproglucagon neurons in the nucleus tractus solitarius (NTS) of the caudal brainstem are activated by cisplatin during the delayed phase of chemotherapy-induced nausea, as cisplatin led to a significant increase in c-Fos immunoreactivity in NTS GLP-1-immunoreactive neurons. These data support a growing body of literature suggesting that the central GLP-1 system may be a potential pharmaceutical target for adjunct anti-emetics used to treat the delayed-phase of nausea and emesis, anorexia, and body weight loss that accompany chemotherapy treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

Novel coumarin modified GLP-1 derivatives with enhanced plasma stability and prolonged in vivo glucose-lowering ability.

PubMed

Han, Jing; Sun, Lidan; Huang, Xun; Li, Zheng; Zhang, Chenyu; Qian, Hai; Huang, Wenlong

2014-12-01

The short biological half-life limits the therapeutic use of glucagon-like peptide-1 (GLP-1) and chemical modification to improve the interaction of peptides with serum albumin represents an effective strategy to develop long-acting peptide analogues. Coumarin, a natural product, is known to bind tightly to plasma proteins and possesses many biological activities. Therefore, we designed and synthesized a series of coumarin-modified GLP-1 derivatives, hypothesizing that conjugation with coumarin would retain the therapeutic effects and prolong the biological half-life of the conjugates. Four cysteine-modified GLP-1 analogues (1-4) were prepared using Gly8 -GLP-1(7-36)-NH2 peptide as a starting point. These analogues were conjugated with two coumarin maleimides to yield eight compounds (conjugates 6-13) for testing. Activation of human GLP-1 receptors, stability to enzymic inactivation in plasma and binding to human albumin were assessed in vitro. In vivo, effects on oral glucose tolerance tests (OGTT) in rats and on blood glucose levels in db/db mice were studied. Most conjugates showed well preserved receptor activation efficacy, enhanced albumin-binding properties and improved in vitro plasma stability and conjugate 7 was selected to undergo further assessment. In rats, conjugate 7 had a longer circulating t1/2 than exendin-4 or liraglutide. A prolonged antidiabetic effect of conjugate 7 was observed after OGTT in rats and a prolonged hypoglycaemic effect in db/db mice. Cysteine-specific coumarin conjugation with GLP-1 offers a useful approach to the development of long-acting incretin-based antidiabetic agents. Conjugate 7 is a promising long-lasting GLP-1 derivative deserving further investigation. © 2014 The British Pharmacological Society.

GlpR is a direct transcriptional repressor of fructose metabolic genes in Haloferax volcanii.

PubMed

Martin, Jonathan H; Rawls, Katie Sherwood; Chan, Jou Chin; Hwang, Sungmin; Martinez-Pastor, Mar; McMillan, Lana J; Prunetti, Laurence; Schmid, Amy K; Maupin-Furlow, Julie A

2018-06-18

DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and occur in select archaea. In the model archaeon Haloferax volcanii , previous work implicated GlpR, a DeoR-type transcriptional regulator, in transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase ( pfkB ) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here we compared transcriptomes of wild type and Δ glpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that Hfx. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators. IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents broad use of archaea as microbial factories for industrial products. Here we characterize how sugar uptake and use is regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in

Liraglutide Modulates Appetite and Body Weight Via GLP-1R-Expressing Glutamatergic Neurons.

PubMed

Adams, Jessica M; Pei, Hongjuan; Sandoval, Darleen A; Seeley, Randy J; Chang, Rui B; Liberles, Stephen D; Olson, David P

2018-05-18

Glucagon-like peptide-1 receptor (GLP-1R) agonists are FDA-approved weight loss drugs. Despite their widespread use, the sites of action through which GLP-1R agonists (GLP1RAs) impact appetite and body weight are still not fully understood. Here, we determined whether GLP-1Rs in either GABAergic or glutamatergic neurons are necessary for the acute and chronic effects of the GLP1RA liraglutide on food intake, visceral illness, body weight and neural network activation. We found that mice lacking GLP-1Rs in vGAT -expressing GABAergic neurons responded identically to controls in all parameters measured, whereas deletion of GLP-1Rs in vGlut2 -expressing glutamatergic neurons eliminated liraglutide-induced weight loss and visceral illness and severely attenuated its effects on feeding. Concomitantly, deletion of GLP-1Rs from glutamatergic neurons completely abolished the neural network activation observed after liraglutide administration. We conclude that liraglutide activates a dispersed but discrete neural network to mediate its physiological effects, and that these effects require GLP-1R expression on glutamatergic but not GABAergic neurons. © 2018 by the American Diabetes Association.

GLP-1 agonists for type 2 diabetes: pharmacokinetic and toxicological considerations.

PubMed

Jespersen, Maria J; Knop, Filip K; Christensen, Mikkel

2013-01-01

Within recent years, glucagon-like peptide 1 receptor agonists (GLP-1-RA) have emerged as a new treatment option for type 2 diabetes. The GLP-1-RA are administered subcutaneously and differ substantially in pharmacokinetic profiles. This review describes the pharmacokinetics and safety aspects of the currently available GLP-1 receptor agonists, liraglutide (based on the structure of native GLP-1), exenatide twice daily and exenatide once weekly (based on exendin-4) in relation to the kinetics and toxicology of native GLP-1. The review is based on electronic literature searches and legal documents in the form of assessment reports from the European Medicines Agency and the United States Food and Drug Administration. GLP-1-based therapy combines several unique mechanisms of action and have the potential to gain widespread use in the fight against diabetes and obesity. The difference in chemical structure have strong implications for key pharmacokinetic parameters such as absorption and clearance, and eventually the safety and efficacy of the individual GLP-1-RA. The main safety concerns are pancreatitis and neoplasms, for which there are no identifiable differences in risk between the available agents. Antibody formation and injection site reactions are more frequent with the exendin-4-based compounds. The efficacy with regard to Hb(A1c) reduction is superior with the longer-acting agonists, whereas the shorter-acting GLP-1-RA seems to provide greater postprandial glucose control and lower tolerability as a possible consequence of less induction of tachyphylaxis. The future place of these agents will depend on the added safety and efficacy data in the several ongoing cardiovascular outcome trials.

A surface plasmon resonance assay for characterisation and epitope mapping of anti-GLP-1 antibodies.

PubMed

Thomsen, Lasse; Gurevich, Leonid

2018-04-19

The incretin hormone glucagon-like peptide-1 (GLP-1) has been subject to substantial pharmaceutical research regarding the treatment of type 2 diabetes mellitus. However, quantification of GLP-1 levels remains complicated due to the low circulation concentration and concurrent existence of numerous metabolites, homologous peptides, and potentially introduced GLP-1 receptor agonists. Surface plasmon resonance (SPR) facilitates real-time monitoring allowing a more detailed characterisation of the interaction compared with conventional enzyme-linked immunosorbent assays (ELISA). In this paper, we describe the development of the first SPR assays for characterisation of anti-GLP-1 antibodies for ELISA purposes. Binding responses were obtained on covalently immobilised anti-GLP-1 antibodies at 12°C, 25°C, and 40°C and fitted to a biomolecular (1:1) interaction model showing association rates of 1.01 × 10 3 to 4.54 × 10 3  M -1  s -1 and dissociation rates of 3.56 × 10 -5 to 1.56 × 10 -3  s -1 leading to affinities of 35.2 to 344 nM, depending on the temperature. Determination of thermodynamic properties revealed an enthalpy driven interaction (ΔH < ΔS < 0) with higher affinities at lower temperatures due to the formation and stabilisation of hydrogen bonds within the binding site primarily composed of polar amino acids (ΔC p  < 0). Pair-wise epitope mapping was performed on captured anti-GLP-1 antibodies followed by subsequent interaction with GLP-1 (7-36) and other anti-GLP-1 antibodies. A global evaluation of every binding response led to an epitope map elucidating the potential of various anti-GLP-1 antibody pairs for sandwich ELISA and hence pinpointing the optimal antibody combinations. The SPR assays proved capable of providing vital information for ELISA development endorsing it as a useful optimisation tool. Copyright © 2018 John Wiley & Sons, Ltd.

Flavonoid-rich extract of Chromolaena odorata modulate circulating GLP-1 in Wistar rats: computational evaluation of TGR5 involvement.

PubMed

Omotuyi, Olaposi Idowu; Nash, Oyekanmi; Inyang, Olumide Kayode; Ogidigo, Joyce; Enejoh, Ojochenemi; Okpalefe, Okiemute; Hamada, Tsuyoshi

2018-02-01

Chromolaena odorata is a major bio-resource in folkloric treatment of diabetes. In the present study, its anti-diabetic component and underlying mechanism were investigated. A library containing 140 phytocompounds previously characterized from C. odorata was generated and docked (Autodock Vina) into homology models of dipeptidyl peptidase (DPP)-4, Takeda-G-protein-receptor-5 (TGR5), glucagon-like peptide 1 (GLP1) receptor, renal sodium dependent glucose transporter (SGLUT)-1/2 and nucleotide-binding oligomerization domain (NOD) proteins 1&2. GLP-1 gene (RT-PCR) modulation and its release (EIA) by C. odorata were confirmed in vivo. From the docking result above, TGR5 was identified as a major target for two key C. odorata flavonoids (5,7-dihydroxy-6-4-dimethoxyflavanone and homoesperetin-7-rutinoside); sodium taurocholate and C. odorata powder included into the diet of the animals both raised the intestinal GLP-1 expression versus control ( p  < 0.05); When treated with flavonoid-rich extract of C. odorata (CoF) or malvidin, circulating GLP-1 increased by 130.7% in malvidin-treated subjects (0 vs. 45 min). CoF treatment also resulted in 128.5 and 275% increase for 10 and 30 mg/kg b.w., respectively. The results of this study support that C. odorata flavonoids may modulate the expression of GLP-1 and its release via TGR5. This finding may underscore its anti-diabetic potency.

Endogenous GLP-1 in lateral septum contributes to stress-induced hypophagia.

PubMed

Terrill, Sarah J; Maske, Calyn B; Williams, Diana L

2018-03-03

Glucagon-like peptide 1 (GLP-1) neurons of the caudal brainstem project to many brain areas, including the lateral septum (LS), which has a known role in stress responses. Previously, we showed that endogenous GLP-1 in the LS plays a physiologic role in the control of feeding under non-stressed conditions, however, central GLP-1 is also involved in behavioral and endocrine responses to stress. Here, we asked whether LS GLP-1 receptors (GLP-1R) contribute to stress-induced hypophagia. Male rats were implanted with bilateral cannulas targeting the dorsal subregion of the LS (dLS). In a within-subjects design, shortly before the onset of the dark phase, rats received dLS injections of saline or the GLP-1R antagonist Exendin (9-39) (Ex9) prior to 30 min restraint stress. Food intake was measured continuously for the next 20 h. The stress-induced hypophagia observed within the first 30 min of dark was not influenced by Ex9 pretreatment, but Ex9 tended to blunt the effect of stress as early as 1 and 2 h into the dark phase. By 4-6 h, there were significant stress X drug interactions, and Ex9 pretreatment blocked the stress-induced suppression of feeding. These effects were mediated entirely through changes in average meal size; stress suppressed meal size while dLS Ex9 attenuated this effect. Using a similar design, we examined the role of dLS GLP-1R in the neuroendocrine response to acute restraint stress. As expected, stress potently increased serum corticosterone, but blockade of dLS GLP-1Rs did not affect this response. Together, these data show that endogenous GLP-1 action in the dLS plays a role in some but not all of the physiologic responses to acute stress. Copyright © 2018 Elsevier Inc. All rights reserved.

The GIP receptor displays higher basal activity than the GLP-1 receptor but does not recruit GRK2 or arrestin3 effectively.

PubMed

Al-Sabah, Suleiman; Al-Fulaij, Munya; Shaaban, Ghina; Ahmed, Hanadi A; Mann, Rosalind J; Donnelly, Dan; Bünemann, Moritz; Krasel, Cornelius

2014-01-01

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients. GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET). GIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding. GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.

Water Permeation Across Biological Membranes: Mechanism and Dynamics of Aquaporin-1 and GlpF

NASA Astrophysics Data System (ADS)

de Groot, Bert L.; Grubmüller, Helmut

2001-12-01

``Real time'' molecular dynamics simulations of water permeation through human aquaporin-1 (AQP1) and the bacterial glycerol facilitator GlpF are presented. We obtained time-resolved, atomic-resolution models of the permeation mechanism across these highly selective membrane channels. Both proteins act as two-stage filters: Conserved fingerprint [asparagine-proline-alanine (NPA)] motifs form a selectivity-determining region; a second (aromatic/arginine) region is proposed to function as a proton filter. Hydrophobic regions near the NPA motifs are rate-limiting water barriers. In AQP1, a fine-tuned water dipole rotation during passage is essential for water selectivity. In GlpF, a glycerol-mediated ``induced fit'' gating motion is proposed to generate selectivity for glycerol over water.

Aspergillus glaucus Aquaglyceroporin Gene glpF Confers High Osmosis Tolerance in Heterologous Organisms.

PubMed

Liu, Xiao-Dan; Wei, Yi; Zhou, Xiao-Yang; Pei, Xue; Zhang, Shi-Hong

2015-10-01

Aquaglyceroporins (GlpFs) that transport glycerol along with water and other uncharged solutes are involved in osmoregulation in myriad species. Fungal species form a large group of eukaryotic organisms, and their GlpFs may be diverse, exhibiting various activities. However, few filamentous fungal GlpFs have been biologically investigated. Here, a glpF gene from the halophilic fungus Aspergillus glaucus (AgglpF) was verified to be a channel of water or glycerol in Xenopus laevis oocytes and was further functionally analyzed in three heterologous systems. In Saccharomyces cerevisiae, cells overexpressing AgglpF possessed significant tolerance of drought, salt, and certain metal ions. AgglpF was then characterized in the filamentous fungus of Neurospora crassa. Based on the N. crassa aquaporin gene (NcAQP) disruption mutant (the Δaqp mutant), a series of complementary strains carrying NcAQP and AgglpF and three asparagine-proline-alanine-gene (NPA)-deleted AgglpF fragments were created. As revealed by salt resistance analysis, the AgglpF complementary strain possessed the highest salt resistance among the tested strains. In addition, the intracellular glycerol content in the AgglpF complementary strain was markedly higher than that in the other strains. The AgGlpF-green fluorescent protein (GFP) fusion protein was subcellularly localized in the plasma membrane of onion epidermal cells, suggesting that AgglpF functions in plants. Indeed, when AgglpF was expressed in Arabidopsis thaliana, transgenic lines survived under conditions of high osmotic stress and under conditions of drought stress in particular. Overall, our results revealed that AgGlpF as a water/glycerol transporter is required for survival of both fungi and plants under conditions of high osmotic stress and may have value in applications in genetic engineering for generating high salt and drought resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Aspergillus glaucus Aquaglyceroporin Gene glpF Confers High Osmosis Tolerance in Heterologous Organisms

PubMed Central

Liu, Xiao-Dan; Wei, Yi; Zhou, Xiao-Yang; Pei, Xue

2015-01-01

Aquaglyceroporins (GlpFs) that transport glycerol along with water and other uncharged solutes are involved in osmoregulation in myriad species. Fungal species form a large group of eukaryotic organisms, and their GlpFs may be diverse, exhibiting various activities. However, few filamentous fungal GlpFs have been biologically investigated. Here, a glpF gene from the halophilic fungus Aspergillus glaucus (AgglpF) was verified to be a channel of water or glycerol in Xenopus laevis oocytes and was further functionally analyzed in three heterologous systems. In Saccharomyces cerevisiae, cells overexpressing AgglpF possessed significant tolerance of drought, salt, and certain metal ions. AgglpF was then characterized in the filamentous fungus of Neurospora crassa. Based on the N. crassa aquaporin gene (NcAQP) disruption mutant (the Δaqp mutant), a series of complementary strains carrying NcAQP and AgglpF and three asparagine-proline-alanine-gene (NPA)-deleted AgglpF fragments were created. As revealed by salt resistance analysis, the AgglpF complementary strain possessed the highest salt resistance among the tested strains. In addition, the intracellular glycerol content in the AgglpF complementary strain was markedly higher than that in the other strains. The AgGlpF-green fluorescent protein (GFP) fusion protein was subcellularly localized in the plasma membrane of onion epidermal cells, suggesting that AgglpF functions in plants. Indeed, when AgglpF was expressed in Arabidopsis thaliana, transgenic lines survived under conditions of high osmotic stress and under conditions of drought stress in particular. Overall, our results revealed that AgGlpF as a water/glycerol transporter is required for survival of both fungi and plants under conditions of high osmotic stress and may have value in applications in genetic engineering for generating high salt and drought resistance. PMID:26209670

Modulation of taste sensitivity by GLP-1 signaling in taste buds.

PubMed

Martin, Bronwen; Dotson, Cedrick D; Shin, Yu-Kyong; Ji, Sunggoan; Drucker, Daniel J; Maudsley, Stuart; Munger, Steven D

2009-07-01

Modulation of sensory function can help animals adjust to a changing external and internal environment. Even so, mechanisms for modulating taste sensitivity are poorly understood. Using immunohistochemical, biochemical, and behavioral approaches, we found that the peptide hormone glucagon-like peptide-1 (GLP-1) and its receptor (GLP-1R) are expressed in mammalian taste buds. Furthermore, we found that GLP-1 signaling plays an important role in the modulation of taste sensitivity: GLP-1R knockout mice exhibit a dramatic reduction in sweet taste sensitivity as well as an enhanced sensitivity to umami-tasting stimuli. Together, these findings suggest a novel paracrine mechanism for the hormonal modulation of taste function in mammals.

Novel coumarin modified GLP-1 derivatives with enhanced plasma stability and prolonged in vivo glucose-lowering ability

PubMed Central

Han, Jing; Sun, Lidan; Huang, Xun; Li, Zheng; Zhang, Chenyu; Qian, Hai; Huang, Wenlong

2014-01-01

Background and Purpose The short biological half-life limits the therapeutic use of glucagon-like peptide-1 (GLP-1) and chemical modification to improve the interaction of peptides with serum albumin represents an effective strategy to develop long-acting peptide analogues. Coumarin, a natural product, is known to bind tightly to plasma proteins and possesses many biological activities. Therefore, we designed and synthesized a series of coumarin-modified GLP-1 derivatives, hypothesizing that conjugation with coumarin would retain the therapeutic effects and prolong the biological half-life of the conjugates. Experimental Approach Four cysteine-modified GLP-1 analogues (1–4) were prepared using Gly8-GLP-1(7–36)-NH2 peptide as a starting point. These analogues were conjugated with two coumarin maleimides to yield eight compounds (conjugates 6–13) for testing. Activation of human GLP-1 receptors, stability to enzymic inactivation in plasma and binding to human albumin were assessed in vitro. In vivo, effects on oral glucose tolerance tests (OGTT) in rats and on blood glucose levels in db/db mice were studied. Key Results Most conjugates showed well preserved receptor activation efficacy, enhanced albumin-binding properties and improved in vitro plasma stability and conjugate 7 was selected to undergo further assessment. In rats, conjugate 7 had a longer circulating t1/2 than exendin-4 or liraglutide. A prolonged antidiabetic effect of conjugate 7 was observed after OGTT in rats and a prolonged hypoglycaemic effect in db/db mice. Conclusions and Implications Cysteine-specific coumarin conjugation with GLP-1 offers a useful approach to the development of long-acting incretin-based antidiabetic agents. Conjugate 7 is a promising long-lasting GLP-1 derivative deserving further investigation. PMID:25039358

Liraglutide, a GLP-1 receptor agonist, inhibits vascular smooth muscle cell proliferation by enhancing AMP-activated protein kinase and cell cycle regulation, and delays atherosclerosis in ApoE deficient mice.

PubMed

Jojima, Teruo; Uchida, Kohsuke; Akimoto, Kazumi; Tomotsune, Takanori; Yanagi, Kazunori; Iijima, Toshie; Suzuki, Kunihiro; Kasai, Kikuo; Aso, Yoshimasa

2017-06-01

Several studies have demonstrated that both native glucagon-like peptide-1 (GLP-1) and GLP-1 receptor agonists suppress the progression of atherosclerosis in animal models. We investigated whether liraglutide, a GLP-1 analogue, could prevent the development of atherosclerosis in apolipoprotein E knockout mice (ApoE -/- ) on a high-fat diet. We also examined the influence of liraglutide on angiotensin II-induced proliferation of rat vascular smooth muscle cells (VSMCs) via enhancement of AMP-activated protein kinase (AMPK) signaling and regulation of cell cycle progression. Treatment of ApoE -/- mice with liraglutide (400 μg/day for 4 weeks) suppressed atherosclerotic lesions and increased AMPK phosphorylation in the aortic wall. Liraglutide also improved the endothelial function of thoracic aortas harvested from ApoE -/- mice in an ex vivo study. Furthermore, liraglutide increased AMPK phosphorylation in rat VSMCs, while liraglutide-induced activation of AMPK was abolished by exendin 9-39, a GLP-1 antagonist. Moreover, angiotensin (Ang) II-induced proliferation of VSMCs was suppressed by liraglutide in a dose-dependent manner, and flow cytometry of Ang II-stimulated VSMCs showed that liraglutide reduced the percentage of cells in G2/M phase (by arrest in G0/G1 phase). These findings suggest that liraglutide may inhibit Ang II-induced VSMC proliferation by activating AMPK signaling and inducing cell cycle arrest, thus delaying the progression of atherosclerosis independently of its glucose-lowering effect. Copyright © 2017 Elsevier B.V. All rights reserved.

PPG neurons of the lower brain stem and their role in brain GLP-1 receptor activation

PubMed Central

Cork, Simon C.

2015-01-01

Within the brain, glucagon-like peptide-1 (GLP-1) affects central autonomic neurons, including those controlling the cardiovascular system, thermogenesis, and energy balance. Additionally, GLP-1 influences the mesolimbic reward system to modulate the rewarding properties of palatable food. GLP-1 is produced in the gut and by hindbrain preproglucagon (PPG) neurons, located mainly in the nucleus tractus solitarii (NTS) and medullary intermediate reticular nucleus. Transgenic mice expressing glucagon promoter-driven yellow fluorescent protein revealed that PPG neurons not only project to central autonomic control regions and mesolimbic reward centers, but also strongly innervate spinal autonomic neurons. Therefore, these brain stem PPG neurons could directly modulate sympathetic outflow through their spinal inputs to sympathetic preganglionic neurons. Electrical recordings from PPG neurons in vitro have revealed that they receive synaptic inputs from vagal afferents entering via the solitary tract. Vagal afferents convey satiation to the brain from signals like postprandial gastric distention or activation of peripheral GLP-1 receptors. CCK and leptin, short- and long-term satiety peptides, respectively, increased the electrical activity of PPG neurons, while ghrelin, an orexigenic peptide, had no effect. These findings indicate that satiation is a main driver of PPG neuronal activation. They also show that PPG neurons are in a prime position to respond to both immediate and long-term indicators of energy and feeding status, enabling regulation of both energy balance and general autonomic homeostasis. This review discusses the question of whether PPG neurons, rather than gut-derived GLP-1, are providing the physiological substrate for the effects elicited by central nervous system GLP-1 receptor activation. PMID:26290108

PPG neurons of the lower brain stem and their role in brain GLP-1 receptor activation.

PubMed

Trapp, Stefan; Cork, Simon C

2015-10-15

Within the brain, glucagon-like peptide-1 (GLP-1) affects central autonomic neurons, including those controlling the cardiovascular system, thermogenesis, and energy balance. Additionally, GLP-1 influences the mesolimbic reward system to modulate the rewarding properties of palatable food. GLP-1 is produced in the gut and by hindbrain preproglucagon (PPG) neurons, located mainly in the nucleus tractus solitarii (NTS) and medullary intermediate reticular nucleus. Transgenic mice expressing glucagon promoter-driven yellow fluorescent protein revealed that PPG neurons not only project to central autonomic control regions and mesolimbic reward centers, but also strongly innervate spinal autonomic neurons. Therefore, these brain stem PPG neurons could directly modulate sympathetic outflow through their spinal inputs to sympathetic preganglionic neurons. Electrical recordings from PPG neurons in vitro have revealed that they receive synaptic inputs from vagal afferents entering via the solitary tract. Vagal afferents convey satiation to the brain from signals like postprandial gastric distention or activation of peripheral GLP-1 receptors. CCK and leptin, short- and long-term satiety peptides, respectively, increased the electrical activity of PPG neurons, while ghrelin, an orexigenic peptide, had no effect. These findings indicate that satiation is a main driver of PPG neuronal activation. They also show that PPG neurons are in a prime position to respond to both immediate and long-term indicators of energy and feeding status, enabling regulation of both energy balance and general autonomic homeostasis. This review discusses the question of whether PPG neurons, rather than gut-derived GLP-1, are providing the physiological substrate for the effects elicited by central nervous system GLP-1 receptor activation. Copyright © 2015 the American Physiological Society.

Central GLP-1 receptor signalling accelerates plasma clearance of triacylglycerol and glucose by activating brown adipose tissue in mice.

PubMed

Kooijman, Sander; Wang, Yanan; Parlevliet, Edwin T; Boon, Mariëtte R; Edelschaap, David; Snaterse, Gido; Pijl, Hanno; Romijn, Johannes A; Rensen, Patrick C N

2015-11-01

Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonism, used in the treatment of type 2 diabetes, has recently been shown to increase thermogenesis via the brain. As brown adipose tissue (BAT) produces heat by burning triacylglycerol (TG) and takes up glucose for de novo lipogenesis, the aim of this study was to evaluate the potential of chronic central GLP-1R activation by exendin-4 to facilitate clearance of lipids and glucose from the circulation by activating BAT. Lean and diet-induced obese (DIO) C57Bl/6J mice were used to explore the effect of a 5 day intracerebroventricular infusion of the GLP-1 analogue exendin-4 or vehicle on lipid and glucose uptake by BAT in both insulin-sensitive and insulin-resistant conditions. Central administration of exendin-4 in lean mice increased sympathetic outflow towards BAT and white adipose tissue (WAT), resulting in increased thermogenesis as evidenced by increased uncoupling protein 1 (UCP-1) protein levels and decreased lipid content, while the uptake of TG-derived fatty acids was increased in both BAT and WAT. Interestingly, in DIO mice, the effects on WAT were blunted, while exendin-4 still increased sympathetic outflow towards BAT and increased the uptake of plasma TG-derived fatty acids and glucose by BAT. These effects were accompanied by increased fat oxidation, lower plasma TG and glucose concentrations, and reduced body weight. Collectively, our results suggest that BAT activation may be a major contributor to the glucose- and TG-lowering effects of GLP-1R agonism.

Epigenetic regulation of RELN and GAD1 in the frontal cortex (FC) of autism spectrum disorder (ASD) subjects.

PubMed

Zhubi, Adrian; Chen, Ying; Guidotti, Alessandro; Grayson, Dennis R

2017-11-01

Both Reelin (RELN) and glutamate decarboxylase 67 (GAD1) have been implicated in the pathophysiology of Autism Spectrum Disorders (ASD). We have previously shown that both mRNAs are reduced in the cerebella (CB) of ASD subjects through a mechanism that involves increases in the amounts of MECP2 binding to the corresponding promoters. In the current study, we examined the expression of RELN, GAD1, GAD2, and several other mRNAs implicated in this disorder in the frontal cortices (FC) of ASD and CON subjects. We also focused on the role that epigenetic processes play in the regulation of these genes in ASD brain. Our goal is to better understand the molecular basis for the down-regulation of genes expressed in GABAergic neurons in ASD brains. We measured mRNA levels corresponding to selected GABAergic genes using qRT-PCR in RNA isolated from both ASD and CON groups. We determined the extent of binding of MECP2 and DNMT1 repressor proteins by chromatin immunoprecipitation (ChIP) assays. The amount of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) present in the promoters of the target genes was quantified by methyl DNA immunoprecipitation (MeDIP) and hydroxyl MeDIP (hMeDIP). We detected significant reductions in the mRNAs associated with RELN and GAD1 and significant increases in mRNAs encoding the Ten-eleven Translocation (TET) enzymes 1, 2, and 3. We also detected increased MECP2 and DNMT1 binding to the corresponding promoter regions of GAD1, RELN, and GAD2. Interestingly, there were decreased amounts of 5mC at both promoters and little change in 5hmC content in these same DNA fragments. Our data demonstrate that RELN, GAD1, and several other genes selectively expressed in GABAergic neurons, are down-regulated in post-mortem ASD FC. In addition, we observed increased DNMT1 and MECP2 binding at the corresponding promoters of these genes. The finding of increased MECP2 binding to the RELN, GAD1 and GAD2 promoters, with reduced amounts of 5mC and unchanged

Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing's sarcoma in vitro.

PubMed

Jully, Babu; Vijayalakshmi, Ramshankar; Gopal, Gopisetty; Sabitha, Kesavan; Rajkumar, Thangarajan

2012-11-12

Ewing's sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251-343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251-280 ectopically in Ewing's sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Our modelling analysis indicated that Junction region (aa 251-343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing's sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing's Sarcoma.

Effects of exendin-4 and selenium on the expression of GLP-1R, IRS-1, and preproinsulin in the pancreas of diabetic rats.

PubMed

Barakat, Ghinwa; Moustafa, Mohamed E; Khalifeh, Ibrahim; Hodroj, Mohammad H; Bikhazi, Anwar; Rizk, Sandra

2016-08-01

The mechanisms by which exendin-4 and selenium exert their antidiabetic actions are still unclear. Here, we investigated the effects of exendin-4 or selenium administration on the expression of glucagon-like peptide-1 receptor (GLP-1R), insulin receptor substrate-1 (IRS-1), and preproinsulin in the pancreas of diabetic rats. Diabetes was induced by streptozotocin administration. Diabetic rats were injected intraperitoneally with 0.03 μg exendin-4/kg body weight/daily or treated with 5 ppm selenium in drinking water for a period of 4 weeks. GLP-1R and IRS-1 levels were decreased while the level of preproinsulin messenger RNA (mRNA) was increased in the pancreas of diabetic untreated rats, as compared to that in control rats. Treatment of diabetic rats with exendin-4 increased protein and mRNA levels of GLP-1R, and IRS-1, and the mRNA level of preproinsulin in the pancreas, as compared to their levels in diabetic untreated rats. Selenium treatment of diabetic rats increased the pancreatic mRNA levels of GLP-1R, IRS-1, and preproinsulin. Exendin-4 or selenium treatment of diabetic rats also increased the numbers of pancreatic islets and GLP-1R molecules in the pancreas. Therefore, exendin-4 and selenium may exert their antidiabetic effects by increasing GLP-1R, IRS-1, and preproinsulin expression in the pancreas and by increasing the number of pancreatic islets.

Critical role for GLP-1 in symptomatic post-bariatric hypoglycaemia.

PubMed

Craig, Colleen M; Liu, Li-Fen; Deacon, Carolyn F; Holst, Jens J; McLaughlin, Tracey L

2017-03-01

Post-bariatric hypoglycaemia (PBH) is a rare, but severe, metabolic disorder arising months to years after bariatric surgery. It is characterised by symptomatic postprandial hypoglycaemia, with inappropriately elevated insulin concentrations. The relative contribution of exaggerated incretin hormone signalling to dysregulated insulin secretion and symptomatic hypoglycaemia is a subject of ongoing inquiry. This study was designed to test the hypothesis that PBH and associated symptoms are primarily mediated by glucagon-like peptide-1 (GLP-1). We conducted a double-blinded crossover study wherein eight participants with confirmed PBH were assigned in random order to intravenous infusion of the GLP-1 receptor (GLP-1r) antagonist. Exendin (9-39) (Ex-9), or placebo during an OGTT on two separate days at the Stanford University Clinical and Translational Research Unit. Metabolic, symptomatic and pharmacokinetic variables were evaluated. Results were compared with a cohort of BMI- and glucose-matched non-surgical controls (NSCs). Infusion of Ex-9 decreased the time to peak glucose and rate of glucose decline during OGTT, and raised the postprandial nadir by over 70%, normalising it relative to NSCs and preventing hypoglycaemia in all PBH participants. Insulin AUC and secretion rate decreased by 57% and 71% respectively, and peak postprandial insulin was normalised relative to NSCs. Autonomic and neuroglycopenic symptoms were significantly reduced during Ex-9 infusion. GLP-1r blockade prevented hypoglycaemia in 100% of individuals, normalised beta cell function and reversed neuroglycopenic symptoms, supporting the conclusion that GLP-1 plays a primary role in mediating hyperinsulinaemic hypoglycaemia in PBH. Competitive antagonism at the GLP-1r merits consideration as a therapeutic strategy. ClinicalTrials.gov NCT02550145.

In vivo dual-delivery of glucagon like peptide-1 (GLP-1) and dipeptidyl peptidase-4 (DPP4) inhibitor through composites prepared by microfluidics for diabetes therapy

NASA Astrophysics Data System (ADS)

Araújo, F.; Shrestha, N.; Gomes, M. J.; Herranz-Blanco, B.; Liu, D.; Hirvonen, J. J.; Granja, P. L.; Santos, H. A.; Sarmento, B.

2016-05-01

Oral delivery of proteins is still a challenge in the pharmaceutical field. Nanoparticles are among the most promising carrier systems for the oral delivery of proteins by increasing their oral bioavailability. However, most of the existent data regarding nanosystems for oral protein delivery is from in vitro studies, lacking in vivo experiments to evaluate the efficacy of these systems. Herein, a multifunctional composite system, tailored by droplet microfluidics, was used for dual delivery of glucagon like peptide-1 (GLP-1) and dipeptidyl peptidase-4 inhibitor (iDPP4) in vivo. Oral delivery of GLP-1 with nano- or micro-systems has been studied before, but the simultaneous nanodelivery of GLP-1 with iDPP4 is a novel strategy presented here. The type 2 diabetes mellitus (T2DM) rat model, induced through the combined administration of streptozotocin and nicotinamide, a non-obese model of T2DM, was used. The combination of both drugs resulted in an increase in the hypoglycemic effects in a sustained, but prolonged manner, where the iDPP4 improved the therapeutic efficacy of GLP-1. Four hours after the oral administration of the system, blood glucose levels were decreased by 44%, and were constant for another 4 h, representing half of the glucose area under the curve when compared to the control. An enhancement of the plasmatic insulin levels was also observed 6 h after the oral administration of the dual-drug composite system and, although no statistically significant differences existed, the amount of pancreatic insulin was also higher. These are promising results for the oral delivery of GLP-1 to be pursued further in a chronic diabetic model study.

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

PubMed Central

Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

2017-01-01

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726–35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. PMID:28213515

Synergism by individual macronutrients explains the marked early GLP-1 and islet hormone responses to mixed meal challenge in mice.

PubMed

Ahlkvist, L; Vikman, J; Pacini, G; Ahrén, B

2012-10-10

Apart from glucose, proteins and lipids also stimulate incretin and islet hormone secretion. However, the glucoregulatory effect of macronutrients in combination is poorly understood. We therefore developed an oral mixed meal model in mice to 1) explore the glucagon-like peptide-1 (GLP-1) and islet hormone responses to mixed meal versus isocaloric glucose, and 2) characterize the relative contribution of individual macronutrients to these responses. Anesthetized C57BL/6J female mice were orally gavaged with 1) a mixed meal (0.285 kcal; glucose, whey protein and peanut oil; 60/20/20% kcal) versus an isocaloric glucose load (0.285 kcal), and 2) a mixed meal (0.285 kcal) versus glucose, whey protein or peanut oil administered individually in their mixed meal caloric quantity, i.e., 0.171, 0.055 and 0.055 kcal, respectively. Plasma was analyzed for glucose, insulin and intact GLP-1 before and during oral challenges. Plasma glucose was lower after mixed meal versus after isocaloric glucose ingestion. In spite of this, the peak insulin response (P=0.02), the peak intact GLP-1 levels (P=0.006) and the estimated β-cell function (P=0.005) were higher. Furthermore, the peak insulin (P=0.004) and intact GLP-1 (P=0.006) levels were higher after mixed meal ingestion than the sum of responses to individual macronutrients. Compared to glucose alone, we conclude that there is a marked early insulin response to mixed meal ingestion, which emanates from a synergistic, rather than an additive, effect of the individual macronutrients in the mixed meal and is in part likely caused by increased levels of GLP-1. Copyright © 2012 Elsevier B.V. All rights reserved.

The recombinant expression and activity detection of MAF-1 fusion protein.

PubMed

Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

2015-10-01

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

PubMed Central

Meng, Jingru; Ma, Xue; Wang, Ning; Jia, Min; Bi, Long; Wang, Yunying; Li, Mingkai; Zhang, Huinan; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Yu, Zhibin; He, Gonghao; Luo, Xiaoxing

2016-01-01

Summary Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis. PMID:26947974

Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

PubMed

Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

2017-04-07

Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Extending residence time and stability of peptides by Protected Graft Copolymer (PGC) excipient: GLP-1 example

PubMed Central

Castillo, Gerardo M.; Reichstetter, Sandra; Bolotin, Elijah M.

2011-01-01

Purpose The purpose of this study is to determine whether a Protected Graft Copolymer (PGC) containing fatty acid can be used as a stabilizing excipient for GLP-1 and whether PGC/GLP-1 given once a week can be an effective treatment for diabetes. Methods To create a PGC excipient, polylysine was grafted with methoxypolyethyleneglycol and fatty acid at the epsilon amino groups. We performed evaluation of 1) the binding of excipient to GLP-1, 2) the DPP IV sensitivity of GLP-1 formulated with PGC as the excipient, 3) the in vitro bio-activity of excipient-formulated GLP-1, 4) the in vivo pharmacokinetics of excipient-formulated GLP-1, and 5) the efficacy of the excipient-formulated GLP-1 in diabetic rats. Results We showed reproducible synthesis of PGC excipient, showed high affinity binding of PGC to GLP-1, slowed protease degradation of excipient-formulated GLP-1, and showed that excipient-formulated GLP-1 induced calcium influx in INS cells. Excipient-formulated GLP-1 stays in the blood for at least 4 days. When excipient-formulated GLP-1 was given subcutaneously once a week to diabetic ZDF rats, a significant reduction of HbA1c compared to control was observed. The reduction is similar to diabetic ZDF rats given exendin twice a day. Conclusions PGC can be an ideal in vivo stabilizing excipient for biologically labile peptides. PMID:21830140

POSTPARTUM GAD IS A RISK FACTOR FOR POSTPARTUM MDD: THE COURSE AND LONGITUDINAL RELATIONSHIPS OF POSTPARTUM GAD AND MDD

PubMed Central

Prenoveau, Jason; Craske, Michelle; Counsell, Nicholas; West, Valerie; Davies, Beverley; Cooper, Peter; Rapa, Elizabeth; Stein, Alan

2013-01-01

Background The objective was to examine the course and longitudinal associations of generalized anxiety disorder (GAD) and major depressive disorder (MDD) in mothers over the postpartum 2 years. Method Using a prospective naturalistic design, 296 mothers recruited from a large community pool were assessed for GAD and MDD at 3, 6, 10, 14, and 24 months postpartum. Structured clinical interviews were used for diagnoses, and symptoms were assessed using self-report questionnaires. Logistic regression analyses were used to examine diagnostic stability and longitudinal relations, and latent variable modeling was employed to examine change in symptoms. Results MDD without co-occurring GAD, GAD without co-occurring MDD, and co-occurring GAD and MDD, displayed significant stability during the postpartum period. Whereas MDD did not predict subsequent GAD, GAD predicted subsequent MDD (in the form of GAD + MDD). Those with GAD + MDD at 3 months postpartum were significantly less likely to be diagnosis free during the follow-up period than those in other diagnostic categories. At the symptom level, symptoms of GAD were more trait-like than those of depression. Conclusions Postpartum GAD and MDD are relatively stable conditions, and GAD is a risk factor for MDD but not vice versa. Given the tendency of MDD and GAD to be persistent, especially when comorbid, and the increased risk for MDD in mothers with GAD, as well as the potential negative effects of cumulative exposure to maternal depression and anxiety on child development, the present findings clearly highlight the need for screening and treatment of GAD in addition to MDD during the postpartum period. PMID:23288653

Insulin and GLP-1 infusions demonstrate the onset of adipose-specific insulin resistance in a large fasting mammal: potential glucogenic role for GLP-1.

PubMed

Viscarra, Jose A; Rodriguez, Ruben; Vazquez-Medina, Jose Pablo; Lee, Andrew; Tift, Michael S; Tavoni, Stephen K; Crocker, Daniel E; Ortiz, Rudy M

2013-08-01

Prolonged food deprivation increases lipid oxidation and utilization, which may contribute to the onset of the insulin resistance associated with fasting. Because insulin resistance promotes the preservation of glucose and oxidation of fat, it has been suggested to be an adaptive response to food deprivation. However, fasting mammals exhibit hypoinsulinemia, suggesting that the insulin resistance-like conditions they experience may actually result from reduced pancreatic sensitivity to glucose/capacity to secrete insulin. To determine whether fasting results in insulin resistance or in pancreatic dysfunction, we infused early- and late-fasted seals (naturally adapted to prolonged fasting) with insulin (0.065 U/kg), and a separate group of late-fasted seals with low (10 pM/kg) or high (100 pM/kg) dosages of glucagon-like peptide-1 (GLP-1) immediately following a glucose bolus (0.5g/kg), and measured the systemic and cellular responses. Because GLP-1 facilitates glucose-stimulated insulin secretion, these infusions provide a method to assess pancreatic insulin-secreting capacity. Insulin infusions increased the phosphorylation of insulin receptor and Akt in adipose and muscle of early and late fasted seals; however the timing of the signaling response was blunted in adipose of late fasted seals. Despite the dose-dependent increases in insulin and increased glucose clearance (high dose), both GLP-1 dosages produced increases in plasma cortisol and glucagon, which may have contributed to the glucogenic role of GLP-1. Results suggest that fasting induces adipose-specific insulin resistance in elephant seal pups, while maintaining skeletal muscle insulin sensitivity, and therefore suggests that the onset of insulin resistance in fasting mammals is an evolved response to cope with prolonged food deprivation.

Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion.

PubMed Central

Morrison, T; McQuain, C; McGinnes, L

1991-01-01

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion. Images PMID:1987376

Assessing generalized anxiety disorder in elderly people using the GAD-7 and GAD-2 scales: results of a validation study.

PubMed

Wild, Beate; Eckl, Anne; Herzog, Wolfgang; Niehoff, Dorothea; Lechner, Sabine; Maatouk, Imad; Schellberg, Dieter; Brenner, Hermann; Müller, Heiko; Löwe, Bernd

2014-10-01

The aim of this study was to evaluate the validity of the seven-item Generalized Anxiety Disorder scale (GAD-7) and its two core items (GAD-2) for detecting GAD in elderly people. A criterion-standard study was performed between May and December of 2010 on a general elderly population living at home. A subsample of 438 elderly persons (ages 58-82) of the large population-based German ESTHER study was included in the study. The GAD-7 was administered to participants as part of a home visit. A telephone-administered structured clinical interview was subsequently conducted by a blinded interviewer. The structured clinical (SCID) interview diagnosis of GAD constituted the criterion standard to determine sensitivity and specificity of the GAD-7 and the GAD-2 scales. Twenty-seven participants met the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for current GAD according to the SCID interview (6.2%; 95% confidence interval [CI]: 3.9%-8.2%). For the GAD-7, a cut point of five or greater appeared to be optimal for detecting GAD. At this cut point the sensitivity of the GAD-7 was 0.63 and the specificity was 0.9. Correspondingly, the optimal cut point for the GAD-2 was two or greater with a sensitivity of 0.67 and a specificity of 0.90. The areas under the curve were 0.88 (95% CI: 0.83-0.93) for the GAD-7 and 0.87 (95% CI: 0.80-0.94) for the GAD-2. The increased scores on both GAD scales were strongly associated with mental health related quality of life (p <0.0001). Our results establish the validity of both the GAD-7 and the GAD-2 in elderly persons. Results of this study show that the recommended cut points of the GAD-7 and the GAD-2 for detecting GAD should be lowered for the elderly general population. Copyright © 2014 American Association for Geriatric Psychiatry. Published by Elsevier Inc. All rights reserved.

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

PubMed

Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

1999-03-01

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

GAD autoantibody affinity in adult patients with latent autoimmune diabetes, the study participants of a GAD65 vaccination trial.

PubMed

Krause, Stephanie; Landherr, Ulrike; Agardh, Carl-David; Hausmann, Simone; Link, Katarina; Hansen, Jesse M; Lynch, Kristian F; Powell, Michael; Furmaniak, Jadwiga; Rees-Smith, Bernard; Bonifacio, Ezio; Ziegler, Anette G; Lernmark, Ake; Achenbach, Peter

2014-06-01

Patients with latent autoimmune diabetes in adults (LADA) express autoantibodies against the 65-kDa isoform of GAD (GADA). Intervention with recombinant human GAD65 formulated with aluminium hydroxide (GAD-alum) given twice subcutaneously to LADA patients at intervals of 4 weeks was safe and did not compromise β-cell function in a Phase II clinical trial. GADA affinity has been shown to predict progression to type 1 diabetes. Here, we asked whether GADA affinity was affected by the GAD65 antigen-specific vaccination and/or associated with β-cell function in participants of this trial. GADA affinity was measured in sera of 46 LADA patients obtained prior to the first week and 20 weeks after the second injection with GAD-alum or placebo using competitive binding experiments with [125I]-labeled and unlabeled human GAD65. At baseline, GADA affinities ranged from 1.9 × 10(7) to 5.0 × 10(12) L/mol (median 2.8 × 10(10) L/mol) and were correlated with GADA titers (r = 0.47; P = 0.0009), fasting (r = -0.37; P = 0.01) and stimulated (r = -0.40; P = 0.006) C-peptide concentrations, and HbA1c (r = 0.39; P = 0.007). No significant changes in affinity were observed from baseline to week 24. Patients with GADA affinities in the lower first quartile (<4 × 10(9) L/mol) had better preserved fasting C-peptide concentrations at baseline than those with higher affinities (mean 1.02 vs. 0.66 nmol/L; P = 0.004) and retained higher concentrations over 30 months of follow-up (mean 1.26 vs. 0.62 nmol/L; P = 0.01). Intervention with GAD-alum in LADA patients had no effect on GADA affinity. Our data suggest that patients with low GADA affinity have a prolonged preservation of residual β-cell function. © 2014 by the American Diabetes Association.

Sepsis-induced activation of endogenous GLP-1 system is enhanced in type 2 diabetes.

PubMed

Perl, Sivan H; Bloch, Olga; Zelnic-Yuval, Dana; Love, Itamar; Mendel-Cohen, Lior; Flor, Hadar; Rapoport, Micha J

2018-05-01

High levels of circulating GLP-1 are associated with severity of sepsis in critically ill nondiabetic patients. Whether patients with type 2 diabetes (T2D) display different activation of the endogenous GLP-1 system during sepsis and whether it is affected by diabetes-related metabolic parameters are not known. Serum levels of GLP-1 (total and active forms) and its inhibitor enzyme sDPP-4 were determined by ELISA on admission and after 2 to 4 days in 37 sepsis patients with (n = 13) and without T2D (n = 24) and compared to normal healthy controls (n = 25). Correlations between GLP-1 system activation and clinical, inflammatory, and diabetes-related metabolic parameters were performed. A 5-fold (P < .001) and 2-fold (P < .05) increase in active and total GLP-1 levels, respectively, were found on admission as compared to controls. At 2 to 4 days from admission, the level of active GLP-1 forms in surviving patients were decreased significantly (P < .005), and positively correlated with inflammatory marker CRP (r = 0.33, P = .05). T2D survivors displayed a similar but more enhanced pattern of GLP-1 response than nondiabetic survivors. Nonsurvivors demonstrate an early extreme increase of both total and active GLP-1 forms, 9.5-fold and 5-fold, respectively (P < .05). The initial and late levels of circulating GLP-1 inhibitory enzyme sDPP-4 were twice lower in all studied groups (P < .001), compared with healthy controls. Taken together, these data indicate that endogenous GLP-1 system is activated during sepsis. Patients with T2D display an enhanced and prolonged activation as compared to nondiabetic patients. Extreme early increased GLP-1 levels during sepsis indicate poor prognosis. Copyright © 2018 John Wiley & Sons, Ltd.

A comparative study of extraction techniques for maximum recovery of glutamate decarboxylase (GAD) from Aspergillus oryzae NSK

PubMed Central

2013-01-01

Background γ-Amino butyric acid (GABA) is a major inhibitory neurotransmitter of the mammalian central nervous system that plays a vital role in regulating vital neurological functions. The enzyme responsible for producing GABA is glutamate decarboxylase (GAD), an intracellular enzyme that both food and pharmaceutical industries are currently using as the major catalyst in trial biotransformation process of GABA. We have successfully isolated a novel strain of Aspergillus oryzae NSK that possesses a relatively high GABA biosynthesizing capability compared to other reported GABA-producing fungal strains, indicating the presence of an active GAD. This finding has prompted us to explore an effective method to recover maximum amount of GAD for further studies on the GAD’s biochemical and kinetic properties. The extraction techniques examined were enzymatic lysis, chemical permeabilization, and mechanical disruption. Under the GAD activity assay used, one unit of GAD activity is expressed as 1 μmol of GABA produced per min per ml enzyme extract (U/ml) while the specific activity was expressed as U/mg protein. Results Mechanical disruption by sonication, which yielded 1.99 U/mg of GAD, was by far the most effective cell disintegration method compared with the other extraction procedures examined. In contrast, the second most effective method, freeze grinding followed by 10% v/v toluene permeabilization at 25°C for 120 min, yielded only 1.17 U/mg of GAD, which is 170% lower than the sonication method. Optimized enzymatic lysis with 3 mg/ml Yatalase® at 60°C for 30 min was the least effective. It yielded only 0.70 U/mg of GAD. Extraction using sonication was further optimized using a one-variable-at-a-time approach (OVAT). Results obtained show that the yield of GAD increased 176% from 1.99 U/mg to 3.50 U/mg. Conclusion Of the techniques used to extract GAD from A. oryzae NSK, sonication was found to be the best. Under optimized conditions, about 176% of GAD

Glucagon-like peptide-1 inhibits vascular smooth muscle cell dedifferentiation through mitochondrial dynamics regulation.

PubMed

Torres, Gloria; Morales, Pablo E; García-Miguel, Marina; Norambuena-Soto, Ignacio; Cartes-Saavedra, Benjamín; Vidal-Peña, Gonzalo; Moncada-Ruff, David; Sanhueza-Olivares, Fernanda; San Martín, Alejandra; Chiong, Mario

2016-03-15

Glucagon-like peptide-1 (GLP-1) is a neuroendocrine hormone produced by gastrointestinal tract in response to food ingestion. GLP-1 plays a very important role in the glucose homeostasis by stimulating glucose-dependent insulin secretion, inhibiting glucagon secretion, inhibiting gastric emptying, reducing appetite and food intake. Because of these actions, the GLP-1 peptide-mimetic exenatide is one of the most promising new medicines for the treatment of type 2 diabetes. In vivo treatments with GLP-1 or exenatide prevent neo-intima layer formation in response to endothelial damage and atherosclerotic lesion formation in aortic tissue. Whether GLP-1 modulates vascular smooth muscle cell (VSMC) migration and proliferation by controlling mitochondrial dynamics is unknown. In this report, we showed that GLP-1 increased mitochondrial fusion and activity in a PKA-dependent manner in the VSMC cell line A7r5. GLP-1 induced a Ser-637 phosphorylation in the mitochondrial fission protein Drp1, and decreased Drp1 mitochondrial localization. GLP-1 inhibited PDGF-BB-induced VSMC migration and proliferation, actions inhibited by overexpressing wild type Drp1 and mimicked by the Drp1 inhibitor Mdivi-1 and by overexpressing dominant negative Drp1. These results show that GLP-1 stimulates mitochondrial fusion, increases mitochondrial activity and decreases PDGF-BB-induced VSMC dedifferentiation by a PKA/Drp1 signaling pathway. Our data suggest that GLP-1 inhibits vascular remodeling through a mitochondrial dynamics-dependent mechanism. Copyright © 2016 Elsevier Inc. All rights reserved.

Sustained expression of GLP-1 receptor differentially modulates β-cell functions in diabetic and nondiabetic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubo, Fumiyo; Miyatsuka, Takeshi, E-mail: miyatsuka-takeshi@umin.net; Department of Medicine, Metabolism and Endocrinology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421

2016-02-26

Glucagon-like peptide 1 (GLP-1) has been shown to play important roles in maintaining β-cell functions, such as insulin secretion and proliferation. While expression levels of GLP-1 receptor (Glp1r) are compromised in the islets of diabetic rodents, it remains unclear when and to what degree Glp1r mRNA levels are decreased during the progression of diabetes. In this study, we performed real-time PCR with the islets of db/db diabetic mice at different ages, and found that the expression levels of Glp1r were comparable to those of the islets of nondiabetic db/misty controls at the age of four weeks, and were significantly decreasedmore » at the age of eight and 12 weeks. To investigate whether restored expression of Glp1r affects the diabetic phenotypes, we generated the transgenic mouse model Pdx1{sup PB}-CreER{sup TM}; CAG-CAT-Glp1r (βGlp1r) that allows for induction of Glp1r expression specifically in β cells. Whereas the expression of exogenous Glp1r had no measurable effect on glucose tolerance in nondiabetic βGlp1r;db/misty mice, βGlp1r;db/db mice exhibited higher glucose and lower insulin levels in blood on glucose challenge test than control db/db littermates. In contrast, four weeks of treatment with exendin-4 improved the glucose profiles and increased serum insulin levels in βGlp1r;db/db mice, to significantly higher levels than those in control db/db mice. These differential effects of exogenous Glp1r in nondiabetic and diabetic mice suggest that downregulation of Glp1r might be required to slow the progression of β-cell failure under diabetic conditions. - Highlights: • Expression levels of incretin receptors were significantly decreased in diabetic db/db islets after the age of eight weeks. • A transgenic mouse model expressing Glp1r specifically in β cells was generated. • Exogenous expression of Glp1r in β cells did not affect metabolic profiles in nondiabetic mice. • Sustained expression of Glp1r in diabetic db/db β cells

Blockade of Central GLP-1 Receptors Deteriorates the Improvement of Diabetes after Ileal Transposition.

PubMed

Chen, Weijie; Xu, Qianqian; Xiao, Yiding; Zhou, Jiaolin; Zhang, Weimin; Lin, Guole; Gong, Fengying

2016-01-01

Background: The mechanism of improvement of type 2 diabetes mellitus induced by ileal transposition (IT) is undefined. Our aim was to investigate the possible role of central glucagon-like peptide 1 (GLP-1) after IT. Methods: Ninety male diabetic rats were randomly divided into the IT, sham IT (S-IT) and control group. The food intake, glucose metabolism and GLP-1 level were measured. Subsequently, we administered GLP-1 antagonist via lateral brain ventricle cannula to block central GLP-1 receptor, and verified whether the food intake, glucose metabolism changed. And the activated pro-opiomelanocortin (POMC) neurons in different groups were compared after sacrifice. Results: IT induced significant diabetic improvement with decreased maximum food intake and higher postprandial GLP-1 level. The GLP-1 level in cerebrospinal fluid increased in correlation with the plasma GLP-1 level. When the central GLP-1 receptor antagonist was given to the IT group rats, the improvement of the glucose level declined. The glucose level surged (169.9 ± 14.2) % during the oral glucose tolerance test, the range was larger than that before central blockade ((67.1 ± 14.2) %, P < 0.001). Moreover, the POMC neuron number in the arcuate nucleus of the hypothalamus were reduced (12.7 ± 6.1 at a magnification of 100×). The relative content level of POMC-derived peptides in the pituitary was lower (0.1 ± 0.05). Conclusions: The central GLP-1 might play an important role in the remission of diabetes after IT. POMC neurons in the hypothalamus may be activated by the enhanced level of GLP-1 after IT.

Choosing between GLP-1 Receptor Agonists and DPP-4 Inhibitors: A Pharmacological Perspective

PubMed Central

Brown, Dominique Xavier; Evans, Marc

2012-01-01

In recent years the incretin therapies have provided a new treatment option for patients with type 2 diabetes mellitus (T2DM). The incretin therapies focus on the increasing levels of the two incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This results in increased glucose dependent insulin synthesis and release. GLP-1 receptor agonists such as liraglutide and exenatide exert an intrinsic biological effect on GLP-1 receptors directly stimulating the release of insulin from pancreatic beta cells. DPP-4 inhibitors such as sitagliptin and linagliptin prevent the inactivation of endogenous GLP-1 and GIP through competitive inhibition of the DPP-4 enzyme. Both incretin therapies have good safety and tolerability profiles and interact minimally with a number of medications commonly prescribed in T2DM. This paper focuses on the pharmacological basis by which the incretin therapies function and how this knowledge can inform and benefit clinical decisions. Each individual incretin agent has benefits and pitfalls relating to aspects such as glycaemic and nonglycaemic efficacy, safety and tolerability, ease of administration, and cost. Overall, a personalized medicine approach has been found to be favourable, tailoring the incretin agent to benefit and suit patient's needs such as renal impairment (RI) or hepatic impairment (HI). PMID:23125920

Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus

PubMed Central

Dicken, Matthew S.; Hughes, Alexander R.; Hentges, Shane T.

2016-01-01

The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. PMID:26370162

Fructose stimulates GLP-1 but not GIP secretion in mice, rats, and humans

PubMed Central

Kuhre, Rune E.; Gribble, Fiona M.; Hartmann, Bolette; Reimann, Frank; Windeløv, Johanne A.; Rehfeld, Jens F.

2014-01-01

Nutrients often stimulate gut hormone secretion, but the effects of fructose are incompletely understood. We studied the effects of fructose on a number of gut hormones with particular focus on glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). In healthy humans, fructose intake caused a rise in blood glucose and plasma insulin and GLP-1, albeit to a lower degree than isocaloric glucose. Cholecystokinin secretion was stimulated similarly by both carbohydrates, but neither peptide YY3–36 nor glucagon secretion was affected by either treatment. Remarkably, while glucose potently stimulated GIP release, fructose was without effect. Similar patterns were found in the mouse and rat, with both fructose and glucose stimulating GLP-1 secretion, whereas only glucose caused GIP secretion. In GLUTag cells, a murine cell line used as model for L cells, fructose was metabolized and stimulated GLP-1 secretion dose-dependently (EC50 = 0.155 mM) by ATP-sensitive potassium channel closure and cell depolarization. Because fructose elicits GLP-1 secretion without simultaneous release of glucagonotropic GIP, the pathways underlying fructose-stimulated GLP-1 release might be useful targets for type 2 diabetes mellitus and obesity drug development. PMID:24525020

Expression, Refolding and Crystallizations of the Grb2-like (GADS) C-Terminal SH3 Domain Complexed with a SLP-76 Motif Peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli,A.; Dimasi, N.

The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.

Exenatide exerts a PKA-dependent positive inotropic effect in human atrial myocardium: GLP-1R mediated effects in human myocardium.

PubMed

Wallner, Markus; Kolesnik, Ewald; Ablasser, Klemens; Khafaga, Mounir; Wakula, Paulina; Ljubojevic, Senka; Thon-Gutschi, Eva Maria; Sourij, Harald; Kapl, Martin; Edmunds, Nicholas J; Kuzmiski, J Brent; Griffith, David A; Knez, Igor; Pieske, Burkert; von Lewinski, Dirk

2015-12-01

Glucagon-like peptide-1 receptor (GLP-1R) agonists are a rapidly growing class of drugs developed for treating type-2 diabetes mellitus. Patients with diabetes carry an up to 5-fold greater mortality risk compared to non-diabetic patients, mainly as a result of cardiovascular diseases. Although beneficial cardiovascular effects have been reported, exact mechanisms of GLP-1R-agonist action in the heart, especially in human myocardium, are poorly understood. The effects of GLP-1R-agonists (exenatide, GLP-1(7-36)NH2, PF-06446009, PF-06446667) on cardiac contractility were tested in non-failing atrial and ventricular trabeculae from 72 patients. The GLP-1(7-36)NH2 metabolite, GLP-1(9-36)NH2, was also examined. In electrically stimulated trabeculae, the effects of compounds on isometric force were measured in the absence and presence of pharmacological inhibitors of signal transduction pathways. The role of β-arrestin signaling was examined using a β-arrestin partial agonist, PF-06446667. Expression levels were tested by immunoblots. Translocation of GLP-1R downstream molecular targets, Epac2, GLUT-1 and GLUT-4, were assessed by fluorescence microscopy. All tested GLP-1R-agonists significantly increased developed force in human atrial trabeculae, whereas GLP-1(9-36)NH2 had no effect. Exendin(9-39)NH2, a GLP-1R-antagonist, and H-89 blunted the inotropic effect of exenatide. In addition, exenatide increased PKA-dependent phosphorylation of phospholamban (PLB), GLUT-1 and Epac2 translocation, but not GLUT-4 translocation. Exenatide failed to enhance contractility in ventricular myocardium. Quantitative real-time PCR (qRT-PCR) revealed a significant higher GLP-1R expression in the atrium compared to ventricle. Exenatide increased contractility in a dose-dependent manner via GLP-1R/cAMP/PKA pathway and induced GLUT-1 and Epac2 translocation in human atrial myocardium, but had no effect in ventricular myocardium. Therapeutic use of GLP-1R-agonists may therefore impart

Gad67 haploinsufficiency reduces amyloid pathology and rescues olfactory memory deficits in a mouse model of Alzheimer's disease.

PubMed

Wang, Yue; Wu, Zheng; Bai, Yu-Ting; Wu, Gang-Yi; Chen, Gong

2017-10-10

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder, affecting millions of people worldwide. Although dysfunction of multiple neurotransmitter systems including cholinergic, glutamatergic and GABAergic systems has been associated with AD progression the underlying mechanisms remain elusive. We and others have recently found that GABA content is elevated in AD brains and linked to cognitive deficits in AD mouse models. The glutamic acid decarboxylase 67 (GAD67) is the major enzyme converting glutamate into GABA and has been implied in a number of neurological disorders such as epilepsy and schizophrenia. However, whether Gad67 is involved in AD pathology has not been well studied. Here, we investigate the functional role of GAD67 in an AD mouse model with Gad67 haploinsufficiency that is caused by replacing one allele of Gad67 with green fluorescent protein (GFP) gene during generation of GAD67-GFP mice. To genetically reduce GAD67 in AD mouse brains, we crossed the Gad67 haploinsufficient mice (GAD67-GFP +/- ) with 5xFAD mice (harboring 5 human familial AD mutations in APP and PS1 genes) to generate a new line of bigenic mice. Immunostaining, ELISA, electrophysiology and behavior test were applied to compare the difference between groups. We found that reduction of GAD67 resulted in a significant decrease of amyloid β production in 5xFAD mice. Concurrently, the abnormal astrocytic GABA and tonic GABA currents, as well as the microglial reactivity were significantly reduced in the 5xFAD mice with Gad67 haploinsufficiency. Importantly, the olfactory memory deficit of 5xFAD mice was rescued by Gad67 haploinsufficiency. Our results demonstrate that GAD67 plays an important role in AD pathology, suggesting that GAD67 may be a potential drug target for modulating the progress of AD.

LINC01121 Inhibits Cell Apoptosis While Facilitating Proliferation, Migration, and Invasion Though Negative Regulation of the Camp/PKA Signaling Pathway via GLP1R.

PubMed

Qian, Yi-Gang; Ye, Zhou; Chen, Hai-Yong; Lv, Zhen; Zhang, Ai-Bin; Fan, Le; Zhou, Jie; Zheng, Shu-Sen; Wang, Wei-Lin

2018-05-24

Pancreatic cancer is an aggressive malignancy as a result of highly metastatic potential. The current study was carried out to alter the expression of LINC01121 in pancreatic cancer, with the aim of elucidating its effects on the biological processes of cell proliferation, migration, invasion, and apoptosis. We hypothesized that both the GLP1R gene and cAMP/PKA signaling pathway participate in the aforementioned process. Microarray data (GSE14245, GSE27890 and GSE16515) and annotating probe files linked to pancreatic cancer were downloaded through the GEO database. The Multi Experiment Matrix (MEM) site was used to predict the target gene of lncRNA. Both pancreatic cancer tissues (n = 56) and paracancerous tissues (n = 45) were collected from patients diagnosed with pancreatic cancer. Immunohistochemistry was applied to identify the positive expression rate of GLP1R protein. Isolated pancreatic cancer cells and PANC-1 cells were independently classified into the blank, negative control (NC), LINC01121 vector, siRNA-LINC01121, siRNA-GLP1R and siRNA-LINC01121 + siRNA-GLP1R groups. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were applied to detect the expressions of LINC01121, GLP1R, cAMP, PKA, CREB, Bcl-2, Bad and PCNA. Cell proliferation, migration, invasion, cycle progression, and apoptosis were examined by MTT assay, scratch test, Transwell assay and flow cytometry analyses of Annexin V-FITC/PI staining. Observations were made indicating that LINC01121 was highly expressed, while low expressions of GLP1R in pancreatic cancer were detected based on microarray data, which was largely in consistent with the data collected of LINC01121 and GLP1R within the tissues. The target prediction program and luciferase activity analysis was testament to the notion suggesting that GLP1R was indeed a target of LINC01121. In contrast to the blank and NC groups, the LINC01121 vector group exhibited increased expressions of LINC

New screening strategy and analysis for identification of allosteric modulators for glucagon-like peptide-1 receptor using GLP-1 (9-36) amide.

PubMed

Nakane, Atsushi; Gotoh, Yusuke; Ichihara, Junji; Nagata, Hidetaka

2015-12-15

The glucagon-like peptide-1 receptor (GLP-1R) is an important physiologic regulator of insulin secretion and a major therapeutic target for diabetes mellitus. GLP-1 (7-36) amide (active form of GLP-1) is truncated to GLP-1 (9-36) amide, which has been described as a weak agonist of GLP-1R and the major form of GLP-1 in the circulation. New classes of positive allosteric modulators (PAMs) for GLP-1R may offer improved therapeutic profiles. To identify these new classes, we developed novel and robust primary and secondary high-throughput screening (HTS) systems in which PAMs were identified to enhance the GLP-1R signaling induced by GLP-1 (9-36) amide. Screening enabled identification of two compounds, HIT-465 and HIT-736, which possessed new patterns of modulation of GLP-1R. We investigated the ability of these compounds to modify GLP-1R signaling enhanced GLP-1 (9-36) amide- and/or GLP-1 (7-36) amide-mediated cyclic adenosine monophosphate (cAMP) accumulation. These compounds also had unique profiles with regard to allosteric modulation of multiple downstream signaling (PathHunter β-arrestin signaling, PathHunter internalization signaling, microscopy-based internalization assay). We found allosteric modulation patterns to be obviously different among HIT-465, HIT-736, and Novo Nordisk compound 2. This work may enable the design of new classes of drug candidates by targeting modulation of GLP-1 (7-36) amide and GLP-1 (9-36) amide. Copyright © 2015 Elsevier Inc. All rights reserved.

Sustained expression of GLP-1 receptor differentially modulates β-cell functions in diabetic and nondiabetic mice

PubMed Central

Kubo, Fumiyo; Miyatsuka, Takeshi; Sasaki, Shugo; Takahara, Mitsuyoshi; Yamamoto, Yuichi; Shimo, Naoki; Watada, Hirotaka; Kaneto, Hideaki; Gannon, Maureen; Matsuoka, Taka-aki; Shimomura, Iichiro

2017-01-01

Glucagon-like peptide 1 (GLP-1) has been shown to play important roles in maintaining β-cell functions, such as insulin secretion and proliferation. While expression levels of GLP-1 receptor (Glp1r) are compromised in the islets of diabetic rodents, it remains unclear when and to what degree Glp1r mRNA levels are decreased during the progression of diabetes. In this study, we performed real-time PCR with the islets of db/db diabetic mice at different ages, and found that the expression levels of Glp1r were comparable to those of the islets of nondiabetic db/misty controls at the age of four weeks, and were significantly decreased at the age of eight and 12 weeks. To investigate whether restored expression of Glp1r affects the diabetic phenotypes, we generated the transgenic mouse model Pdx1PB-CreER™; CAG-CAT-Glp1r (βGlp1r) that allows for induction of Glp1r expression specifically in β cells. Whereas the expression of exogenous Glp1r had no measurable effect on glucose tolerance in nondiabetic βGlp1r;db/misty mice, βGlp1r;db/db mice exhibited higher glucose and lower insulin levels in blood on glucose challenge test than control db/db littermates. In contrast, four weeks of treatment with exendin-4 improved the glucose profiles and increased serum insulin levels in βGlp1r;db/db mice, to significantly higher levels than those in control db/db mice. These differential effects of exogenous Glp1r in nondiabetic and diabetic mice suggest that downregulation of Glp1r might be required to slow the progression of β-cell failure under diabetic conditions. PMID:26854076

Molecular mechanisms redirecting the GLP-1 receptor signalling profile in pancreatic β-cells during type 2 diabetes.

PubMed

Roussel, Morgane; Mathieu, Julia; Dalle, Stéphane

2016-05-01

Treatments with β-cell preserving properties are essential for the management of type 2 diabetes (T2D), and the new therapeutic avenues, developed over the last years, rely on the physiological role of glucagon-like peptide-1 (GLP-1). Sustained pharmacological levels of GLP-1 are achieved by subcutaneous administration of GLP-1 analogues, while transient and lower physiological levels of GLP-1 are attained following treatment with inhibitors of dipeptidylpeptidase 4 (DPP4), an endoprotease which degrades the peptide. Both therapeutic classes display a sustained and durable hypoglycaemic action in patients with T2D. However, the GLP-1 incretin effect is known to be reduced in patients with T2D, and GLP-1 analogues and DPP4 inhibitors were shown to lose their effectiveness over time in some patients. The pathological mechanisms behind these observations can be either a decrease in GLP-1 secretion from intestinal L-cells and, as a consequence, a reduction in GLP-1 plasma concentrations, combined or not with a reduced action of GLP-1 in the β-cell, the so-called GLP-1 resistance. Much evidence for a GLP-1 resistance of the β-cell in subjects with T2D have emerged. Here, we review the potential roles of the genetic background, the hyperglycaemia, the hyperlipidaemia, the prostaglandin E receptor 3, the nuclear glucocorticoid receptor, the GLP-1R desensitization and internalisation processes, and the β-arrestin-1 expression levels on GLP-1 resistance in β-cells during T2D.

IgG-Paraoxonase-1 Fusion Protein for Targeted Drug Delivery Across the Human Blood-Brain Barrier

PubMed Central

Boado, Ruben J.; Zhang, Yun; Zhang, Yufeng; Wang, Yuntao; Pardridge, William M.

2009-01-01

Paraoxonase (PON)-1 is the most potent human protein with organophosphatase activity against organophosphate (OP) toxins. OP compounds readily cross the blood-brain barrier (BBB), and have lethal mechanisms of action within the brain. The production of a brain penetrating form of human PON1, which crosses the BBB, is possible with the re-engineering of the enzyme as a fusion protein with a monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry the PON1 into brain. The human PON1 was fused to the carboxyl terminus of the heavy chain of the chimeric HIRMAb. COS cells were dual transfected with the heavy chain gene and the light chain gene, and the HIRMAb-PON1 fusion protein was affinity purified with protein A chromatography. Western blotting with antibodies to human IgG or human PON1 showed the heavy chain of the HIRMAb-PON1 fusion protein was 40 kDa larger than the heavy chain of the chimeric HIRMAb. The ED50 of binding to the HIR extracellular domain was 0.55 ± 0.07 nM and 1.1 ±0.1 nM, respectively, for the chimeric HIRMAb and the HIRMAb-PON1 fusion protein. The PON1 enzyme activity of the fusion protein was approximately 25% of the enzyme activity in human plasma, based on a fluorometric enzymatic assay. In conclusion, human PON1 has been re-engineered as an IgG-organophosphatase fusion protein that penetrates the human BBB. PMID:19434854

Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus.

PubMed

Dicken, Matthew S; Hughes, Alexander R; Hentges, Shane T

2015-11-01

The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

[Effects of GLP-1 receptor agonists on carbohydrate metabolism control].

PubMed

Fernández-García, José Carlos; Colomo, Natalia; Tinahones, Francisco José

2014-01-01

Glucagon-like peptide-1 (GLP-1) receptor agonists are a new group of drugs for the treatment of type 2 diabetes mellitus (DM2). In the present article, we review the available evidence on the efficacy of GLP-1 receptor agonists as glucose-lowering agents, their place in therapeutic algorithms, and the clinical factors associated with a favorable treatment response. Finally, we describe the clinical characteristics of patients who may benefit from these drugs.

GLP-1 agonism stimulates brown adipose tissue thermogenesis and browning through hypothalamic AMPK.

PubMed

Beiroa, Daniel; Imbernon, Monica; Gallego, Rosalía; Senra, Ana; Herranz, Daniel; Villarroya, Francesc; Serrano, Manuel; Fernø, Johan; Salvador, Javier; Escalada, Javier; Dieguez, Carlos; Lopez, Miguel; Frühbeck, Gema; Nogueiras, Ruben

2014-10-01

GLP-1 receptor (GLP-1R) is widely located throughout the brain, but the precise molecular mechanisms mediating the actions of GLP-1 and its long-acting analogs on adipose tissue as well as the brain areas responsible for these interactions remain largely unknown. We found that central injection of a clinically used GLP-1R agonist, liraglutide, in mice stimulates brown adipose tissue (BAT) thermogenesis and adipocyte browning independent of nutrient intake. The mechanism controlling these actions is located in the hypothalamic ventromedial nucleus (VMH), and the activation of AMPK in this area is sufficient to blunt both central liraglutide-induced thermogenesis and adipocyte browning. The decreased body weight caused by the central injection of liraglutide in other hypothalamic sites was sufficiently explained by the suppression of food intake. In a longitudinal study involving obese type 2 diabetic patients treated for 1 year with GLP-1R agonists, both exenatide and liraglutide increased energy expenditure. Although the results do not exclude the possibility that extrahypothalamic areas are also modulating the effects of GLP-1R agonists, the data indicate that long-acting GLP-1R agonists influence body weight by regulating either food intake or energy expenditure through various hypothalamic sites and that these mechanisms might be clinically relevant. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Combination therapy with GLP-1 receptor agonists and basal insulin: a systematic review of the literature

PubMed Central

Balena, R; Hensley, I E; Miller, S; Barnett, A H

2013-01-01

Treatment algorithms for type 2 diabetes call for intensification of therapy over time as the disease progresses and glycaemic control worsens. If diet, exercise and oral antihyperglycaemic medications (OAMs) fail to maintain glycaemic control then basal insulin is added and ultimately prandial insulin may be required. However, such an intensification strategy carries risk of increased hypoglycaemia and weight gain, both of which are associated with worse long-term outcomes. An alternative strategy is to intensify therapy by the addition of a short-acting glucagon-like peptide-1 receptor agonist (GLP-1 RA) rather than prandial insulin. Short-acting GLP-1 RAs such as exenatide twice daily are particularly effective at reducing postprandial glucose while basal insulin has a greater effect on fasting glucose, providing a physiological rationale for this complementary approach. This review analyzes the latest randomized controlled clinical trials of insulin/GLP-1 RA combination therapy and examines results from ‘real-world’ use of the combinations as reported through observational and clinical practice studies. The most common finding across all types of studies was that combination therapy improved glycaemic control without weight gain or an increased risk of hypoglycaemia. Many studies reported weight loss and a reduction in insulin use when a GLP-1 RA was added to existing insulin therapy. Overall, the relative degree of benefit to glycaemic control and weight was influenced by the insulin titration employed in conjunction with the GLP-1 RA. The greatest glycaemic benefits were observed in studies with structured titration of insulin to glycaemic targets while the greatest weight benefits were observed in studies with a protocol-specified focus on insulin sparing. The adverse event profile of GLP-1 RAs in the reviewed trials was similar to that reported with GLP-1 RAs as monotherapy or in combination with OAMs with gastrointestinal events being the most commonly

β cell membrane remodelling and procoagulant events occur in inflammation-driven insulin impairment: a GLP-1 receptor dependent and independent control.

PubMed

Gleizes, Céline; Kreutter, Guillaume; Abbas, Malak; Kassem, Mohamad; Constantinescu, Andrei Alexandru; Boisramé-Helms, Julie; Yver, Blandine; Toti, Florence; Kessler, Laurence

2016-02-01

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell-derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon-like peptide (GLP)-1 analogue, is known to promote insulin secretion and β-cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin-m5f β-cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross-talk model. Methyl-β-cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N-éthylmaleimide-sensitive-factor Attachment protein Receptor (SNARE)-dependent exocytosis. Cytokines induced a two-fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two-fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD-treated cells showed similar patterns. Cells pre-treated by saturating concentration of the GLP-1r antagonist exendin (9-39), showed a partial abolishment of the liraglutide-driven insulin secretion and liraglutide-decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP-1r-dependent and -independent pathways. Our results confirm an integrative β-cell response to GLP-1 that targets receptor-mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation-driven procoagulant events. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and

Dietary resistant starch upregulates total GLP-1 and PYY in a sustained day-long manner through fermentation in rodents.

PubMed

Zhou, June; Martin, Roy J; Tulley, Richard T; Raggio, Anne M; McCutcheon, Kathleen L; Shen, Li; Danna, Samuel Colby; Tripathy, Sasmita; Hegsted, Maren; Keenan, Michael J

2008-11-01

Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are anti-diabetes/obesity hormones secreted from the gut after meal ingestion. We have shown that dietary-resistant starch (RS) increased GLP-1 and PYY secretion, but the mechanism remains unknown. RS is a fermentable fiber that lowers the glycemic index of the diet and liberates short-chain fatty acids (SCFAs) through fermentation in the gut. This study investigates the two possible mechanisms by which RS stimulates GLP-1 and PYY secretion: the effect of a meal or glycemic index, and the effect of fermentation. Because GLP-1 and PYY secretions are stimulated by nutrient availability in the gut, the timing of blood sample collections could influence the outcome when two diets with different glycemic indexes are compared. Thus we examined GLP-1 and PYY plasma levels at various time points over a 24-h period in RS-fed rats. In addition, we tested proglucagon (a precursor to GLP-1) and PYY gene expression patterns in specific areas of the gut of RS-fed rats and in an enteroendocrine cell line following exposure to SCFAs in vitro. Our findings are as follows. 1) RS stimulates GLP-1 and PYY secretion in a substantial day-long manner, independent of meal effect or changes in dietary glycemia. 2) Fermentation and the liberation of SCFAs in the lower gut are associated with increased proglucagon and PYY gene expression. 3) Glucose tolerance, an indicator of increased active forms of GLP-1 and PYY, was improved in RS-fed diabetic mice. We conclude that fermentation of RS is most likely the primary mechanism for increased endogenous secretions of total GLP-1 and PYY in rodents. Thus any factor that affects fermentation should be considered when dietary fermentable fiber is used to stimulate GLP-1 and PYY secretion.

Minireview: Signal Bias, Allosterism, and Polymorphic Variation at the GLP-1R: Implications for Drug Discovery

PubMed Central

Koole, Cassandra; Savage, Emilia E.; Christopoulos, Arthur; Miller, Laurence J.

2013-01-01

The glucagon-like peptide-1 receptor (GLP-1R) controls the physiological responses to the incretin hormone glucagon-like peptide-1 and is a major therapeutic target for the treatment of type 2 diabetes, owing to the broad range of effects that are mediated upon its activation. These include the promotion of glucose-dependent insulin secretion, increased insulin biosynthesis, preservation of β-cell mass, improved peripheral insulin action, and promotion of weight loss. Regulation of GLP-1R function is complex, with multiple endogenous and exogenous peptides that interact with the receptor that result in the activation of numerous downstream signaling cascades. The current understanding of GLP-1R signaling and regulation is limited, with the desired spectrum of signaling required for the ideal therapeutic outcome still to be determined. In addition, there are several single-nucleotide polymorphisms (used in this review as defining a natural change of single nucleotide in the receptor sequence; clinically, this is viewed as a single-nucleotide polymorphism only if the frequency of the mutation occurs in 1% or more of the population) distributed within the coding sequence of the receptor protein that have the potential to produce differential responses for distinct ligands. In this review, we discuss the current understanding of GLP-1R function, in particular highlighting recent advances in the field on ligand-directed signal bias, allosteric modulation, and probe dependence and the implications of these behaviors for drug discovery and development. PMID:23864649

High fat diet and GLP-1 drugs induce pancreatic injury in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rouse, Rodney, E-mail: rodney.rouse@fda.hhs.gov; Xu, Lin; Stewart, Sharron

Glucagon Like Peptide-1 (GLP-1) drugs are currently used to treat type-2 diabetes. Safety concerns for increased risk of pancreatitis and pancreatic ductal metaplasia have accompanied these drugs. High fat diet (HFD) is a type-2 diabetes risk factor that may affect the response to GLP-1 drug treatment. The objective of the present study was to investigate the effects of diet and GLP-1 based drugs on the exocrine pancreas in mice. Experiments were designed in a mouse model of insulin resistance created by feeding a HFD or standard diet (STD) for 6 weeks. The GLP-1 drugs, sitagliptin (SIT) and exenatide (EXE) weremore » administered once daily for additional 6 weeks in both mice fed HFD or STD. The results showed that body weight, blood glucose levels, and serum levels of pro-inflammatory cytokines (TNFα, IL-1β, and KC) were significantly greater in HFD mice than in STD mice regardless of GLP-1 drug treatment. The semi-quantitative grading showed that pancreatic changes were significantly greater in EXE and SIT-treated mice compared to control and that HFD exacerbated spontaneous exocrine pancreatic changes seen in saline-treated mice on a standard diet. Exocrine pancreatic changes identified in this study included acinar cell injury (hypertrophy, autophagy, apoptosis, necrosis, and atrophy), vascular injury, interstitial edema and inflammation, fat necrosis, and duct changes. These findings support HFD as a risk factor to increased susceptibility/severity for acute pancreatitis and indicate that GLP-1 drugs cause pancreatic injury that can be exacerbated in a HFD environment.« less

Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

PubMed

Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars

2006-02-17

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

GLP-1 response to a mixed meal: what happens 10 years after Roux-en-Y gastric bypass (RYGB)?

PubMed

Dar, Moahad S; Chapman, William H; Pender, John R; Drake, Almond J; O'Brien, Kevin; Tanenberg, Robert J; Dohm, G Lynis; Pories, Walter J

2012-07-01

Oral meal consumption increases glucagon-like peptide 1 (GLP-1) release which maintains euglycemia by increasing insulin secretion. This effect is exaggerated during short-term follow-up of Roux-en-y gastric bypass (RYGB). We examined the durability of this effect in patient with type 2 diabetes (T2DM) >10 years after RYGB. GLP-1 response to a mixed meal in the 10-year post-RYGB group (n = 5) was compared to lean (n = 9), obese (n = 6), and type 2 diabetic (n = 10) controls using a cross-sectional study design. Analysis of variance (ANOVA) was used to evaluate GLP-1 response to mixed meal consumption from 0 to 300 min, 0-20 min, 20-60 min, and 60-300 min, respectively. Weight, insulin resistance, and T2DM were also assessed. GLP-1 response 0-300 min in the 10-year post-RYGB showed a statistically significant overall difference (p =  0.01) compared to controls. Furthermore, GLP-1 response 0-20 min in the 10-year post-RYGB group showed a very rapid statistically significant rise (p = 0.035) to a peak of 40 pM. GLP-1 response between 20 and 60 min showed a rapid statistically significant (p = 0.041) decline in GLP-1 response from ~40 pM to 10 pM. GLP-1 response in the 10-year post-RYGB group from 60 to 300 min showed no statistically significant difference from controls. BMI, HOMA, and fasting serum glucose before and >10 years after RYGB changed from 59.9 → 40.4, 8.7 → 0.88, and 155.2 → 87.6 mg/dl, respectively, and were statistically significant (p < 0.05). An exaggerated GLP-1 response was noted 10 years after RYGB, strongly suggesting a durability of this effect. This phenomenon may play a key role in maintaining type 2 diabetes remission and weight loss after RYGB.

Neuroprotective effects of (Val8)GLP-1-Glu-PAL in the MPTP Parkinson's disease mouse model.

PubMed

Zhang, YanFang; Chen, YiMei; Li, Lin; Hölscher, Christian

2015-10-15

Glucagon-like peptide 1 (GLP-1) is a hormone and a growth factor. GLP-1 mimetics are currently on the market as treatments for type 2 diabetes. They also have shown neuroprotective properties in animal models of neurodegenerative disorders. In addition, the GLP-1 mimetic exendin-4 has shown protective effects in animal models of Parkinson's disease (PD), and a first clinical trial in PD patients showed promising results. (Val8)GLP-1-glu-PAL is a new GLP-1 analogue which has a longer biological half-life than exendin-4. We previously showed that (Val8)GLP-1-glu-PAL has neuroprotective properties. Here we tested the drug in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MPTP was injected (30mg/kg i.p.) along with (Val8)GLP-1-glu-PAL (25nmol/kg i.p.) once-daily for 8 days. (Val8)GLP-1-glu-PAL showed good effects in preventing the MPTP-induced motor impairment (Rotarod, open field locomotion, swim test), reduction in tyrosine hydroxylase levels (dopamine synthesis) in the substantia nigra, a reduction of activated caspase 3 levels, of TUNEL positive cell numbers, of the pro-apoptotic signaling molecule BAX and an increase in the growth signaling molecule Bcl-2. The results demonstrate that (Val8)GLP-1-glu-PAL shows promise as a novel treatment of PD. Copyright © 2015 Elsevier B.V. All rights reserved.

A review of the new GLP-1 receptor agonist/basal insulin fixed-ratio combination products.

PubMed

Nuffer, Wesley; Guesnier, Ashley; Trujillo, Jennifer M

2018-03-01

There have been several new treatment approaches established for the management of hyperglycemia in type 2 diabetes (T2D), with treatment guidelines listing both glucagon-like peptide 1 receptor agonists (GLP-1 RAs) and basal insulin therapies as considerations for patients who have failed to control their blood glucose with oral antidiabetic agents. New studies have highlighted the importance of initiating combination therapy earlier in the T2D disease process to avoid clinical inertia and prevent the long-term complications arising from uncontrolled diabetes. Until recently, both GLP-1 RAs and basal insulin therapies were only available as single agents, but there are now two combination pen devices that deliver both a GLP-1 RA and basal insulin simultaneously. This article reviews the current clinical evidence evaluating the use of these combination GLP-1 RA/basal insulin preparations to treat T2D, presents both potential benefits as well as possible downsides with the use of these agents, and discusses the current place in therapy these products represent in the management of T2D.

Dynamin-related protein-1 controls fusion pore dynamics during platelet granule exocytosis.

PubMed

Koseoglu, Secil; Dilks, James R; Peters, Christian G; Fitch-Tewfik, Jennifer L; Fadel, Nathalie A; Jasuja, Reema; Italiano, Joseph E; Haynes, Christy L; Flaumenhaft, Robert

2013-03-01

Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known. We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and α-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1. These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

Differences in acute anorectic effects of long-acting GLP-1 receptor agonists in rats.

PubMed

Sisley, Stephanie; Smith, Kathleen; Sandoval, Darleen A; Seeley, Randy J

2014-08-01

Long-acting glucagon-like peptide-1 receptor (GLP-1R) agonists have both glucose- and weight-lowering effects. The brain is poised to mediate both of these actions since GLP-1Rs are present in key areas known to control weight and glucose. Although some research has been performed on the effects of exendin-4 in the brain, little data exists on the central effects of liraglutide, a long-acting GLP-1R agonist with much closer structural homology to native GLP-1. In lean, Long-Evans rats, we found that direct intra-third cerebroventricular (i3vt) administration of 0.26 nmol liraglutide caused a 50% reduction in food intake. However, exendin-4 produced the same reduction in food intake with 10-fold greater potency (0.02 nmol). These data are supported by similar c-Fos immunoreactivity in the hypothalamic paraventricular nuclei by exendin-4 as compared to liraglutide despite differing doses. The anorectic effects of both drugs were blocked with i3vt pre-treatment of a GLP-1R competitive antagonist, exendin(9-39), indicating that both drugs required the GLP-1R for their effects. Exendin-4, and not liraglutide, caused hyperglycemia when given i3vt prior to an oral glucose tolerance test, although liraglutide did not lower glucose. Thus, these data show that GLP-1R agonists have differing anorectic potencies in the CNS, which may account for some of their clinical differences. Additionally, we show here that the glucose lowering properties of acute administration of GLP-1R agonists are not accounted for by their central effects. Copyright © 2014 Elsevier Inc. All rights reserved.

Preparation of GST Fusion Proteins.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-04-01

INTRODUCTIONThis protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

cAMP-secretion coupling is impaired in diabetic GK/Par rat β-cells: a defect counteracted by GLP-1.

PubMed

Dolz, Manuel; Movassat, Jamileh; Bailbé, Danielle; Le Stunff, Hervé; Giroix, Marie-Hélène; Fradet, Magali; Kergoat, Micheline; Portha, Bernard

2011-11-01

cAMP-raising agents with glucagon-like peptide-1 (GLP-1) as the first in class, exhibit multiple actions that are beneficial for the treatment of type 2 diabetic (T2D) patients, including improvement of glucose-induced insulin secretion (GIIS). To gain additional insight into the role of cAMP in the disturbed stimulus-secretion coupling within the diabetic β-cell, we examined more thoroughly the relationship between changes in islet cAMP concentration and insulin release in the GK/Par rat model of T2D. Basal cAMP content in GK/Par islets was significantly higher, whereas their basal insulin release was not significantly different from that of Wistar (W) islets. Even in the presence of IBMX or GLP-1, their insulin release did not significantly change despite further enhanced cAMP accumulation in both cases. The high basal cAMP level most likely reflects an increased cAMP generation in GK/Par compared with W islets since 1) forskolin dose-dependently induced an exaggerated cAMP accumulation; 2) adenylyl cyclase (AC)2, AC3, and G(s)α proteins were overexpressed; 3) IBMX-activated cAMP accumulation was less efficient and PDE-3B and PDE-1C mRNA were decreased. Moreover, the GK/Par insulin release apparatus appears less sensitive to cAMP, since GK/Par islets released less insulin at submaximal cAMP levels and required five times more cAMP to reach a maximal secretion rate no longer different from W. GLP-1 was able to reactivate GK/Par insulin secretion so that GIIS became indistinguishable from that of W. The exaggerated cAMP production is instrumental, since GLP-1-induced GIIS reactivation was lost in the presence the AC blocker 2',5'-dideoxyadenosine. This GLP-1 effect takes place in the absence of any improvement of the [Ca(2+)](i) response and correlates with activation of the cAMP-dependent PKA-dependent pathway.

[Effects of GLP-1 receptor agonists on carbohydrate metabolism control].

PubMed

Fernández-García, José Carlos; Colomo, Natalia; Tinahones, Francisco José

2014-09-01

Glucagon-like peptide-1 (GLP-1) receptor agonists are a new group of drugs for the treatment of type 2 diabetes mellitus (DM2). In the present article, we review the available evidence on the efficacy of GLP-1 receptor agonists as glucose-lowering agents, their place in therapeutic algorithms, and the clinical factors associated with a favorable treatment response. Finally, we describe the clinical characteristics of patients who may benefit from these drugs. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer

PubMed Central

Trabucco, S E; Priedigkeit, N; Parachoniak, C A; Vanden Borre, P; Morley, S; Rosenzweig, M; Gay, L M; Goldberg, M E; Suh, J; Ali, S M; Ross, J; Leyland-Jones, B; Young, B; Williams, C; Park, B; Tsai, M; Haley, B; Peguero, J; Callahan, R D; Sachelarie, I; Cho, J; Atkinson, J M; Bahreini, A; Nagle, A M; Puhalla, S L; Watters, R J; Erdogan-Yildirim, Z; Cao, L; Oesterreich, S; Mathew, A; Lucas, P C; Davidson, N E; Brufsky, A M; Frampton, G M; Stephens, P J; Chmielecki, J; Lee, A V

2018-01-01

Abstract Background Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations

Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer.

PubMed

Hartmaier, R J; Trabucco, S E; Priedigkeit, N; Chung, J H; Parachoniak, C A; Vanden Borre, P; Morley, S; Rosenzweig, M; Gay, L M; Goldberg, M E; Suh, J; Ali, S M; Ross, J; Leyland-Jones, B; Young, B; Williams, C; Park, B; Tsai, M; Haley, B; Peguero, J; Callahan, R D; Sachelarie, I; Cho, J; Atkinson, J M; Bahreini, A; Nagle, A M; Puhalla, S L; Watters, R J; Erdogan-Yildirim, Z; Cao, L; Oesterreich, S; Mathew, A; Lucas, P C; Davidson, N E; Brufsky, A M; Frampton, G M; Stephens, P J; Chmielecki, J; Lee, A V

2018-04-01

Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287-395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3' partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Collectively, these data indicate that N-terminal ESR1

A novel human-based receptor antagonist of sustained action reveals body weight control by endogenous GLP-1.

PubMed

Patterson, James T; Ottaway, Nickki; Gelfanov, Vasily M; Smiley, David L; Perez-Tilve, Diego; Pfluger, Paul T; Tschöp, Matthias H; Dimarchi, Richard D

2011-02-18

Ex-4 (9-39)a is a well characterized GLP-1 receptor antagonist that suffers from two notable limitations, its nonhuman amino acid sequence and its relatively short in vivo duration of action. Comparable N-terminal shortening of human GLP-1 lessens agonism but does not provide a high potency antagonist. Through a series of GLP-1/Ex-4 hybrid peptides, the minimal structural changes required to generate a pure GLP-1-based antagonist were identified as Glu16, Val19, and Arg20, yielding an antagonist of approximately 3-fold greater in vitro potency compared with Ex-4 (9-39)a. The structural basis of antagonism appears to result from stabilization of the α helix combined with enhanced electrostatic and hydrophobic interactions with the extracellular domain of the receptor. Site-specific acylation of the human-based antagonist yielded a peptide of increased potency as a GLP-1 receptor antagonist and 10-fold greater selectivity relative to the GIP receptor. The acylated antagonist demonstrated sufficient duration of action to maintain inhibitory activity when administered as a daily subcutaneous injection. The sustained pharmacokinetics and enhanced human sequence combine to form an antagonist optimized for clinical study. Daily administration of this antagonist by subcutaneous injection to diet-induced obese mice for 1 week caused a significant increase in food intake, body weight, and glucose intolerance, demonstrating endogenous GLP-1 as a relevant hormone in mammalian energy balance in the obese state.

Rubella virus: first calcium-requiring viral fusion protein.

PubMed

Dubé, Mathieu; Rey, Felix A; Kielian, Margaret

2014-12-01

Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca(2+) during virus entry. Other tested cations did not substitute. Ca(2+) was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca(2+) was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca(2+)-dependent viral fusion protein and has a unique membrane interaction mechanism.

Partial blockade of Kv2.1 channel potentiates GLP-1's insulinotropic effects in islets and reduces its dose required for improving glucose tolerance in type 2 diabetic male mice.

PubMed

Sukma Rita, Rauza; Dezaki, Katsuya; Kurashina, Tomoyuki; Kakei, Masafumi; Yada, Toshihiko

2015-01-01

Glucagon-like peptide-1 (GLP-1)-based medicines have recently been widely used to treat type 2 diabetic patients, whereas adverse effects of nausea and vomiting have been documented. Inhibition of voltage-gated K(+) channel subtype Kv2.1 in pancreatic β-cells has been suggested to contribute to mild depolarization and promotion of insulin release. This study aimed to determine whether the blockade of Kv2.1 channels potentiates the insulinotropic effect of GLP-1 agonists. Kv2.1 channel blocker guangxitoxin-1E (GxTx) and GLP-1 agonist exendin-4 at subthreshold concentrations, when combined, markedly increased the insulin release and cytosolic Ca(2+) concentration ([Ca(2+)]i) in a glucose-dependent manner in mouse islets and β-cells. Exendin-4 at subthreshold concentration alone increased islet insulin release and β-cell [Ca(2+)]i in Kv2.1(+/-) mice. The [Ca(2+)]i response to subthreshold exendin-4 and GxTx in combination was attenuated by pretreatment with protein kinase A inhibitor H-89, indicating the protein kinase A dependency of the cooperative effect. Furthermore, subthreshold doses of GxTx and GLP-1 agonist liraglutide in combination markedly increased plasma insulin and improved glucose tolerance in diabetic db/db mice and NSY mice. These results demonstrate that a modest suppression of Kv2.1 channels dramatically raises insulinotropic potency of GLP-1-based drugs, which opens a new avenue to reduce their doses and associated adverse effects while achieving the same glycemic control in type 2 diabetes.

Prevalence of anxiety disorders among Finnish primary care high utilizers and validation of Finnish translation of GAD-7 and GAD-2 screening tools.

PubMed

Kujanpää, Tero; Ylisaukko-Oja, Tero; Jokelainen, Jari; Hirsikangas, Sari; Kanste, Outi; Kyngäs, Helvi; Timonen, Markku

2014-06-01

To analyse the prevalence of GAD and other anxiety disorders, as well as sensitivity and specificity of GAD-7 among high utilizers of health care. Four municipal health centres in Northern Finland. A psychiatric interview was conducted for 150 high utilizers of health care. Prevalence of GAD as well as sensitivity and specificity of GAD-7. The prevalence of GAD was 4% in this study group of Finnish high utilizers of health care. The sensitivity of GAD-7 was 100.0% (95% CI 54.1-100.0) and the specificity of GAD-7 was 82.6% (95% CI 75.4-88.4) with a cut-off point of 7 or more. GAD is rather common among high utilizers of primary care, although the prevalence of 4% is lower than that previously reported. GAD-7 is a valid and useful tool for detecting GAD among primary health care patients.

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

PubMed

Porotto, Matteo; Devito, Ilaria; Palmer, Samantha G; Jurgens, Eric M; Yee, Jia L; Yokoyama, Christine C; Pessi, Antonello; Moscona, Anne

2011-12-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

Glucagon-Like Peptide-1 (GLP-1) Receptor Agonists in the Treatment of Obese Women with Polycystic Ovary Syndrome.

PubMed

Tzotzas, Themistoklis; Karras, Spyridon N; Katsiki, Niki

2017-01-01

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in females and is often associated with a number of cardiometabolic disorders such as central obesity, dyslipidaemia, hypertension, insulin resistance, hyperinsulinaemia, glucose intolerance and type 2 diabetes mellitus (T2DM). Glucagon-like peptide-1 (GLP-1), a gut hormone secreted after a meal, enhances glucosestimulated insulin secretion and additionally suppresses appetite and gastric motility. Most studies found impaired GLP-1 kinetics in obese individuals, whereas small studies in PCOS reported reduced, normal or even elevated GLP-1 levels. Apart from their efficacy in patients with T2DM, some GLP-1 receptor agonists (GLP-1 RAs) have been successfully tested in terms of both efficiency and safety in obese individuals without diabetes and liraglutide 3 mg once daily has been approved as an antiobesity drug in the USA and the European Union. Recently, some small trials of short duration using GLP-1 RAs as monotherapy or combined with metformin in obese PCOS women showed positive results regarding weight reduction and a decrease in testosterone levels but without significant effects on insulin levels, insulin sensitivity and menstrual patterns. Longer term studies with more patients and higher doses of liraglutide (as this drug is already approved for obese individuals) are required to determine the precise indications of GLP-1 RAs in PCOS and to evaluate safety issues. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

GAD vaccine reduces insulin loss in recently diagnosed type 1 diabetes: findings from a Bayesian meta-analysis.

PubMed

Beam, Craig A; MacCallum, Colleen; Herold, Kevan C; Wherrett, Diane K; Palmer, Jerry; Ludvigsson, Johnny

2017-01-01

GAD is a major target of the autoimmune response that occurs in type 1 diabetes mellitus. Randomised controlled clinical trials of a GAD + alum vaccine in human participants have so far given conflicting results. In this study, we sought to see whether a clearer answer to the question of whether GAD65 has an effect on C-peptide could be reached by combining individual-level data from the randomised controlled trials using Bayesian meta-analysis to estimate the probability of a positive biological effect (a reduction in C-peptide loss compared with placebo approximately 1 year after the GAD vaccine). We estimate that there is a 98% probability that 20 μg GAD with alum administered twice yields a positive biological effect. The effect is probably a 15-20% reduction in the loss of C-peptide at approximately 1 year after treatment. This translates to an annual expected loss of between -0.250 and -0.235 pmol/ml in treated patients compared with an expected 2 h AUC loss of -0.294 pmol/ml at 1 year for untreated newly diagnosed patients. The biological effect of this vaccination should be developed further in order to reach clinically desirable reductions in insulin loss in patients recently diagnosed with type 1 diabetes.

Expression of GAD67 and Dlx5 in the taste buds of mice genetically lacking Mash1.

PubMed

Kito-Shingaki, Ayae; Seta, Yuji; Toyono, Takashi; Kataoka, Shinji; Kakinoki, Yasuaki; Yanagawa, Yuchio; Toyoshima, Kuniaki

2014-06-01

It has been reported that a subset of type III taste cells express glutamate decarboxylase (GAD)67, which is a molecule that synthesizes gamma-aminobutyric acid (GABA), and that Mash1 could be a potential regulator of the development of GABAnergic neurons via Dlx transcription factors in the central nervous system. In this study, we investigated the expression of GAD67 and Dlx in the embryonic taste buds of the soft palate and circumvallate papilla using Mash1 knockout (KO)/GAD67-GFP knock-in mice. In the wild-type animal, a subset of type III taste cells contained GAD67 in the taste buds of the soft palate and the developing circumvallate papilla, whereas GAD67-expressing taste bud cells were missing from Mash1 KO mice. A subset of type III cells expressed mRNA for Dlx5 in the wild-type animals, whereas Dlx5-expressing cells were not evident in the apical part of the circumvallate papilla and taste buds in the soft palate of Mash1 KO mice. Our results suggest that Mash1 is required for the expression of GAD67 and Dlx5 in taste bud cells. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwasnicka-Crawford, Dorota A.; Carson, Andrew R.; Scherer, Stephen W.

The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNAmore » is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions.« less

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

PubMed Central

Jin, Chunsheng; Liu, Jining; Karlsson, Niclas G.; Holgersson, Jan

2016-01-01

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases. PMID:27456831

The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

PubMed

Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

2016-10-28

Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A) + RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A) + RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export*

PubMed Central

Port, Sarah A.; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H.

2016-01-01

Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. PMID:27613868

GLP-1 and GIP Levels in Patients With Hyperthyroidism: The Effect of Antithyroid Treatment.

PubMed

Cira, Duygu Kalkan; Sari, Ramazan; Ozdem, Sebahat; Yilmaz, Nusret; Bozkurt, Selen

2017-08-01

Incretin hormones (glucagon-like peptide-1 [GLP-1] and gastric inhibitory polypeptide [GIP]) may play a role in the development of glucose intolerance and hyperglycemia in patients with hyperthyroidism. We aimed to assess both incretin levels and treatment-induced changes in incretin levels in those with hyperthyroidism. A total of 24 subjects (12 with hyperthyroidism and 12 healthy) were enrolled in the study. Oral glucose tolerance test was performed and serum glucose, insulin GLP1, and GIP levels were evaluated at 0 (baseline), 30, 60, 90, and 120 minutes using ELISA. Measurements were repeated after euthyroidism was reached in subjects with hyperthyroidism. The baseline glucose level was higher in those with hyperthyroidism compared with controls ( P = 0.03). GLP-1 and GIP responses to oral glucose load did not differ significantly between those with hyperthyroidism and controls. Peak GLP-1 and GIP levels were reached in both groups at 60 and 90 minutes, respectively. Areas under the curve (AUCs) for GLP1 and GIP were similar in those with hyperthyroidism and controls. Although GLP-1 and GIP levels did not change before and after antithyroid treatment in subjects with hyperthyroidism, time to peak GLP-1 and GIP levels were reached at 30 minutes after euthyroid state was achieved. Reversal of hyperthyroid to euthyroid status did not induce significant changes in AUCs for incretins. The findings of the present study suggest that the total incretin response to oral glucose load is preserved in patients with hypertyhroidism, but peak incretin responses may change after achieving euthyroid state.

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

Bariatric surgery may reduce the risk of Alzheimer's diseases through GLP-1 mediated neuroprotective effects.

PubMed

Keshava, Hari B; Mowla, Ashkan; Heinberg, Leslie J; Schauer, Philip R; Brethauer, Stacy A; Aminian, Ali

2017-07-01

Obesity and diabetes are associated with deficits in multiple neurocognitive domains and increased risk for dementia. Over the last two decades, there has been a significant increase in bariatric and metabolic surgery worldwide, driven by rising intertwined pandemics of obesity and diabetes, along with improvement in surgical techniques. Patients undergoing bariatric surgery achieve a significant decrease in their excess weight and a multitude of sequela associated with obesity, diabetes, and metabolic syndrome. Glucagon-like peptide 1 (GLP-1) is an intestinal peptide that has been implicated as one of the weight loss-independent mechanisms in how bariatric surgery affects type 2 diabetes. GLP-1 improves insulin secretion, inhibits apoptosis and induce pancreatic islet neogenesis, promotes satiety, and can regulate heart rate and blood pressure. Moreover, numerous studies have demonstrated potential neuroprotective and neurotrophic effects of GLP-1. Increased GLP-1 activity has been shown to increase cortical activity, promote neuronal growth, and inhibit neuronal degeneration. Specifically, in experimental studies on Alzheimer's disease, GLP-1 decreases amyloid deposition and neurofibrillary tangles. Furthermore, recent studies have also suggested that GLP-1 based therapies, new class of antidiabetic drugs, have favorable effects on neurodegenerative disorders such as Alzheimer's disease. We present a hypothesis that bariatric surgery can help delay or even prevent the onset of Alzheimer's disease in long-term by increasing the levels of GLP-1. This hypothesis has a potential for many studies from basic science projects to large population studies to fully understand the neurological and cognitive consequences of bariatric surgery and associated rise in GLP-1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Physiological Gut Oxygenation Alters GLP-1 Secretion from the Enteroendocrine Cell Line STC-1.

PubMed

Kondrashina, Alina; Papkovsky, Dmitri; Giblin, Linda

2018-02-01

Enteroendocrine cell lines are routinely assayed in simple buffers at ≈20% oxygen to screen foods for bioactives that boost satiety hormone levels. However, in vivo, enteroendocrine cells are exposed to different phases of food digestion and function at low oxygen concentration, ranging from 7.5% in the stomach to 0.5% in the colon-rectal junction. The objective of this study is to investigate the effect of physiologically relevant O 2 concentrations of the gut on the production and secretion of the satiety hormone, glucagon-like peptide 1 (GLP-1), from the murine enteroendocrine cell line, secretin tumor cell line (STC-1), in response to dairy macronutrients as they transit the gut. GLP-1 exocytosis from STC-1 cells is influenced by both oxygen concentration and by individual macronutrients. At low oxygen, STC-1 cell viability is significantly improved for all macronutrient stimulations and cyclic adenosine monophosphate levels are dampened. GLP-1 secretion from STC-1 cells is influenced by both the phase of yogurt digestion and corresponding O 2 concentration. Atmospheric oxygen at 4.5% combined with upper gastric digesta, which simulates ileum conditions, yields the highest GLP-1 response. This demonstrates the importance of considering physiological oxygen levels and food digestion along gastrointestinal tract for reliable in vitro analysis of gut hormone secretion. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

PubMed

Rizo, Josep; Südhof, Thomas C

2012-01-01

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyuhas, Ronit; Noy, Hava; Fishman, Sigal

2009-08-21

HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited {approx}1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect onmore » 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.« less

Gender Differences in Associations of Glutamate Decarboxylase 1 Gene (GAD1) Variants with Panic Disorder

PubMed Central

Weber, Heike; Scholz, Claus Jürgen; Domschke, Katharina; Baumann, Christian; Klauke, Benedikt; Jacob, Christian P.; Maier, Wolfgang; Fritze, Jürgen; Bandelow, Borwin; Zwanzger, Peter Michael; Lang, Thomas; Fehm, Lydia; Ströhle, Andreas; Hamm, Alfons; Gerlach, Alexander L.; Alpers, Georg W.; Kircher, Tilo; Wittchen, Hans-Ulrich; Arolt, Volker; Pauli, Paul; Deckert, Jürgen; Reif, Andreas

2012-01-01

Background Panic disorder is common (5% prevalence) and females are twice as likely to be affected as males. The heritable component of panic disorder is estimated at 48%. Glutamic acid dehydrogenase GAD1, the key enzyme for the synthesis of the inhibitory and anxiolytic neurotransmitter GABA, is supposed to influence various mental disorders, including mood and anxiety disorders. In a recent association study in depression, which is highly comorbid with panic disorder, GAD1 risk allele associations were restricted to females. Methodology/Principal Findings Nineteen single nucleotide polymorphisms (SNPs) tagging the common variation in GAD1 were genotyped in two independent gender and age matched case-control samples (discovery sample n = 478; replication sample n = 584). Thirteen SNPs passed quality control and were examined for gender-specific enrichment of risk alleles associated with panic disorder by using logistic regression including a genotype×gender interaction term. The latter was found to be nominally significant for four SNPs (rs1978340, rs3762555, rs3749034, rs2241165) in the discovery sample; of note, the respective minor/risk alleles were associated with panic disorder only in females. These findings were not confirmed in the replication sample; however, the genotype×gender interaction of rs3749034 remained significant in the combined sample. Furthermore, this polymorphism showed a nominally significant association with the Agoraphobic Cognitions Questionnaire sum score. Conclusions/Significance The present study represents the first systematic evaluation of gender-specific enrichment of risk alleles of the common SNP variation in the panic disorder candidate gene GAD1. Our tentative results provide a possible explanation for the higher susceptibility of females to panic disorder. PMID:22662185

Glucagon-like peptide 1 receptor (GLP-1R) expression by nerve fibres in inflammatory bowel disease and functional effects in cultured neurons.

PubMed

Anand, Uma; Yiangou, Yiangos; Akbar, Ayesha; Quick, Tom; MacQuillan, Anthony; Fox, Mike; Sinisi, Marco; Korchev, Yuri E; Jones, Ben; Bloom, Steve R; Anand, Praveen

2018-01-01

Glucagon like-peptide 1 receptor (GLP-1R) agonists diminish appetite and may contribute to the weight loss in inflammatory bowel disease (IBD). The aim of this study was to determine, for the first time, the expression of GLP-1R by colon nerve fibres in patients with IBD, and functional effects of its agonists in cultured rat and human sensory neurons. GLP-1R and other nerve markers were studied by immunohistochemistry in colon biopsies from patients with IBD (n = 16) and controls (n = 8), human dorsal root ganglia (DRG) tissue, and in GLP-1R transfected HEK293 cells. The morphological effects of incretin hormones oxyntomodulin, exendin-4 and glucagon were studied on neurite extension in cultured DRG neurons, and their functional effects on capsaicin and ATP signalling, using calcium imaging. Significantly increased numbers of colonic mucosal nerve fibres were observed in IBD biopsies expressing GLP-1R (p = 0.0013), the pan-neuronal marker PGP9.5 (p = 0.0008), and sensory neuropeptide CGRP (p = 0.0014). An increase of GLP-1R positive nerve fibres in IBD colon was confirmed with a different antibody to GLP-1R (p = 0.016). GLP-1R immunostaining was intensely positive in small and medium-sized neurons in human DRG, and in human and rat DRG cultured neurons. Co-localization of GLP-1R expression with neuronal markers in colon and DRG confirmed the neural expression of GLP-1R, and antibody specificity was confirmed in HEK293 cells transfected with the GLP-1R. Treatment with oxyntomodulin, exendin-4 and GLP-1 increased neurite length in cultured neurons compared with controls, but did not stimulate calcium influx directly, or affect capsaicin responses. However, exendin-4 significantly enhanced ATP responses in human DRG neurons. Our results show that increased GLP-1R innervation in IBD bowel could mediate enhanced visceral afferent signalling, and provide a peripheral target for therapeutic intervention. The differential effect of GLP-1R agonists on capsaicin and ATP

Glucagon-like peptide 1 receptor (GLP-1R) expression by nerve fibres in inflammatory bowel disease and functional effects in cultured neurons

PubMed Central

Yiangou, Yiangos; Akbar, Ayesha; Quick, Tom; MacQuillan, Anthony; Fox, Mike; Sinisi, Marco; Korchev, Yuri E.; Jones, Ben; Bloom, Steve R.; Anand, Praveen

2018-01-01

Introduction Glucagon like-peptide 1 receptor (GLP-1R) agonists diminish appetite and may contribute to the weight loss in inflammatory bowel disease (IBD). Objectives The aim of this study was to determine, for the first time, the expression of GLP-1R by colon nerve fibres in patients with IBD, and functional effects of its agonists in cultured rat and human sensory neurons. Methods GLP-1R and other nerve markers were studied by immunohistochemistry in colon biopsies from patients with IBD (n = 16) and controls (n = 8), human dorsal root ganglia (DRG) tissue, and in GLP-1R transfected HEK293 cells. The morphological effects of incretin hormones oxyntomodulin, exendin-4 and glucagon were studied on neurite extension in cultured DRG neurons, and their functional effects on capsaicin and ATP signalling, using calcium imaging. Results Significantly increased numbers of colonic mucosal nerve fibres were observed in IBD biopsies expressing GLP-1R (p = 0.0013), the pan-neuronal marker PGP9.5 (p = 0.0008), and sensory neuropeptide CGRP (p = 0.0014). An increase of GLP-1R positive nerve fibres in IBD colon was confirmed with a different antibody to GLP-1R (p = 0.016). GLP-1R immunostaining was intensely positive in small and medium-sized neurons in human DRG, and in human and rat DRG cultured neurons. Co-localization of GLP-1R expression with neuronal markers in colon and DRG confirmed the neural expression of GLP-1R, and antibody specificity was confirmed in HEK293 cells transfected with the GLP-1R. Treatment with oxyntomodulin, exendin-4 and GLP-1 increased neurite length in cultured neurons compared with controls, but did not stimulate calcium influx directly, or affect capsaicin responses. However, exendin-4 significantly enhanced ATP responses in human DRG neurons. Conclusion Our results show that increased GLP-1R innervation in IBD bowel could mediate enhanced visceral afferent signalling, and provide a peripheral target for therapeutic intervention. The differential

Antigen-based therapy with glutamic acid decarboxylase (GAD) vaccine in patients with recent-onset type 1 diabetes: a randomised double-blind trial.

PubMed

Wherrett, Diane K; Bundy, Brian; Becker, Dorothy J; DiMeglio, Linda A; Gitelman, Stephen E; Goland, Robin; Gottlieb, Peter A; Greenbaum, Carla J; Herold, Kevan C; Marks, Jennifer B; Monzavi, Roshanak; Moran, Antoinette; Orban, Tihamer; Palmer, Jerry P; Raskin, Philip; Rodriguez, Henry; Schatz, Desmond; Wilson, Darrell M; Krischer, Jeffrey P; Skyler, Jay S

2011-07-23

Glutamic acid decarboxylase (GAD) is a major target of the autoimmune response that occurs in type 1 diabetes mellitus. In animal models of autoimmunity, treatment with a target antigen can modulate aggressive autoimmunity. We aimed to assess whether immunisation with GAD formulated with aluminum hydroxide (GAD-alum) would preserve insulin production in recent-onset type 1 diabetes. Patients aged 3-45 years who had been diagnosed with type 1 diabetes for less than 100 days were enrolled from 15 sites in the USA and Canada, and randomly assigned to receive one of three treatments: three injections of 20 μg GAD-alum, two injections of 20 μg GAD-alum and one of alum, or 3 injections of alum. Injections were given subcutaneously at baseline, 4 weeks later, and 8 weeks after the second injection. The randomisation sequence was computer generated at the TrialNet coordinating centre. Patients and study personnel were masked to treatment assignment. The primary outcome was the baseline-adjusted geometric mean area under the curve (AUC) of serum C-peptide during the first 2 h of a 4-h mixed meal tolerance test at 1 year. Secondary outcomes included changes in glycated haemoglobin A(1c) (HbA(1c)) and insulin dose, and safety. Analysis included all randomised patients with known measurements. This trial is registered with ClinicalTrials.gov, number NCT00529399. 145 patients were enrolled and treated with GAD-alum (n=48), GAD-alum plus alum (n=49), or alum (n=48). At 1 year, the 2-h AUC of C-peptide, adjusted for age, sex, and baseline C-peptide value, was 0·412 nmol/L (95% CI 0·349-0·478) in the GAD-alum group, 0·382 nmol/L (0·322-0·446) in the GAD-alum plus alum group, and 0·413 nmol/L (0·351-0·477) in the alum group. The ratio of the population mean of the adjusted geometric mean 2-h AUC of C-peptide was 0·998 (95% CI 0·779-1·22; p=0·98) for GAD-alum versus alum, and 0·926 (0·720-1·13; p=0·50) for GAD-alum plus alum versus alum. HbA(1c), insulin use, and

Linear {GLP}-algebras and their elementary theories

NASA Astrophysics Data System (ADS)

Pakhomov, F. N.

2016-12-01

The polymodal provability logic {GLP} was introduced by Japaridze in 1986. It is the provability logic of certain chains of provability predicates of increasing strength. Every polymodal logic corresponds to a variety of polymodal algebras. Beklemishev and Visser asked whether the elementary theory of the free {GLP}-algebra generated by the constants \\mathbf{0}, \\mathbf{1} is decidable [1]. For every positive integer n we solve the corresponding question for the logics {GLP}_n that are the fragments of {GLP} with n modalities. We prove that the elementary theory of the free {GLP}_n-algebra generated by the constants \\mathbf{0}, \\mathbf{1} is decidable for all n. We introduce the notion of a linear {GLP}_n-algebra and prove that all free {GLP}_n-algebras generated by the constants \\mathbf{0}, \\mathbf{1} are linear. We also consider the more general case of the logics {GLP}_α whose modalities are indexed by the elements of a linearly ordered set α: we define the notion of a linear algebra and prove the latter result in this case.

T-cell immunotherapy for human MK-1-expressing tumors using a fusion protein of the superantigen SEA and anti-MK-1 scFv antibody.

PubMed

Ueno, Aruto; Arakawa, Fumiko; Abe, Hironori; Matsumoto, Hisanobu; Kudo, Toshio; Asano, Ryutaro; Tsumoto, Kohei; Kumagai, Izumi; Kuroki, Motomu; Kuroki, Masahide

2002-01-01

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we constructed a recombinant fusion protein of SEA and the single-chain variable fragment (scFv) of the FU-MK-1 antibody, which recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas. We employed recombinant DNA techniques to fuse recombinant mutant SEA to an scFv antibody derived from FU-MK-1 and the resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system, purified with a metal-affinity column, and characterized for its MK-1-binding specificity and its antitumor activity. The SEA/FUscFv fusion protein retained the reactivity with MK-1-expressing tumor cells, introduced a specific cytotoxicity of lymphokine-activated killer T-cells to the tumor cells, and consequently suppressed the tumor growth in a SCID mouse xenograft model. This genetically engineered SEA/FUscFv fusion protein may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

Cultural-based biases of the GAD-7.

PubMed

Parkerson, Holly A; Thibodeau, Michel A; Brandt, Charles P; Zvolensky, Michael J; Asmundson, Gordon J G

2015-04-01

The GAD-7 is a popular measure of generalized anxiety disorder (GAD) symptoms that has been used across many cultural groups. Existing evidence demonstrates that the prevalence of GAD varies across self-identified ethnic/cultural groups, a phenomenon that some researchers attribute to cross-cultural measurement error rather than to actual differences in rates of GAD. Nonetheless, the effect of culture on factor structure and response patterns to the GAD-7 have not been examined and could result over- or under-estimated GAD-7 scores across different cultural groups. The current investigation assessed the factor structure of the GAD-7 in White/Caucasian, Hispanic, and Black/African American undergraduates and tested for cultural-based biases. A modified one-factor model exhibited good fit across subsamples. Results revealed that Black/African American participants with high GAD symptoms scored lower on the GAD-7 than other participants with similar GAD symptoms. Results highlight the need for culturally sensitive GAD screening tools. Copyright © 2015 Elsevier Ltd. All rights reserved.

Design and testing for a nontagged F1-V fusion protein as vaccine antigen against bubonic and pneumonic plague.

PubMed

Powell, Bradford S; Andrews, Gerard P; Enama, Jeffrey T; Jendrek, Scott; Bolt, Chris; Worsham, Patricia; Pullen, Jeffrey K; Ribot, Wilson; Hines, Harry; Smith, Leonard; Heath, David G; Adamovicz, Jeffrey J

2005-01-01

A two-component recombinant fusion protein antigen was re-engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine-tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea-denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N-terminal methionine. Purity, quality, and higher-order structure were compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy, and multi-angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V protein also protected mice from wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1-V as the active pharmaceutical ingredient of the next plague vaccine.

GLP-1-oestrogen attenuates hyperphagia and protects from beta cell failure in diabetes-prone New Zealand obese (NZO) mice.

PubMed

Schwenk, Robert W; Baumeier, Christian; Finan, Brian; Kluth, Oliver; Brauer, Christine; Joost, Hans-Georg; DiMarchi, Richard D; Tschöp, Matthias H; Schürmann, Annette

2015-03-01

Oestrogens have previously been shown to exert beta cell protective, glucose-lowering effects in mouse models. Therefore, the recent development of a glucagon-like peptide-1 (GLP-1)-oestrogen conjugate, which targets oestrogen into cells expressing GLP-1 receptors, offers an opportunity for a cell-specific and enhanced beta cell protection by oestrogen. The purpose of this study was to compare the effects of GLP-1 and GLP-1-oestrogen during beta cell failure under glucolipotoxic conditions. Male New Zealand obese (NZO) mice were treated with daily s.c. injections of GLP-1 and GLP-1-oestrogen, respectively. Subsequently, the effects on energy homeostasis and beta cell integrity were measured. In order to clarify the targeting of GLP-1-oestrogen, transcription analyses of oestrogen-responsive genes in distinct tissues as well as microarray analyses in pancreatic islets were performed. In contrast to GLP-1, GLP-1-oestrogen significantly decreased food intake resulting in a substantial weight reduction, preserved normoglycaemia, increased glucose tolerance and enhanced beta cell protection. Analysis of hypothalamic mRNA profiles revealed elevated expression of Pomc and Leprb. In livers from GLP-1-oestrogen-treated mice, expression of lipogenic genes was attenuated and hepatic triacylglycerol levels were decreased. In pancreatic islets, GLP-1-oestrogen altered the mRNA expression to a pattern that was similar to that of diabetes-resistant NZO females. However, conventional oestrogen-responsive genes were not different, indicating rather indirect protection of pancreatic beta cells. GLP-1-oestrogen efficiently protects NZO mice against carbohydrate-induced beta cell failure by attenuation of hyperphagia. In this regard, targeted delivery of oestrogen to the hypothalamus by far exceeds the anorexigenic capacity of GLP-1 alone.

Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

PubMed

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-12-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

Altered glucose metabolism after bariatric surgery: What's GLP-1 got to do with it?

PubMed

Smith, Eric P; Polanco, Georgina; Yaqub, Abid; Salehi, Marzieh

2018-06-01

Bariatric surgery is an effective treatment for obesity. The two widely performed weight-loss procedures, Roux-en-Y gastric bypass (GB) and sleeve gastrectomy (SG), alter postprandial glucose pattern and enhance gut hormone secretion immediately after surgery before significant weight loss. This weight-loss independent glycemic effects of GB has been attributed to an accelerated nutrient transit from stomach pouch to the gut and enhanced secretion of insulinotropic gut factors; in particular, glucagon-like peptide-1 (GLP-1). Meal-induced GLP-1 secretion is as much as tenfold higher in patients after GB compared to non-surgical individuals and inhibition of GLP-1 action during meals reduces postprandial hyperinsulinemia after GB two to three times more than that in persons without surgery. Moreover, in a subgroup of patients with the late complication of postprandial hyperinsulinemic hypoglycemia after GB, GLP1R blockade reverses hypoglycemia by reducing meal stimulated insulin secretion. The role of enteroinsular axis activity after SG, an increasingly popular alternative to GB, is less understood but, similar to GB, SG accelerates nutrient delivery to the intestine, improves glucose tolerance, and increases postprandial GLP-1 secretion. This review will focus on the current evidence for and against the role of GLP-1 on glycemic effects of GB and will also highlight differences between GB and SG. Copyright © 2017 Elsevier Inc. All rights reserved.

Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ding, Donglin; Qu, Xiying; Li, Lin

Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was foundmore » to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency.« less

A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation▿

PubMed Central

Gardner, Amanda E.; Dutch, Rebecca E.

2007-01-01

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion. PMID:17507474

A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion Proteins is Critical for Proper F Protein Folding†

PubMed Central

Gardner, Amanda E.; Martin, Kimberly L.; Dutch, Rebecca E.

2008-01-01

Paramyxoviruses are a diverse family which utilizes a fusion (F) protein to enter cells via fusion of the viral lipid bilayer with a target cell membrane. Although certain regions of F are known to play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequence alignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region of conserved amino acid sequence in a large domain between the heptad repeats of F1, designated CBF1. We employed site-directed mutagenesis to analyze the function of completely conserved residues of CBF1 in both the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF1 point mutants were deficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For some SV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30°C, and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 F V402A displayed a hyperfusogenic phenotype at both 30°C and 37°C, indicating this mutation allows for efficient fusion with only an extremely small amount of cleaved, active protein. The recently published prefusogenic structure of PIV5/SV5 F [Yin, H.S., et al. (2006) Nature 439, 38–44] indicates that residues within and flanking CBF1 interact with the fusion peptide domain. Together, these data suggest that CBF1-fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from across this important viral family, and can also modulate subsequent membrane fusion promotion. PMID:17417875

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins

PubMed Central

Raran-Kurussi, Sreejith; Keefe, Karina; Waugh, David S.

2015-01-01

Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that 1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and 2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins. PMID:25782741

Immunogenicity of a recombinant fusion protein of tandem repeat epitopes of foot-and-mouth disease virus type Asia 1 for guinea pigs.

PubMed

Zhang, Q; Yang, Y Q; Zhang, Z Y; Li, L; Yan, W Y; Jiang, W J; Xin, A G; Lei, C X; Zheng, Z X

2002-01-01

In this study, the sequences of capsid protein VPI regions of YNAs1.1 and YNAs1.2 isolates of foot-and-mouth disease virus (FMDV) were analyzed and a peptide containing amino acids (aa) 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia I was assumed to contain B and T cell epitopes, because it is hypervariable and includes a cell attachment site RGD located in the G-H loop. The DNA fragments encoding aa 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia 1 were chemically synthesized and ligated into a tandem repeat of aa 133-158-20 approximately 34-133-158. In order to enhance its immunogenicity, the tandem repeat was inserted downstream of the beta-galactosidase gene in the expression vector pWR590. This insertion yielded a recombinant expression vector pAS1 encoding the fusion protein. The latter reacted with sera from FMDV type Asia 1-infected animals in vitro and elicited high levels of neutralizing antibodies in guinea pigs. The T cell proliferation in immunized animals increased following stimulation with the fusion protein. It is reported for the first time that a recombinant fusion protein vaccine was produced using B and T cell epitopes of FMDV type Asia 1 and that this fusion protein was immunogenic. The fusion protein reported here can serve as a candidate of fusion epitopes for design of a vaccine against FMDV type Asia 1.

ChiPPI: a novel method for mapping chimeric protein-protein interactions uncovers selection principles of protein fusion events in cancer.

PubMed

Frenkel-Morgenstern, Milana; Gorohovski, Alessandro; Tagore, Somnath; Sekar, Vaishnovi; Vazquez, Miguel; Valencia, Alfonso

2017-07-07

Fusion proteins, comprising peptides deriving from the translation of two parental genes, are produced in cancer by chromosomal aberrations. The expressed fusion protein incorporates domains of both parental proteins. Using a methodology that treats discrete protein domains as binding sites for specific domains of interacting proteins, we have cataloged the protein interaction networks for 11 528 cancer fusions (ChiTaRS-3.1). Here, we present our novel method, chimeric protein-protein interactions (ChiPPI) that uses the domain-domain co-occurrence scores in order to identify preserved interactors of chimeric proteins. Mapping the influence of fusion proteins on cell metabolism and pathways reveals that ChiPPI networks often lose tumor suppressor proteins and gain oncoproteins. Furthermore, fusions often induce novel connections between non-interactors skewing interaction networks and signaling pathways. We compared fusion protein PPI networks in leukemia/lymphoma, sarcoma and solid tumors finding distinct enrichment patterns for each disease type. While certain pathways are enriched in all three diseases (Wnt, Notch and TGF β), there are distinct patterns for leukemia (EGFR signaling, DNA replication and CCKR signaling), for sarcoma (p53 pathway and CCKR signaling) and solid tumors (FGFR and EGFR signaling). Thus, the ChiPPI method represents a comprehensive tool for studying the anomaly of skewed cellular networks produced by fusion proteins in cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Active metabolite of GLP-1 mediates myocardial glucose uptake and improves left ventricular performance in conscious dogs with dilated cardiomyopathy.

PubMed

Nikolaidis, Lazaros A; Elahi, Dariush; Shen, You-Tang; Shannon, Richard P

2005-12-01

We have shown previously that the glucagon-like peptide-1 (GLP-1)-(7-36) amide increases myocardial glucose uptake and improves left ventricular (LV) and systemic hemodynamics in both conscious dogs with pacing-induced dilated cardiomyopathy (DCM) and humans with LV systolic dysfunction after acute myocardial infarction. However, GLP-1-(7-36) is rapidly degraded in the plasma to GLP-1-(9-36) by dipeptidyl peptidase IV (DPP IV), raising the issue of which peptide is the active moiety. By way of methodology, we compared the efficacy of a 48-h continuous intravenous infusion of GLP-1-(7-36) (1.5 pmol.kg(-1).min(-1)) to GLP-1-(9-36) (1.5 pmol.kg(-1).min(-1)) in 28 conscious, chronically instrumented dogs with pacing-induced DCM by measuring LV function and transmyocardial substrate uptake under basal and insulin-stimulated conditions using hyperinsulinemic-euglycemic clamps. As a result, dogs with DCM demonstrated myocardial insulin resistance under basal and insulin-stimulated conditions. Both GLP-1-(7-36) and GLP-1-(9-36) significantly reduced (P < 0.01) LV end-diastolic pressure [GLP-1-(7-36), 28 +/- 1 to 15 +/- 2 mmHg; GLP-1-(9-36), 29 +/- 2 to 16 +/- 1 mmHg] and significantly increased (P < 0.01) the first derivative of LV pressure [GLP-1-(7-36), 1,315 +/- 81 to 2,195 +/- 102 mmHg/s; GLP-1-(9-36), 1,336 +/- 77 to 2,208 +/- 68 mmHg] and cardiac output [GLP-1-(7-36), 1.5 +/- 0.1 to 1.9 +/- 0.1 l/min; GLP-1-(9-36), 2.0 +/- 0.1 to 2.4 +/- 0.05 l/min], whereas an equivolume infusion of saline had no effect. Both peptides increased myocardial glucose uptake but without a significant increase in plasma insulin. During the GLP-1-(9-36) infusion, negligible active (NH2-terminal) peptide was measured in the plasma. In conclusion, in DCM, GLP-1-(9-36) mimics the effects of GLP-1-(7-36) in stimulating myocardial glucose uptake and improving LV and systemic hemodynamics through insulinomimetic as opposed to insulinotropic effects. These data suggest that GLP-1-(9-36) amide is

Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy in Chlamydomonas.

PubMed

Liu, Yanjie; Misamore, Michael J; Snell, William J

2010-05-01

The plasma membranes of gametes are specialized for fusion, yet, once fusion occurs, in many organisms the new zygote becomes incapable of further membrane fusion reactions. The molecular mechanisms that underlie this loss of fusion capacity (block to polygamy) remain unknown. During fertilization in the green alga Chlamydomonas, the plus gamete-specific membrane protein FUS1 is required for adhesion between the apically localized sites on the plasma membranes of plus and minus gametes that are specialized for fusion, and the minus-specific membrane protein HAP2 is essential for completion of the membrane fusion reaction. HAP2 (GCS1) family members are also required for fertilization in Arabidopsis, and for the membrane fusion reaction in the malaria organism Plasmodium berghei. Here, we tested whether Chlamydomonas gamete fusion triggers alterations in FUS1 and HAP2 and renders the plasma membranes of the cells incapable of subsequent fusion. We find that, even though the fusogenic sites support multi-cell adhesions, triploid zygotes are rare, indicating a fusion-triggered block to the membrane fusion reaction. Consistent with the extinction of fusogenic capacity, both FUS1 and HAP2 are degraded upon fusion. The rapid, fusion-triggered cleavage of HAP2 in zygotes is distinct from degradation occurring during constitutive turnover in gametes. Thus, gamete fusion triggers specific degradation of fusion-essential proteins and renders the zygote incapable of fusion. Our results provide the first molecular explanation for a membrane block to polygamy in any organism.

Myocardial regeneration in adriamycin cardiomyopathy by nuclear expression of GLP1 using ultrasound targeted microbubble destruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Shuyuan; Chen, Jiaxi; Huang, Pintong

Recently GLP-1 was found to have cardioprotective effects independent of those attributable to tight glycemic control. Methods and results: We employed ultrasound targeted microbubble destruction (UTMD) to deliver piggybac transposon plasmids encoding the GLP-1 gene with a nuclear localizing signal to rat hearts with adriamycin cardiomyopathy. After a single UTMD treatment, overexpression of transgenic GLP-1 was found in nuclei of rat heart cells with evidence that transfected cardiac cells had undergone proliferation. UTMD-GLP-1 gene therapy restored LV mass, fractional shortening index, and LV posterior wall diameter to nearly normal. Nuclear overexpression of GLP-1 by inducing phosphorylation of FoxO1-S256 and translocationmore » of FoxO1 from the nucleus to the cytoplasm significantly inactivated FoxO1 and activated the expression of cyclin D1 in nuclei of cardiac muscle cells. Reversal of adriamycin cardiomyopathy appeared to be mediated by dedifferentiation and proliferation of nuclear FoxO1-positive cardiac muscle cells with evidence of embryonic stem cell markers (OCT4, Nanog, SOX2 and c-kit), cardiac early differentiation markers (NKX2.5 and ISL-1) and cellular proliferation markers (BrdU and PHH3) after UTMD with GLP-1 gene therapy. Conclusions: Intranuclear myocardial delivery of the GLP-1gene can reverse established adriamycin cardiomyopathy by stimulating myocardial regeneration. - Highlights: • The activation of nuclear FoxO1 in cardiac muscle cells associated with adriamycin cardiomyopathy. • Myocardial nuclear GLP-1 stimulates myocardial regeneration and reverses adriamycin cardiomyopathy. • The process of myocardial regeneration associated with dedifferentiation and proliferation.« less

Transduced PEP-1-Heme Oxygenase-1 Fusion Protein Attenuates Lung Injury in Septic Shock Rats

PubMed Central

Yan, Xue-Tao; Wang, Yan-Lin; Zhang, Zong-Ze; Tang, Jun-Jiao

2018-01-01

Oxidative stress and inflammation have been identified to play a vital role in the pathogenesis of lung injury induced by septic shock. Heme oxygenase-1 (HO-1), an effective antioxidant and anti-inflammatory and antiapoptotic substance, has been used for the treatment of heart, lung, and liver diseases. Thus, we postulated that administration of exogenous HO-1 protein transduced by cell-penetrating peptide PEP-1 has a protective role against septic shock-induced lung injury. Septic shock produced by cecal ligation and puncture caused severe lung damage, manifested in the increase in the lung wet/dry ratio, oxidative stress, inflammation, and apoptosis. However, these changes were reversed by treatment with the PEP-1-HO-1 fusion protein, whereas lung injury in septic shock rats was alleviated. Furthermore, the septic shock upregulated the expression of Toll-like receptor 4 (TLR4) and transcription factor NF-κB, accompanied by the increase of lung injury. Administration of PEP-1-HO-1 fusion protein reversed septic shock-induced lung injury by downregulating the expression of TLR4 and NF-κB. Our study indicates that treatment with HO-1 protein transduced by PEP-1 confers protection against septic shock-induced lung injury by its antioxidant, anti-inflammatory, and antiapoptotic effects. PMID:29682161

Glucagon-like peptide-1 acutely affects renal blood flow and urinary flow rate in spontaneously hypertensive rats despite significantly reduced renal expression of GLP-1 receptors.

PubMed

Ronn, Jonas; Jensen, Elisa P; Wewer Albrechtsen, Nicolai J; Holst, Jens Juul; Sorensen, Charlotte M

2017-12-01

Glucagon-like peptide-1 (GLP-1) is an incretin hormone increasing postprandial insulin release. GLP-1 also induces diuresis and natriuresis in humans and rodents. The GLP-1 receptor is extensively expressed in the renal vascular tree in normotensive rats where acute GLP-1 treatment leads to increased mean arterial pressure (MAP) and increased renal blood flow (RBF). In hypertensive animal models, GLP-1 has been reported both to increase and decrease MAP. The aim of this study was to examine expression of renal GLP-1 receptors in spontaneously hypertensive rats (SHR) and to assess the effect of acute intrarenal infusion of GLP-1. We hypothesized that GLP-1 would increase diuresis and natriuresis and reduce MAP in SHR. Immunohistochemical staining and in situ hybridization for the GLP-1 receptor were used to localize GLP-1 receptors in the kidney. Sevoflurane-anesthetized normotensive Sprague-Dawley rats and SHR received a 20 min intrarenal infusion of GLP-1 and changes in MAP, RBF, heart rate, dieresis, and natriuresis were measured. The vasodilatory effect of GLP-1 was assessed in isolated interlobar arteries from normo- and hypertensive rats. We found no expression of GLP-1 receptors in the kidney from SHR. However, acute intrarenal infusion of GLP-1 increased MAP, RBF, dieresis, and natriuresis without affecting heart rate in both rat strains. These results suggest that the acute renal effects of GLP-1 in SHR are caused either by extrarenal GLP-1 receptors activating other mechanisms (e.g., insulin) to induce the renal changes observed or possibly by an alternative renal GLP-1 receptor. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

GAD1 gene polymorphisms are associated with hyperactivity in Attention-Deficit/Hyperactivity Disorder.

PubMed

Bruxel, Estela M; Akutagava-Martins, Glaucia C; Salatino-Oliveira, Angélica; Genro, Julia P; Zeni, Cristian P; Polanczyk, Guilherme V; Chazan, Rodrigo; Schmitz, Marcelo; Rohde, Luis A; Hutz, Mara H

2016-12-01

Attention-Deficit/Hyperactivity Disorder (ADHD) is one of the most common neurodevelopmental disorders of childhood. Recent studies suggest a role for γ-aminobutyric acid (GABA) on ADHD hyperactive/impulsive symptoms due to behavioral disinhibition resulting from inappropriate modulation of both glutamatergic and GABAergic signaling. The glutamic acid decarboxylase (GAD1) gene encodes a key enzyme of GABA biosynthesis. The aim of the present study was to investigate the possible influence of GAD1 SNPs rs3749034 and rs11542313 on ADHD susceptibility. The clinical sample consisted of 547 families with ADHD probands recruited at the ADHD Outpatient Clinics from Hospital de Clínicas de Porto Alegre. Hyperactive/impulsive symptoms were evaluated based on parent reports from the Swanson, Nolan, and Pelham Scale-version IV (SNAP-IV). The C allele of rs11542313 was significantly overtransmitted from parents to ADHD probands (P = 0.02). Hyperactive/impulsive score was higher in rs3749034G allele (P = 0.005, Cohen's D = 0.19) and rs11542313C allele (P = 0.03; Cohen's D = 0.16) carriers. GAD1 haplotypes were also associated with higher hyperactive/impulsive scores in ADHD youths (global P-value = 0.01). In the specific haplotype test, the GC haplotype was the one with the highest hyperactive/impulsive scores (P = 0.03). Our results suggest that the GAD1 gene is associated with ADHD susceptibility, contributing particularly to the hyperactive/impulsive symptom domain. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

GLP-1 and estrogen conjugate acts in the supramammillary nucleus to reduce food-reward and body weight.

PubMed

Vogel, Heike; Wolf, Stefanie; Rabasa, Cristina; Rodriguez-Pacheco, Francisca; Babaei, Carina S; Stöber, Franziska; Goldschmidt, Jürgen; DiMarchi, Richard D; Finan, Brian; Tschöp, Matthias H; Dickson, Suzanne L; Schürmann, Annette; Skibicka, Karolina P

2016-11-01

The obesity epidemic continues unabated and currently available pharmacological treatments are not sufficiently effective. Combining gut/brain peptide, GLP-1, with estrogen into a conjugate may represent a novel, safe and potent, strategy to treat diabesity. Here we demonstrate that the central administration of GLP-1-estrogen conjugate reduced food reward, food intake, and body weight in rats. In order to determine the brain location of the interaction of GLP-1 with estrogen, we avail of single-photon emission computed tomography imaging of regional cerebral blood flow and pinpoint a brain site unexplored for its role in feeding and reward, the supramammillary nucleus (SUM) as a potential target of the conjugated GLP-1-estrogen. We confirm that conjugated GLP-1 and estrogen directly target the SUM with site-specific microinjections. Additional microinjections of GLP-1-estrogen into classic energy balance controlling nuclei, the lateral hypothalamus (LH) and the nucleus of the solitary tract (NTS) revealed that the metabolic benefits resulting from GLP-1-estrogen injections are mediated through the LH and to some extent by the NTS. In contrast, no additional benefit of the conjugate was noted on food reward when the compound was microinjected into the LH or the NTS, identifying the SUM as the only neural substrate identified here to underlie the reward reducing benefits of GLP-1 and estrogen conjugate. Collectively we discover a surprising neural substrate underlying food intake and reward effects of GLP-1 and estrogen and uncover a new brain area capable of regulating energy balance and reward. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Adding prandial GLP-1 receptor agonists to basal insulin: a promising option for type 2 diabetes therapy.

PubMed

Goldenberg, Ronald M; Berard, Lori

2018-01-01

Diabetes mellitus is a serious and increasingly prevalent condition in Canada and around the world. Treatment strategies have become increasingly complex, with a widening array of pharmacological agents available for glycemic management in type 2 diabetes mellitus (T2DM). New therapies that act in concert with available basal insulins may represent alternatives to basal insulin intensification with prandial or pre-mixed insulin. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) have recently shown promise as useful additions to basal insulin, with significant reductions in glycated hemoglobin and potentially beneficial effects on body weight. This review will focus on pivotal clinical trials to assess the potential benefits of adding prandial GLP-1 RAs to basal insulin in patients with T2DM. Clinical studies combining prandial GLP-1 RAs and basal insulin (published between 2011 and July 2017) were identified and reviewed in PubMed, the Cochrane Central Register of Clinical Trials (Issue 6, June 2017), and clinicaltrials.gov. Most of the studies presented in this review show that the addition of a prandial GLP-1 RA to basal insulin results in equal or slightly superior efficacy compared to the addition of prandial insulin, together with weight loss and less hypoglycemia. The results of the studies suggest that a prandial GLP-1 RA as an add-on to basal insulin may be a safe and effective treatment intensification option (vs basal-plus or basal-bolus insulin).

Acute peripheral administration of synthetic human GLP-1 (7-36 amide) decreases circulating IL-6 in obese patients with type 2 diabetes mellitus: a potential role for GLP-1 in modulation of the diabetic pro-inflammatory state?

PubMed

Daousi, Christina; Pinkney, Jonathan H; Cleator, Jacqueline; Wilding, John P; Ranganath, L R

2013-05-10

To explore the effects of acute administration of GLP-1 and GIP on circulating levels of key adipocyte-derived hormones and gut-brain peptides with established roles in energy and appetite regulation, modulation of insulin sensitivity and inflammation. Six obese male patients with diet-treated type 2 diabetes (T2DM) and 6 healthy lean subjects were studied. The protocol included 4 experiments for each participant that were carried out in randomised order and comprised: GLP-1 infusion at a rate of 1 pmol/kg/min for 4h, GIP at a rate of 2 pmol/kg/min, GLP-1+GIP and placebo infusion. Plasma leptin, adiponectin, IL-6, insulin, ghrelin and obestatin were measured at baseline, 15, 60, 120, 180 and 240 min following the start of infusion. Patients with T2DM had higher baseline IL-6 compared with healthy [day of placebo infusion: T2DM IL-6 mean (SEM) 1.3 (0.3) pg/ml vs 0.3 (0.1)pg/ml, p=0.003]. GLP-1 infusion in T2DM was associated with a significant reduction in circulating IL-6 [baseline IL-6 1.2 pg/ml vs IL-6=0.7 at 120 min, p=0.0001; vs IL-6=0.8 at 180 min, p=0.001]. There was no significant change in leptin, adiponectin, ghrelin or obestatin compared to baseline on all 4 experimental days in both groups. Short-term infusion of supraphysiological concentrations of GLP-1 in T2DM results in suppression of IL-6, a key inflammatory mediator strongly linked to development of obesity and T2DM-related insulin resistance. It remains to be confirmed whether GLP-1-based diabetes therapies can impact favourably on cardiovascular outcomes. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging.

PubMed

Zhang, Liang; Thurber, Greg M

2016-02-01

Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type 1 diabetes. The glucagon-like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice. Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified. Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower-clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively. Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, down-regulation of the GLP-1 receptor and non-specific background uptake result in a higher target-to-background ratio for fast-clearing agents.

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging

PubMed Central

Zhang, Liang; Thurber, Greg M.

2016-01-01

Purpose Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type-1 diabetes. The glucagon like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice. Procedures Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified. Results Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively. Conclusions Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, downregulation of the GLP-1 receptor and non-specific background uptake result in a higher TBR for fast-clearing agents. PMID:26194012

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins.

PubMed

Raran-Kurussi, Sreejith; Keefe, Karina; Waugh, David S

2015-06-01

Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that (1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and (2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins. Published by Elsevier Inc.

Exo-endo cellulase fusion protein

DOEpatents

Bower, Benjamin S [Palo Alto, CA; Larenas, Edmund A [Palo Alto, CA; Mitchinson, Colin [Palo Alto, CA

2012-01-17

The present invention relates to a heterologous exo-endo cellulase fusion construct, which encodes a fusion protein having cellulolytic activity comprising a catalytic domain derived from a fungal exo-cellobiohydrolase and a catalytic domain derived from an endoglucanase. The invention also relates to vectors and fungal host cells comprising the heterologous exo-endo cellulase fusion construct as well as methods for producing a cellulase fusion protein and enzymatic cellulase compositions.

S-nitrosylation of GAD65 is implicated in decreased GAD activity and oxygen-induced seizures.

PubMed

Gasier, Heath G; Demchenko, Ivan T; Tatro, Lynn G; Piantadosi, Claude A

2017-07-13

Breathing oxygen at partial pressures ≥2.5 atmospheres absolute, which can occur in diving and hyperbaric oxygen (HBO 2 ) therapy, can rapidly become toxic to the central nervous system (CNS). This neurotoxicity culminates in generalized EEG epileptiform discharges, tonic-clonic convulsions and ultimately death. Increased production of neuronal nitric oxide (NO) has been implicated in eliciting hyperoxic seizures by altering the equilibrium between glutamatergic and GABAergic synaptic transmission. Inhibition of glutamic acid decarboxylase (GAD) activity in HBO 2 promotes this imbalance; however, the mechanisms by which this occurs is unknown. Therefore, we conducted a series of experiments using mice, a species that is highly susceptible to CNS oxygen toxicity, to explore the possibility that NO modulates GABA metabolism. Mice were exposed to 100% oxygen at 4 ATA for various durations, and brain GAD and GABA transaminase (GABA-T) activity, as well as S-nitrosylation of GAD65 and GAD67 were determined. HBO 2 inhibited GAD activity by 50% and this was negatively correlated with S-nitrosylation of GAD65, whereas GABA-T activity and S-nitrosylation of GAD67 were unaltered. These results suggest a new mechanism by which NO alters GABA metabolism, leading to neuroexcitation and seizures in HBO 2 . Published by Elsevier B.V.

Structural characterization of Mumps virus fusion protein core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Yueyong; Xu Yanhui; Lou Zhiyong

2006-09-29

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus,more » forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.« less

Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

PubMed

Wu, Hsing Chieh; Chen, Yu San; Shen, Pin Chun; Shien, Jui Hung; Lee, Long Huw; Chiu, Hua Hsien

2015-01-01

The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12. © 2015 American Institute of Chemical Engineers.

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kar, Rekha; Department of Biochemistry, UT Health Science Center at San Antonio, San Antonio, TX 78229; Mishra, Nandita

2010-09-03

Research highlights: {yields} Chemical inhibition of fission protein Drp1 leads to mitochondrial fusion. {yields} Increased fusion stimulates molecular changes in mitochondrial fusion protein OPA1. {yields} Proteolysis of larger isoforms, new synthesis and ubiquitination of OPA1 occur. {yields} Loss of mitochondrial tubular rigidity and disorganization of cristae. {yields} Generation of large swollen dysfunctional mitochondria. -- Abstract: We showed earlier that 15 deoxy {Delta}{sup 12,14} prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion . However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria bymore » 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.« less

Feedback suppression of meal-induced glucagon-like peptide-1 (GLP-1) secretion mediated through elevations in intact GLP-1 caused by dipeptidyl peptidase-4 inhibition: a randomized, prospective comparison of sitagliptin and vildagliptin treatment.

PubMed

Baranov, Oleg; Kahle, Melanie; Deacon, Carolyn F; Holst, Jens J; Nauck, Michael A

2016-11-01

To compare directly the clinical effects of vildagliptin and sitagliptin in patients with type 2 diabetes, with a special emphasis on incretin hormones and L-cell feedback inhibition induced by dipeptidyl peptidase (DPP-4) inhibition. A total of 24 patients (12 on a diet/exercise regimen, 12 on metformin) were treated, in randomized order, for 7-9 days, with either vildagliptin (50 mg twice daily = 100 mg/d), sitagliptin (100 mg once daily in those on diet, 50 mg twice daily in those on metformin treatment = 100 mg/d) or placebo (twice daily). A mixed-meal test was performed. Intact glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide concentrations were doubled by both DPP-4 inhibitors. Meal-related total GLP-1 responses were reduced by vildagliptin and sitagliptin treatment alike in the majority of patients (vildagliptin: p = 0.0005; sitagliptin: p = 0.019), but with substantial inter-individual variation. L-cell feedback appeared to be more pronounced in those whose intact GLP-1 relative to total GLP-1 increased more, and who had greater reductions in fasting plasma glucose after DPP-4 inhibition. K-cell feedback inhibition overall was not significant. There were no differences in any of the clinical variables (glycaemia, insulin and glucagon secretory responses) between vildagliptin and sitagliptin treatment. Vildagliptin and sitagliptin affected incretin hormones, glucose concentrations, insulin and glucagon secretion in a similar manner. Inter-individual variations in L-cell feedback inhibition may indicate heterogeneity in the clinical response to DPP-4 inhibition. © 2016 John Wiley & Sons Ltd.

GAD1 Encodes a Secreted Peptide That Regulates Grain Number, Grain Length, and Awn Development in Rice Domestication[OPEN

PubMed Central

Hua, Lei; Zhao, Xinhui; Zhang, Weifeng; Liu, Fengxia; Fu, Yongcai; Cai, Hongwei; Sun, Xianyou; Gu, Ping; Xie, Daoxin

2016-01-01

Cultivated rice (Oryza sativa) was domesticated from wild rice (Oryza rufipogon), which typically displays fewer grains per panicle and longer grains than cultivated rice. In addition, wild rice has long awns, whereas cultivated rice has short awns or lacks them altogether. These changes represent critical events in rice domestication. Here, we identified a major gene, GRAIN NUMBER, GRAIN LENGTH AND AWN DEVELOPMENT1 (GAD1), that regulates those critical changes during rice domestication. GAD1 is located on chromosome 8 and is predicted to encode a small secretary signal peptide belonging to the EPIDERMAL PATTERNING FACTOR-LIKE family. A frame-shift insertion in gad1 destroyed the conserved cysteine residues of the peptide, resulting in a loss of function, and causing the increased number of grains per panicle, shorter grains, and awnless phenotype characteristic of cultivated rice. Our findings provide a useful paradigm for revealing functions of peptide signal molecules in plant development and helps elucidate the molecular basis of rice domestication. PMID:27634315

A comparative safety review between GLP-1 receptor agonists and SGLT2 inhibitors for diabetes treatment.

PubMed

Consoli, Agostino; Formoso, Gloria; Baldassarre, Maria Pompea Antonia; Febo, Fabrizio

2018-03-01

Glucagon-like peptide-1 receptor agonists (GLP-1RA) and sodium glucose cotransporter 2 inhibitors (SGLT2i) are of particular interest in type 2 diabetes treatment strategies, due to their efficacy in reducing HbA1c with a low risk of hypoglycaemia, to their positive effects on body weight and blood pressure and in light of their effects on cardiovascular risk and on nephroprotection emerged from the most recent cardiovascular outcome trials. Since it is therefore very likely that GLP-1RA and SGLT2i use will become more and more common, it is more and more important to gather and discuss information about their safety profile. Area Covered: adverse events and the safety concerns most often emerged in trials with GLP-1RA namely, exenatide long acting release (LAR), dulaglutide, liraglutide, semaglutide, lixisenatide or SGLT2i, namely empagliflozin, dapagliflozin, canagliflozin and SGLT2i with an attempt at comparing the safety profiles of molecules of these two classes. Expert opinion: GLP-1RA and SGLT2i, although each associated with different specific side effects, share a 'similar' safety profile and are both drugs relatively easy to handle. The potentially complementary mechanisms of action, the cardio and nephroprotective effects demonstrated by molecules of both classes, make these drugs potentially useful even in add on to each other.

Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.

2006-07-05

The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra ormore » SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.« less

Fusion proteins of flagellin and the major birch pollen allergen Bet v 1 show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity.

PubMed

Kitzmüller, Claudia; Kalser, Julia; Mutschlechner, Sonja; Hauser, Michael; Zlabinger, Gerhard J; Ferreira, Fatima; Bohle, Barbara

2018-01-01

Recombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins can enhance the efficacy of allergen-specific immunotherapy. We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines. A truncated version of flagellin (NtCFlg) was genetically fused to the N- or C-terminus of Bet v 1. Toll-like receptor (TLR) 5 binding was assessed with HEK293 cells expressing TLR5. Upregulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells from allergic patients was analyzed by using flow cytometry. The T cell-stimulatory capacity of the fusion proteins was assessed with naive and Bet v 1-specific T cells. IgE binding was tested in inhibition ELISAs and basophil activation tests. Mice were immunized with the fusion proteins in the absence and presence of aluminum hydroxide. Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in basophil activation tests. Both fusion proteins matured monocyte-derived dendritic cells through TLR5. Compared with Bet v 1, the fusion proteins showed stronger T cell-stimulatory and reduced IgE-binding capacity and induced murine Bet v 1-specific antibodies in the absence of aluminum hydroxide. However, only antibodies induced by means of immunization with NtCFlg fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1. Bet v 1-flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity and thus represent promising vaccines for birch pollen allergen-specific immunotherapy. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunologic characteristics. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Equivalent Increases in Circulating GLP-1 Following Jejunal Delivery of Intact and Hydrolysed Casein: Relevance to Satiety Induction Following Bariatric Surgery.

PubMed

le Roux, Carel W; Engström, My; Björnfot, Niclas; Fändriks, Lars; Docherty, Neil G

2016-08-01

Both Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) decrease the latency of food delivery to the proximal small intestine. This is implicated in exaggerated post-prandial release of glucagon-like peptide 1 (GLP-1), which provokes early satiety and reductions in food intake. Altered stomach anatomy also creates a deficit in enzymatic pre-processing. The impact of this state effect as a modulator of gut hormone responses remains underexplored. A double-blind cross-over trial study was conducted in 13 healthy subjects assigned to receive in the fasted state and in random order at 1 week apart, a direct jejunal infusion of either intact casein or a casein hydrolysate. Downstream effects on GLP-1 release, ratings of hunger and fullness and food and water intake on each study day were recorded when an ad libitum meal was provided 30 min after the infusion. Circulating GLP-1 was increased 25 min after infusions and peaked to a similar degree at 15 min post-meal initiation. The hormone surge had no impact on ratings of hunger and fullness ahead of the ad libitum meal. The kinetic and magnitude of satiation following each infusion was not significantly different. Food and water intake were likewise not differentially impacted by the two infusion types. Protein macronutrient state upon arrival in the small intestine does not in isolation impact upon GLP-1 responses and subsequent onset of satiety. This potentially points to rate of delivery being the dominant factor in exaggerated post-prandial GLP-1 responses in patients post-RYGB and VSG.

Circulating GLP-1 in infants born small-for-gestational-age: breast-feeding versus formula-feeding.

PubMed

Díaz, M; Bassols, J; Sebastiani, G; López-Bermejo, A; Ibáñez, L; de Zegher, F

2015-10-01

Prenatal growth restraint associates with the risk for later diabetes, particularly if such restraint is followed by postnatal formula-feeding (FOF) rather than breast-feeding (BRF). Circulating incretins can influence the neonatal programming of hypothalamic setpoints for appetite and energy expenditure, and are thus candidate mediators of the long-term effects exerted by early nutrition. We have tested this concept by measuring (at birth and at age 4 months) the circulating concentrations of glucagon-like peptide-1 (GLP-1) in BRF infants born appropriate-for-gestational-age (AGA; n=63) and in small-for-gestational-age (SGA) infants receiving either BRF (n=28) or FOF (n=26). At birth, concentrations of GLP-1 were similar in AGA and SGA infants. At 4 months, pre-feeding GLP-1 concentrations were higher than at birth; SGA-BRF infants had GLP-1 concentrations similar to those in AGA-BRF infants but SGA-FOF infants had higher concentrations. In conclusion, nutrition appears to influence the circulating GLP-1 concentrations in SGA infants and may thereby modulate long-term diabetes risk.

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, G.; Singer, A.; Lunin, V. V.

2009-02-06

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed amore » compact, globular shape with two {alpha}/{beta}-sandwich domains. The core fold of GlpX is structurally similar to that of Li{sup +}-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr{sup 90} is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.« less

Exendin-4 and GLP-1 decreases induced expression of ICAM-1, VCAM-1 and RAGE in human retinal pigment epithelial cells.

PubMed

Dorecka, Mariola; Siemianowicz, Krzysztof; Francuz, Tomasz; Garczorz, Wojciech; Chyra, Agnieszka; Klych, Agnieszka; Romaniuk, Wanda

2013-01-01

Advanced glycation end products (AGEs) take part in the development of diabetic retinopathy. Hyperglycemia triggers an inflammatory response in the retina. These mechanisms may lead to an enhanced expression of adhesion molecules (ICAM-1 and VCAM-1) in human retinal pigment epithelium (HRPE). Glucagon-like peptide 1 (GLP-1) functions as an incretin hormone with antidiabetogenic properties. GLP-1 also possesses vasoprotective properties. The aim of our study was to evaluate the influence of glycated albumin (GlyAlb; 100; 500 and 1000 mg/l) and pro-inflammatory cytokine, TNF-α (2.5 and 10 ng/ml), on expression of RAGE, ICAM-1 and VCAM-1 and to evaluate the influence of GLP-1 (100 nM) and its analogue, exendin-4 (10 nM), on the expression of RAGE, ICAM-1 and VCAM-1 in stimulated HRPE. TNF-α increased RAGE expression in HRPE cells. The addition of GlyAlb (500 and 1000 mg/l) resulted in a decrease of RAGE expression. Both TNF-α and GlyAlb increased the secretion of both adhesion molecules. In cells co-treated with GLP-1 or exendin-4 both incretins decreased RAGE expression in TNF-α treated cells, and in GlyAlb group. The ICAM-1 expression was lowered by exendin-4 and GLP-1 in cells stimulated by TNF-α and GlyAlb. The similar results were obtained for VCAM-1. All observed alterations were statistically significant. The obtained results indicate that both GLP-1 and exendin-4 by decreasing the expression of RAGE in HRPE can make these cells more resistant to circulating AGEs, and decreased expression of circulating VCAM-1 and ICAM-1, can be the result of anti-inflammatory properties of incretins and decreased expression of RAGE.

A Paleointensity-Based Test of the Geocentric Axial Dipole (GAD) Hypothesis

NASA Astrophysics Data System (ADS)

Heimpel, M. H.; Veikkolainen, T.; Evans, M. E.; Pesonen, L. J.; Korhonen, K.

2016-12-01

The GAD model is central to many aspects of geophysics, including plate tectonics and paleoclimate. However, significant departures from a GAD field over geologic time have not been ruled out, particularly for the Precambrian. Here, we investigate a test of the GAD model using published paleointensity data. Our goals are to determine if paleointensities can shed light on the validity of the GAD model, and hence to see if they provide constraints on the evolution of the geodynamo throughout earth history. Using numerical dynamo models, we show that intensity distributions can be fairly well characterized by the first three zonal Gauss coefficients (dipole, quadrupole and octupole), although time-averaging tends to broaden the range of intensities. The dynamo models indicate that the ancient core, prior to nucleation of the inner core, may have had a significant (up to 10%) contribution of the zonal octupole. We then investigate the connection between the measured paleointensities assembled in the PINT database and the GAD model by means of predicted theoretical frequency distributions for various simple models (GAD, GAD ± small zonal quadrupole or octupole components). Hitherto, paleointensities have often been analysed in terms of corresponding virtual dipole moments (VDMs). But this rather begs the question because a GAD model is assumed in order to derive a VDM. By using raw field values reported from each sampling site we eliminate dependence on the GAD hypothesis. We find that models consisting of one or two different GADs cannot explain the data, but 3- or 4-GAD models can fit the data surprisingly well, and adding a ±5% octupole significantly improves the fit.

The Aversive Agent Lithium Chloride Suppresses Phasic Dopamine Release Through Central GLP-1 Receptors.

PubMed

Fortin, Samantha M; Chartoff, Elena H; Roitman, Mitchell F

2016-02-01

Unconditioned rewarding stimuli evoke phasic increases in dopamine concentration in the nucleus accumbens (NAc) while discrete aversive stimuli elicit pauses in dopamine neuron firing and reductions in NAc dopamine concentration. The unconditioned effects of more prolonged aversive states on dopamine release dynamics are not well understood and are investigated here using the malaise-inducing agent lithium chloride (LiCl). We used fast-scan cyclic voltammetry to measure phasic increases in NAc dopamine resulting from electrical stimulation of dopamine cell bodies in the ventral tegmental area (VTA). Systemic LiCl injection reduced electrically evoked dopamine release in the NAc of both anesthetized and awake rats. As some behavioral effects of LiCl appear to be mediated through glucagon-like peptide-1 receptor (GLP-1R) activation, we hypothesized that the suppression of phasic dopamine by LiCl is GLP-1R dependent. Indeed, peripheral pretreatment with the GLP-1R antagonist exendin-9 (Ex-9) potently attenuated the LiCl-induced suppression of dopamine. Pretreatment with Ex-9 did not, however, affect the suppression of phasic dopamine release by the kappa-opioid receptor agonist, salvinorin A, supporting a selective effect of GLP-1R stimulation in LiCl-induced dopamine suppression. By delivering Ex-9 to either the lateral or fourth ventricle, we highlight a population of central GLP-1 receptors rostral to the hindbrain that are involved in the LiCl-mediated suppression of NAc dopamine release.

Novel role of GLP-1 receptor signaling in energy expenditure during chronic high fat diet feeding in rats.

PubMed

Krieger, Jean-Philippe; Langhans, Wolfgang; Lee, Shin J

2018-04-11

Glucagon-like peptide-1 (GLP-1) secreted from intestinal L-cells plays a major role in meal termination and glucose-dependent insulin secretion. Several lines of evidence indicate, however, that the acute satiating and incretin effects of GLP-1 are attenuated with high fat diet (HFD) exposure. Here we tested the hypothesis that endogenous GLP-1 differentially affects energy balance and glucose homeostasis dependent on whether rats are fed chow or HFD (60% energy from fat). We blocked GLP-1 receptor (GLP-1R) signaling by daily intraperitoneal (IP) injection of the GLP-1R antagonist exendin (9-39) (Ex9, 10 μg/kg) or vehicle for 5 weeks in male Sprague-Dawley rats fed either chow or HFD, recorded body weight (BW) and food intake throughout, and assessed energy expenditure (3rd week) and glucose tolerance (4th week). Five week daily Ex9 injections reduced BW gain in HFD-fed rats, but did not affect BW in chow-fed rats. On the other hand, chronic Ex9 treatment did not affect daily food intake in either chow or HFD-fed rats during the entire study. The reduced BW gain in HFD-fed rats was associated with an increase in energy expenditure. Interestingly, chronic Ex9 treatment induced glucose intolerance in chow-fed rats, but not in HFD-fed rats, suggesting a differential role of GLP-1R signaling in glucose metabolism during chow and HFD feeding. Our findings reveal a novel role of GLP-1R signaling, modulating energy expenditure rather than eating behavior during HFD feeding. Furthermore, these results suggest a previously unrecognized contribution of GLP-1R signaling to the pathophysiology of obesity. Copyright © 2018 Elsevier Inc. All rights reserved.

Venous blood provides lower GLP-1 concentrations than arterialised blood in the postprandial, but not fasted state: Consequences of sampling methods.

PubMed

Chen, Yung-Chih; Edinburgh, Robert M; Hengist, Aaron; Smith, Harry A; Walhin, Jean-Philippe; Betts, James A; Thompson, Dylan; Gonzalez, Javier T

2018-06-27

What is the central question of this study? Glucagon-like peptide-1 (GLP-1) is an important obesity/diabetes target, with effects dependent on circulating GLP-1 concentrations. Peripheral tissues extract GLP-1, therefore sampling venous versus arterialised blood may provide different GLP-1 concentrations. This study examined whether arterialisation alters GLP-1 concentrations during fasting and feeding. What is the main finding and its importance? This study demonstrates that venous blood provides lower postprandial, but not fasting, GLP-1 concentrations versus arterialised blood. Therefore, when accurate assessment of postprandial peripheral availability of GLP-1 is required, blood sampling methods should be carefully considered, clearly reported, and arterialisation is recommended. Glucagon-like peptide-1 (GLP-1) displays concentration-dependent effects on metabolism, appetite and angiogenesis, so accurate determination of circulating GLP-1 concentrations is important. This study compared GLP-1 concentrations in venous versus arterialised blood under both fasted and fed conditions. Venous and arterialised blood samples were simultaneously drawn from ten, young, healthy men before, and 30, 60 and 120 min after, ingestion of 75 g glucose. Plasma GLP-1 concentrations increased in response to glucose ingestion (time effect: p < 0.01) and to a lesser extend in venous versus arterialised plasma (time x arterialisation interaction: p < 0.01). Accordingly, the plasma incremental area under the curve was lower in venous versus arterialised plasma (974 ± 88 versus 1214 ± 115 pmol·L x 120 min -1 , respectively, p = 0.049). In the postprandial state, there was a positive relationship between arterialised GLP-1 concentrations and the venous-arterialised difference in GLP-1 concentrations (r 2  = 0.51; p < 0.01). Both arterialised and venous peak GLP-1 concentrations showed positive relationships with peak arterialised insulin concentrations (both r 2 �

[Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21].

PubMed

Li, Guo-Hui; Fan, Yu-Zhen; Huang, Si-Yong; Liu, Qiang; Yin, Dan-Dan; Liu, Li; Chen, Ren-An; Hao, Miao-Wang; Liang, Ying-Min

2014-06-01

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

PubMed

Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

2010-01-01

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

Fc-fusion Proteins in Therapy: An Updated View.

PubMed

Jafari, Reza; Zolbanin, Naime M; Rafatpanah, Houshang; Majidi, Jafar; Kazemi, Tohid

2017-01-01

Fc-fusion proteins are composed of Fc region of IgG antibody (Hinge-CH2-CH3) and a desired linked protein. Fc region of Fc-fusion proteins can bind to neonatal Fc receptor (FcRn) thereby rescuing it from degradation. The first therapeutic Fc-fusion protein was introduced for the treatment of AIDS. The molecular designing is the first stage in production of Fc-fusion proteins. The amino acid residues in the Fc region and linked protein are very important in the bioactivity and affinity of the fusion proteins. Although, therapeutic monoclonal antibodies are the top selling biologics but the application of therapeutic Fc-fusion proteins in clinic is in progress and among these medications Etanercept is the most effective in therapy. At present, eleven Fc-fusion proteins have been approved by FDA. There are novel Fc-fusion proteins which are in pre-clinical and clinical development. In this article, we review the molecular and biological characteristics of Fc-fusion proteins and then further discuss the features of novel therapeutic Fc-fusion proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Can GLP-1 preparations be used in children and adolescents with diabetes mellitus?

PubMed

Shehadeh, Naim; Daich, Eena; Zuckerman-Levin, Nehama

2014-03-01

The number of young diabetics is increasing and therapeutic options for these patients are limited. Glucagon-like peptide-1 (GLP-1) is secreted from the gut after meals and enhances glucose-induced insulin secretion, inhibits glucagon secretion, suppresses appetite, and delays the gastric-emptying rate. GLP-1 analogs are already widely used in the adult population to improve glycemic control and induce weight loss in overweight subjects with type 2 diabetes. The glucose-lowering effects resulting from the inhibition of glucagon secretion and the gastric-emptying rate could be of clinical importance in type 1 diabetes. In this article we review clinical data regarding the use of GLP-1 receptor agonists in youth and address the potential benefits and safety aspects of these compounds. Large scale clinical trials are still needed in the pediatric population.

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

PubMed Central

Clifton, Matthew C.; Dranow, David M.; Leed, Alison; Fulroth, Ben; Fairman, James W.; Abendroth, Jan; Atkins, Kateri A.; Wallace, Ellen; Fan, Dazhong; Xu, Guoping; Ni, Z. J.; Daniels, Doug; Van Drie, John; Wei, Guo; Burgin, Alex B.; Golub, Todd R.; Hubbard, Brian K.; Serrano-Wu, Michael H.

2015-01-01

Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors. PMID:25909780

Silencing of a Germin-Like Protein Gene (CchGLP) in Geminivirus-Resistant Pepper (Capsicum chinense Jacq.) BG-3821 Increases Susceptibility to Single and Mixed Infections by Geminiviruses PHYVV and PepGMV

PubMed Central

Mejía-Teniente, Laura; Joaquin-Ramos, Ahuizolt de Jesús; Torres-Pacheco, Irineo; Rivera-Bustamante, Rafael F.; Guevara-Olvera, Lorenzo; Rico-García, Enrique; Guevara-Gonzalez, Ramon G.

2015-01-01

Germin-like proteins (GLPs) are encoded by a family of genes found in all plants, and in terms of function, the GLPs are implicated in the response of plants to biotic and abiotic stresses. CchGLP is a gene encoding a GLP identified in a geminivirus-resistant Capsicum chinense Jacq accession named BG-3821, and it is important in geminivirus resistance when transferred to susceptible tobacco in transgenic experiments. To characterize the role of this GLP in geminivirus resistance in the original accession from which this gene was identified, this work aimed at demonstrating the possible role of CchGLP in resistance to geminiviruses in Capsicum chinense Jacq. BG-3821. Virus-induced gene silencing studies using a geminiviral vector based in PHYVV component A, displaying that silencing of CchGLP in accession BG-3821, increased susceptibility to geminivirus single and mixed infections. These results suggested that CchGLP is an important factor for geminivirus resistance in C. chinense BG-3821 accession. PMID:26610554

Silencing of a Germin-Like Protein Gene (CchGLP) in Geminivirus-Resistant Pepper (Capsicum chinense Jacq.) BG-3821 Increases Susceptibility to Single and Mixed Infections by Geminiviruses PHYVV and PepGMV.

PubMed

Mejía-Teniente, Laura; Joaquin-Ramos, Ahuizolt de Jesús; Torres-Pacheco, Irineo; Rivera-Bustamante, Rafael F; Guevara-Olvera, Lorenzo; Rico-García, Enrique; Guevara-Gonzalez, Ramon G

2015-11-25

Germin-like proteins (GLPs) are encoded by a family of genes found in all plants, and in terms of function, the GLPs are implicated in the response of plants to biotic and abiotic stresses. CchGLP is a gene encoding a GLP identified in a geminivirus-resistant Capsicum chinense Jacq accession named BG-3821, and it is important in geminivirus resistance when transferred to susceptible tobacco in transgenic experiments. To characterize the role of this GLP in geminivirus resistance in the original accession from which this gene was identified, this work aimed at demonstrating the possible role of CchGLP in resistance to geminiviruses in Capsicum chinense Jacq. BG-3821. Virus-induced gene silencing studies using a geminiviral vector based in PHYVV component A, displaying that silencing of CchGLP in accession BG-3821, increased susceptibility to geminivirus single and mixed infections. These results suggested that CchGLP is an important factor for geminivirus resistance in C. chinense BG-3821 accession.

Complications, revision fusions, readmissions, and utilization over a 1-year period after bone morphogenetic protein use during primary cervical spine fusions.

PubMed

Goode, Adam P; Richardson, William J; Schectman, Robin M; Carey, Timothy S

2014-09-01

Nationwide estimates examining bone morphogenetic protein (BMP) use with cervical spine fusions have been limited to perioperative outcomes. To determine the 1-year risk of complications, cervical revision fusions, hospital readmissions, and health care services utilization. A retrospective cohort study from 2002 to 2009 using a nationwide claims database. There were 61,937 primary cervical spine fusions of which 1,677 received BMP. Complications, revision fusions, 30-day hospital readmission, and health care utilization. Data for these analyses come from the Thomson Reuters MarketScan Commercial Claims and Encounters Database 2010. Patients were aged 18 to 64 years, receiving and not receiving BMP with a primary (C2-C7) cervical spine fusion. All outcomes were defined by International Classification of Diseases, 9th edition Clinical Modification and Current Procedural and Terminology, 4th edition codes. Complications were analyzed as any complication and stratified by nervous system, wound, and dysphagia or hoarseness. Cervical revision fusions were determined in the 1-year follow-up. Hospital readmission discharge records defined 30-day hospital readmission and reason for the readmission. The utilization of at least one health care service of cervical spine imaging, epidural usage or rehabilitation service was examined. Poisson regression models were used to estimate the relative risk and 95% confidence interval (CI). Linear regression was used to determine the time to hospital readmission. Results were stratified by anterior or posterior and circumferential approaches. Patients receiving BMP were 29% more likely to have a complication (adjusted relative risk [aRR]=1.29 [95% CI, 1.14-1.46]) and a nervous system complication (aRR=1.42 [95% CI, 1.10-1.83]). Cervical revision fusions were more likely among patients receiving BMP (aRR=1.69 [95% CI, 1.35-2.13]). The risk of 30-day readmission was greater with BMP use (aRR=1.37 [95% CI, 1.07-1.73]) and readmission

Effect of oral contraceptives and/or metformin on GLP-1 secretion and reactive hypoglycaemia in polycystic ovary syndrome.

PubMed

Glintborg, Dorte; Mumm, Hanne; Holst, Jens Juul; Andersen, Marianne

2017-05-01

Insulin resistance in polycystic ovary syndrome (PCOS) may increase the risk of reactive hypoglycaemia (RH) and decrease glucagon-like peptide-1 (GLP-1) secretion. The possible effects of treatment with oral contraceptives (OCP) and/or metformin on GLP-1 secretion and risk of RH in PCOS is undetermined. Outpatient clinic. Randomized, controlled clinical trial. Ninety women with PCOS were randomized to 12-month treatment with OCP (150 mg desogestrel + 30 mg ethinylestradiol), metformin (2 g/day) or metformin + OCP. Five-hour oral glucose tolerance tests (5-h OGTT) measuring fasting and area under the curve (AUC) for GLP-1, glucose, insulin and C-peptide were performed before and after the intervention period. Sixty-five women completed the study and 34 weight-matched healthy women were included as controls. Changes in GLP-1, glucose, insulin and C-peptide during 5-h OGTT. Fasting GLP-1 levels increased during metformin + OCP vs OCP treatment, whereas AUC GLP-1 levels were unchanged during medical treatment. The prevalence of reactive hypoglycemia increased from 9/65 to 14/65 after intervention ( P  < 0.01) and was more common after treatment with metformin + OCP (increase from 3/23 to 6/23, P  = 0.01). Reactive hypoglycaemia was associated with higher insulin and C-peptide levels during 5-h OGTT, but was unassociated with BMI and AUC GLP-1. GLP-1 levels were comparable in PCOS vs controls. AUC GLP-1 levels were significantly lower in obese vs lean patients and were inversely associated with BMI. AUC GLP-1 levels were unchanged during treatment. Increased risk of hypoglycemia during metformin + OCP could be associated with increased insulin secretion. © 2017 The authors.

Differences in acute anorectic effects of long-acting GLP-1 receptor agonists in rats

USDA-ARS?s Scientific Manuscript database

Long-acting glucagon-like peptide-1 receptor (GLP-1R) agonists have both glucose- and weight-lowering effects. The brain is poised to mediate both of these actions since GLP-1Rs are present in key areas known to control weight and glucose. Although some research has been performed on the effects of ...

Serodiagnosis of Louse-Borne Relapsing Fever with Glycerophosphodiester Phosphodiesterase (GlpQ) from Borrelia recurrentis

PubMed Central

Porcella, Stephen F.; Raffel, Sandra J.; Schrumpf, Merry E.; Schriefer, Martin E.; Dennis, David T.; Schwan, Tom G.

2000-01-01

Human louse-borne relapsing fever occurs in sporadic outbreaks in central and eastern Africa that are characterized by significant morbidity and mortality. Isolates of the causative agent, Borrelia recurrentis, were obtained from the blood of four patients during a recent epidemic of the disease in southern Sudan. The glpQ gene, encoding glycerophosphodiester phosphodiesterase, from these isolates was sequenced and compared with the glpQ sequences obtained from other relapsing-fever spirochetes. Previously we showed that GlpQ of Borrelia hermsii is an immunogenic protein with utility as a serological test antigen for discriminating tick-borne relapsing fever from Lyme disease. In the present work, we cloned and expressed the glpQ gene from B. recurrentis and used recombinant GlpQ in serological tests. Acute- and convalescent-phase serum samples obtained from 42 patients with louse-borne relapsing fever were tested with an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) that used whole cells of B. recurrentis and with immunoblotting to whole-cell lysates of the spirochete and Escherichia coli producing recombinant GlpQ. The geometric mean titers of the acute- and convalescent-phase serum samples measured by IFA were 1:83 and 1:575, respectively. The immunoblot analysis identified a high level of reactivity and seroconversion to GlpQ, and the assay was more sensitive than the whole-cell IFA and ELISA using purified, recombinant histidine-tagged GlpQ. Serum antibodies to GlpQ and other antigens persisted for 27 years in one patient. We conclude that assessment of anti-GlpQ antibodies will allow serological confirmation of louse-borne relapsing fever and determination of disease prevalence. PMID:11015364

Effects of E2HSA, a Long-Acting Glucagon Like Peptide-1 Receptor Agonist, on Glycemic Control and Beta Cell Function in Spontaneous Diabetic db/db Mice.

PubMed