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  • Abstract: Diabetes x
  • Abstract: Islets x
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Thomas H Claus, Clark Q Pan, Joanne M Buxton, Ling Yang, Jennifer C Reynolds, Nicole Barucci, Michael Burns, Astrid A Ortiz, Steve Roczniak, James N Livingston, Kevin B Clairmont and James P Whelan

Type 2 diabetes is characterized by reduced insulin secretion from the pancreas and overproduction of glucose by the liver. Glucagon-like peptide-1 (GLP-1) promotes glucose-dependent insulin secretion from the pancreas, while glucagon promotes glucose output from the liver. Taking advantage of the homology between GLP-1 and glucagon, a GLP-1/glucagon hybrid peptide, dual-acting peptide for diabetes (DAPD), was identified with combined GLP-1 receptor agonist and glucagon receptor antagonist activity. To overcome its short plasma half-life DAPD was PEGylated, resulting in dramatically prolonged activity in vivo. PEGylated DAPD (PEG-DAPD) increases insulin and decreases glucose in a glucose tolerance test, evidence of GLP-1 receptor agonism. It also reduces blood glucose following a glucagon challenge and elevates fasting glucagon levels in mice, evidence of glucagon receptor antagonism. The PEG-DAPD effects on glucose tolerance are also observed in the presence of the GLP-1 antagonist peptide, exendin(9–39). An antidiabetic effect of PEG-DAPD is observed in db/db mice. Furthermore, PEGylation of DAPD eliminates the inhibition of gastrointestinal motility observed with GLP-1 and its analogues. Thus, PEG-DAPD has the potential to be developed as a novel dual-acting peptide to treat type 2 diabetes, with prolonged in vivo activity, and without the GI side-effects.

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Yoko Fujiwara, Masami Hiroyama, Atsushi Sanbe, Junji Yamauchi, Gozoh Tsujimoto and Akito Tanoue

[Arg8]-vasopressin (AVP) and oxytocin (OT) are neurohypophysial hormones which exert various actions, including the control of blood glucose, in some peripheral tissues. To investigate the type of receptors involved in AVP- and OT-induced glucagon secretion, we investigated the effect of these peptides on glucagon secretion in islets of wild-type (V1bR+/+) and vasopressin V1b receptor knockout (V1bR/−) mice. AVP-induced glucagon secretion was significantly inhibited by the selective V1b receptor antagonist, SSR149415 (30%), and OT-induced glucagon secretion by the specific OT receptor antagonist, d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH2 9]OVT (CL-14-26) (45%), in islets of V1bR+/+mice. AVP- and OT-induced glucagon secretions were not by the antagonist of each, but co-incubation with both 10−6 M SSR149415 and 10−6 M CL-14-26 further inhibited AVP- and OT-induced glucagon secretions in islets of V1bR+/+ mice (57 and 69% of the stimulation values respectively). In addition, both AVP and OT stimulated glucagon secretion with the same efficacy in V1bR/− mice as in V1bR+/+ mice. AVP- and OT-induced glucagon secretion in V1bR/− mice was significantly inhibited by CL-14-26. These results demonstrate that V1b receptors can mediate OT-induced glucagon secretion and OT receptors can mediate AVP-induced glucagon secretion in islets from V1bR+/+mice in the presence of a heterologous antagonist, while AVP and OT can stimulate glucagon secretion through the OT receptors in V1bR/−mice, suggesting that the other receptor can compensate when one receptor is absent.

Free access

Zhongxiuzi Gao, Li Zhang, Wenting Xie, Siqi Wang, Xiaorui Bao, Yuli Guo, Houjian Zhang, Qingzhong Hu, Yi Chen, Zeen Wang, Maoqiang Xue and Guanghui Jin

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited syndrome characterized by multiple tumors in the parathyroid glands, endocrine pancreas and anterior pituitary. Recent clinical studies have revealed a strong association between MEN1 syndrome and the risk of developing diabetes mellitus; however, the underlying mechanisms remain unknown. In this study, heterozygous Men1 knockout (Men1 +/−) mice were used as MEN1 models to investigate MEN1-associated glucose metabolic phenotypes and mechanisms. Heterozygous deficiency of Men1 in 12-month-old male mice induced fasting hyperglycemia, along with increased serum insulin levels. However, male Men1 +/− mice did not show insulin resistance, as evidenced by Akt activation in hepatic tissues and an insulin tolerance test. Increased glucose levels following pyruvate challenge and expression of key gluconeogenic genes suggested increased hepatic glucose output in the male Men1 +/− mice. This effect could be partly due to higher basal serum glucagon levels, which resulted from pancreatic islet cell proliferation induced by heterozygous loss of Men1. Taken together, our results indicate that fasted male Men1 +/− mice, in the early stage of development of MEN1, display glucose metabolic disorders. These disorders are caused not by direct induction of insulin resistance, but via increased glucagon secretion and the consequent stimulation of hepatic glucose production.

Free access

L M McShane, N Irwin, D O’Flynn, Z J Franklin, C M Hewage and F P M O’Harte

Ablation of glucagon receptor signaling represents a potential treatment option for type 2 diabetes (T2DM). Additionally, activation of glucose-dependent insulinotropic polypeptide (GIP) receptor signaling also holds therapeutic promise for T2DM. Therefore, this study examined both independent and combined metabolic actions of desHis1Pro4Glu9(Lys12PAL)-glucagon (glucagon receptor antagonist) and d-Ala2GIP (GIP receptor agonist) in diet-induced obese mice. Glucagon receptor binding has been linked to alpha-helical structure and desHis1Pro4Glu9(Lys12PAL)-glucagon displayed enhanced alpha-helical content compared with native glucagon. In clonal pancreatic BRIN-BD11 beta-cells, desHis1Pro4Glu9(Lys12PAL)-glucagon was devoid of any insulinotropic or cAMP-generating actions, and did not impede d-Ala2GIP-mediated (P<0.01 to P<0.001) effects on insulin and cAMP production. Twice-daily injection of desHis1Pro4Glu9(Lys12PAL)-glucagon or d-Ala2GIP alone, and in combination, in high-fat-fed mice failed to affect body weight or energy intake. Circulating blood glucose levels were significantly (P<0.05 to P<0.01) decreased by all treatments regimens, with plasma and pancreatic insulin elevated (P<0.05 to P<0.001) in all mice receiving d-Ala2GIP. Interestingly, plasma glucagon concentrations were decreased (P<0.05) by sustained glucagon inhibition (day 28), but increased (P<0.05) by d-Ala2GIP therapy, with a combined treatment resulting in glucagon concentration similar to saline controls. All treatments improved (P<0.01) intraperitoneal and oral glucose tolerance, and peripheral insulin sensitivity. d-Ala2GIP-treated mice showed increased glucose-induced insulin secretion in response to intraperitoneal and oral glucose. Metabolic rate and ambulatory locomotor activity were increased (P<0.05 to P<0.001) in all desHis1Pro4Glu9(Lys12PAL)-glucagon-treated mice. These studies highlight the potential of glucagon receptor inhibition alone, and in combination with GIP receptor activation, for T2DM treatment.

Free access

Birgitte N Friedrichsen, Nicole Neubauer, Ying C Lee, Vivian K Gram, Niels Blume, Jacob S Petersen, Jens H Nielsen and Annette Møldrup

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), have been suggested to act as β-cell growth factors and may therefore be of critical importance for the maintenance of a proper β-cell mass. We have investigated the molecular mechanism of incretin-induced β-cell replication in primary monolayer cultures of newborn rat islet cells. GLP-1, GIP and the long-acting GLP-1 derivative, lira-glutide, increased β-cell replication 50–80% at 10–100 nM upon a 24 h stimulus, whereas glucagon at a similar concentration had no significant effect. The stimulatory effect of GLP-1 and GIP was efficiently mimicked by the adenylate cyclase activator, forskolin, at 10 nM (~90% increase) and was additive (~170–250% increase) with the growth response to human growth hormone (hGH), indicating the use of distinct intracellular signalling pathways leading to mitosis by incretins and cytokines, respectively. The response to both GLP-1 and GIP was completely blocked by the protein kinase A (PKA) inhibitor, H89. In addition, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin and the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, both inhibited GLP-1- and GIP-stimulated proliferation. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, had no inhibitory effect on either GLP-1 or GIP stimulated proliferation. Cyclin Ds act as molecular switches for the G0/G1-S phase transition in many cell types and we have previously demonstrated hGH-induced cyclin D2 expression in the insulinoma cell line, INS-1. GLP-1 time-dependently induced the cyclin D1 mRNA and protein levels in INS-1E, whereas the cyclin D2 levels were unaffected. However, minor effect of GLP-1 stimulation was observed on the cyclin D3 mRNA levels. Transient transfection of a cyclin D1 promoter-luciferase reporter construct into islet monolayer cells or INS-1 cells revealed approximately a 2–3 fold increase of transcriptional activity in response to GLP-1 and GIP, and a 4–7 fold increase in response to forskolin. However, treatment of either cell type with hGH had no effect on cyclin D1 promoter activity. The stimulation of the cyclin D1 promoter by GLP-1 was inhibited by H89, wortmannin, and PD98059. We conclude that incretin-induced β-cell replication is dependent on cAMP/PKA, p42 MAPK and PI3K activities, which may involve transcriptional induction of cyclin D1. GLP-1, GIP and liraglutide may have the potential to increase β-cell replication in humans which would have significant impact on long-term diabetes treatment.

Free access

N M Whalley, L E Pritchard, D M Smith and A White

Proglucagon is cleaved to glucagon by prohormone convertase 2 (PC2) in pancreatic α-cells, but is cleaved to glucagon-like peptide-1 (GLP-1) by PC1 in intestinal L-cells. The aim of this study was to identify mechanisms which switch processing of proglucagon to generate GLP-1 in the pancreas, given that GLP-1 can increase insulin secretion and β-cell mass. The α-cell line, αTC1-6, expressed PC1 at low levels and GLP-1 was detected in cells and in culture media. GLP-1 was also found in isolated human islets and in rat islets cultured for 7 days. High glucose concentrations increased Pc1 gene expression and PC1 protein in rat islets. High glucose (25 mM) also increased GLP-1 but decreased glucagon secretion from αTC1-6 cells suggesting a switch in processing to favour GLP-1. Three G protein-coupled receptors, GPR120, TGR5 and GPR119, implicated in the release of GLP-1 from L-cells are expressed in αTC1-6 cells. Incubation of these cells with an agonist of TGR5 increased PC1 promoter activity and GLP-1 secretion suggesting that this is a mechanism for switching processing to GLP-1 in the pancreas. Treatment of isolated rat islets with streptozotocin caused β-cell toxicity as evidenced by decreased glucose-stimulated insulin secretion. This increased GLP-1 but not glucagon in the islets. In summary, proglucagon can be processed to GLP-1 in pancreatic cells. This process is upregulated by elevated glucose, activation of TGR5 and β-cell destruction. Understanding this phenomenon may lead to advances in therapies to protect β-cell mass, and thereby slow progression from insulin resistance to type 2 diabetes.

Free access

Linda Ahlkvist, Bilal Omar, Anders Valeur, Keld Fosgerau and Bo Ahrén

Stimulation of insulin secretion by short-term glucagon receptor (GCGR) activation is well characterized; however, the effect of long-term GCGR activation on β-cell function is not known, but of interest, since hyperglucagonemia occurs early during development of type 2 diabetes. Therefore, we examined whether chronic GCGR activation affects insulin secretion in glucose intolerant mice. To induce chronic GCGR activation, high-fat diet fed mice were continuously (2 weeks) infused with the stable glucagon analog ZP-GA-1 and challenged with oral glucose and intravenous glucose±glucagon-like peptide 1 (GLP1). Islets were isolated to evaluate the insulin secretory response to glucose±GLP1 and their pancreas were collected for immunohistochemical analysis. Two weeks of ZP-GA-1 infusion reduced insulin secretion both after oral and intravenous glucose challenges in vivo and in isolated islets. These inhibitory effects were corrected for by GLP1. Also, we observed increased β-cell area and islet size. We conclude that induction of chronic ZP-GA-1 levels in glucose intolerant mice markedly reduces insulin secretion, and thus, we suggest that chronic activation of the GCGR may contribute to the failure of β-cell function during development of type 2 diabetes.

Free access

Weiwei Xu, Jamie Morford and Franck Mauvais-Jarvis

One of the most sexually dimorphic aspects of metabolic regulation is the bidirectional modulation of glucose homeostasis by testosterone in male and females. Severe testosterone deficiency predisposes men to type 2 diabetes (T2D), while in contrast, androgen excess predisposes women to hyperglycemia. The role of androgen deficiency and excess in promoting visceral obesity and insulin resistance in men and women respectively is well established. However, although it is established that hyperglycemia requires β cell dysfunction to develop, the role of testosterone in β cell function is less understood. This review discusses recent evidence that the androgen receptor (AR) is present in male and female β cells. In males, testosterone action on AR in β cells enhances glucose-stimulated insulin secretion by potentiating the insulinotropic action of glucagon-like peptide-1. In females, excess testosterone action via AR in β cells promotes insulin hypersecretion leading to oxidative injury, which in turn predisposes to T2D.

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BD Green, MH Mooney, VA Gault, N Irwin, CJ Bailey, P Harriott, B Greer, FP O'Harte and PR Flatt

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His(7)-modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC(50) values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC(50) 0.37 nM). Similarly, both analogues stimulated cAMP production with EC(50) values of 16.3 and 27 nM respectively compared with GLP-1 (EC(50) 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P<0.05 to P<0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.

Free access

EG Siegel, A Seidenstucker, B Gallwitz, F Schmitz, A Reinecke-Luthge, G Kloppel, UR Folsch and WE Schmidt

Liver cirrhosis is often accompanied by a disturbed carbohydrate metabolism similar to type 2 diabetes. To investigate the severity of the defect in insulin secretion in this form of diabetes, we measured insulin release from isolated pancreatic islets of rats with CCl(4)-phenobarbital-induced liver cirrhosis. Cirrhosis was confirmed by clinical signs, elevated liver enzymes and histology. Fasting venous plasma glucose concentrations were equal in rats with liver cirrhosis and in controls. Plasma insulin and glucagon concentrations were significantly greater (P<0.01) in cirrhotic rats than in control animals. Glucose (16.7 mM)-induced stimulation of insulin release from pancreatic islets revealed a twofold increase in control and cirrhotic rats. Basal and stimulated insulin secretion, however, were significantly lower in cirrhotic animals. The incretin hormone, glucagon-like peptide-1 (GLP-1), has therapeutic potential for the treatment of type 2 diabetes. Therefore, islets from control and cirrhotic animals were incubated with GLP-1 in concentrations from 10(-)(11) to 10(-)(6) M. GLP-1 stimulated insulin release in a concentration-dependent manner. In islets from cirrhotic rats, basal and stimulated insulin secretion was blunted compared with controls. These data show that the hyperinsulinemia observed in liver cirrhosis is not due to an increase of insulin secretion from islets, but could be explained by decreased hepatic clearance of insulin. GLP-1 may ameliorate diabetes in patients with liver cirrhosis.