Glucagon-like peptide 1 agonists and the development and growth of pancreatic beta-cells

Glucagon-like peptide 1 (GLP-1) is an intestine-derived insulinotropic hormone that stimulates glucose-dependent insulin production and secretion from pancreatic beta-cells. Other recognized actions of GLP-1 are to suppress glucagon secretion and hepatic glucose output, delay gastric emptying, reduce food intake, and promote glucose disposal in peripheral tissues. All of these actions are potentially beneficial for the treatment of type 2 diabetes mellitus. Several GLP-1 agonists are in clinical trials for the treatment of diabetes. More recently, GLP-1 agonists have been shown to stimulate the growth and differentiation of pancreatic beta-cells, as well as to exert cytoprotective, antiapoptotic effects on beta-cells. Recent evidence indicates that GLP-1 agonists act on receptors on pancreas-derived stem/progenitor cells to prompt their differentiation into beta-cells. These new findings suggest an approach to create beta-cells in vitro by expanding stem/progenitor cells and then to convert them into beta-cells by treatment with GLP-1. Thus GLP-1 may be a means by which to create beta-cells ex vivo for transplantation into patients with insulinopenic type 1 diabetes and severe forms of type 2 diabetes.     

Glucagon like Peptide-1-induced glucose metabolism in differentiated human muscle satellite cells is attenuated by hyperglycemia

Background

Glucagon like peptide-1 (GLP-1) stimulates insulin secretion from the pancreas but also has extra-pancreatic effects. GLP-1 may stimulate glucose uptake in cultured muscle cells but the mechanism is not clearly defined. Furthermore, while the pancreatic effects of GLP-1 are glucose-dependent, the glucose-dependency of its extra-pancreatic effects has not been examined.

Methods

Skeletal muscle satellite cells isolated from young (22.5±0.97 yr), lean (BMI 22.5±0.6 kg/m²), healthy males were differentiated in media containing either 22.5 mM (high) or 5 mM (normal) glucose for 7 days in the absence or presence of insulin and/or various GLP-1 concentrations. Myocellular effects of GLP-1, insulin and glucose were assessed by western-blot, glucose uptake and glycogen synthesis.

Results

We firstly show that the GLP-1 receptor protein is expressed in differentiated human muscle satellite cells (myocytes). Secondly, we show that in 5 mM glucose media, exposure of myocytes to GLP-1 results in a dose dependent increase in glucose uptake, GLUT4 amount and subsequently glycogen synthesis in a PI3K dependent manner, independent of the insulin signaling cascade. Importantly, we provide evidence that differentiation of human satellite cells in hyperglycemic (22.5 mM glucose) conditions increases GLUT1 expression, and renders the cells insulin resistant and interestingly GLP-1 resistant in terms of glucose uptake and glycogen synthesis. Hyperglycemic conditions did not affect the ability of insulin to phosphorylate downstream targets, PKB or GSK3. Interestingly we show that at 5 mM glucose, GLP-1 increases GLUT4 protein levels and that this effect is abolished by hyperglycemia.

Conclusions

GLP-1 increases glucose uptake and glycogen synthesis into fully-differentiated human satellite cells in a PI3-K dependent mechanism potentially through increased GLUT4 protein levels. The latter occurs independently of the insulin signaling pathway. Attenuation of both GLP-1 and insulin-induced glucose metabolism by hyperglycemia is likely to occur downstream of PI3K.     

The physiological role of GLP-1 in human: incretin, ileal brake or more?

The proglucagon-derived peptide glucagon-like peptide-1 (GLP-1) is an intestinal signal peptide postprandially released from the L cells of the lower gut. Exogenously administered the synthetic hormone exerts a glucose-dependent insulinotropic effect at the pancreatic beta-cells and lowers plasma glucagon by an inhibitory effect against the alpha-cells. It delays gastric emptying by relaxation of the gastric fundus, inhibition of antral contractility, and stimulation of both the tonic and phasic motility of the pyloric sphincter. Enhancement of insulin, suppression of glucagon, and inhibition of gastric emptying are the main determinants controlling glucose homeostasis with GLP-1. Human studies employing the specific GLP-1 receptor antagonist exendin(9-39) show that endogenously released GLP-1 likewise controls fasting plasma glucagon, stimulates insulin, and influences all the motoric mechanisms known to control gastric emptying. Therefore, GLP-1 is discussed as an incretin hormone and as an enterogastrone in man. Synthetic GLP-1 also suppresses gastric acid and pancreatic enzyme secretion. The inhibitory effects on upper gastrointestinal functions are at least partly mediated by vagal-cholinergic inhibition and may involve interactions with vagal afferent pathways and/or circumventricular regions within the CNS. GLP-1 is a candidate humoral mediator of the 'ileal brake' exerting inhibition of upper gastrointestinal function preventing malabsorption and postprandial metabolic disturbances. As human studies indicate a central action of GLP-1 in reduction of food intake, it is uncertain if this is a consequence of induction of satiety or of transduction of visceral aversive stress signals.     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Glucagon-like peptide 1 stimulates post-translational activation of glucokinase in pancreatic beta cells

Glucagon-like peptide 1 (GLP-1) potentiates glucose-stimulated insulin secretion from pancreatic β cells, yet does not directly stimulate secretion. The mechanisms underlying this phenomenon are incompletely understood. Here, we report that GLP-1 augments glucose-dependent rises in NAD(P)H autofluorescence in both βTC3 insulinoma cells and islets in a manner consistent with post-translational activation of glucokinase (GCK). GLP-1 treatment increased GCK activity and enhanced GCK S-nitrosylation in βTC3 cells. A 2-fold increase in S-nitrosylated GCK was also observed in mouse islets. Furthermore, GLP-1 activated a FRET-based GCK reporter in living cells. Activation of this reporter was sensitive to inhibition of nitric-oxide synthase (NOS), and incorporating the S-nitrosylation-blocking V367M mutation into this sensor prevented activation by GLP-1. GLP-1 potentiation of the glucose-dependent increase in islet NAD(P)H autofluorescence was also sensitive to a NOS inhibitor, whereas NOS inhibition did not affect the response to glucose alone. Expression of the GCK(V367M) mutant also blocked GLP-1 potentiation of the NAD(P)H response to glucose in βTC3 cells, but did not significantly affect metabolism of glucose in the absence of GLP-1. Co-expression of WT or mutant GCK proteins with a sensor for insulin secretory granule fusion also revealed that blockade of post-translational GCK S-nitrosylation diminished the effects of GLP-1 on granule exocytosis by ～40% in βTC3 cells. These results suggest that post-translational activation of GCK is an important mechanism for mediating the insulinotropic effects of GLP-1.     

Engineered beta-cells secreting dipeptidyl peptidase IV-resistant glucagon-like peptide-1 show enhanced glucose-responsiveness

Type 2 diabetes is a polygenic disorder characterized by increased insulin resistance, and impaired insulin secretion leading to abnormalities of glucose and lipid metabolism. Reduced responsiveness of the beta-cells to glucose is a critical feature of this syndrome. Glucagon-like peptide 1, a product of the pro-glucagon gene makes beta-cells competent and has many other anti-diabetic properties. We speculated whether GLP-1-based gene therapy could be an approach for treatment of type 2 diabetes. We started with a clone of rat insulinoma cells (S4 cells), which showed reduced responsiveness to glucose in terms of insulin secretion. We transfected these cells with a plasmid encoding a mutated form of GLP-1 (GLP-1-Gly8), which is resistant to the degrading enzyme dipeptidyl-peptidase IV. Activity of secreted GLP-1-Gly8 was assayed using Chinese hamster lung fibroblasts (CHL) cells that expressed cloned GLP-1 receptor and that were transfected with CRE-Luc. Stable cell lines (Glipsulin cells) obtained by this means produced and stored immunoreactive GLP-1-Gly8. In addition to insulin, the Glipsulin cells secreted the GLP-1-Gly8. The secreted GLP-1-Gly8 was active as evidenced by the ability of the conditioned media to elevate cAMP levels in CHL cells expressing GLP-1 receptors. Glipsulin cells responded to glucose with a 6.8 fold increase in insulin secretion compared to a 2.2 fold increase in the control cells. Our results demonstrate that prolonged exposure to GLP-1-Gly8 secreted by increases glucose-responsiveness of these cells. We speculate that engineering GLP-1-Gly8 secretion by beta-cells is a potential gene therapeutic strategy to treat diabetes.     

Insulin contributes to fine-tuning of the pancreatic beta-cell response to glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion from pancreatic β-cells in a glucose-dependent manner. However, factors other than glucose that regulate the β-cell response to GLP-1 remain poorly understood. In this study, we examined the possible involvement of insulin and receptor tyrosine kinase signaling in regulation of the GLP-1 responsiveness of β-cells. Pretreatment of β-cells with HNMPA, an insulin receptor inhibitor, and AG1478, an epidermal growth factor receptor inhibitor, further increased the cAMP level and Erk phosphorylation in the presence of exendin-4 (exe-4), a GLP-1 agonist. When β-cells were exposed to a high concentration of glucose (25 mM), which stimulates insulin secretion, exe-4-induced cAMP formation declined gradually as exposure time was increased. This decreased cAMP formation was not observed in the presence of HNMPA. HNMPA was able to further increase the exe-4-induced insulin secretion when β-cells were exposed to high glucose for 18 h. Treatment of β-cells with insulin significantly decreased exe-4-induced cAMP formation in a dose-dependent manner. Lowering the phospho-Akt level by HNMPA or LY294002, a PI3K inhibitor, further augmented exe-4-induced cAMP formation and Erk phosphorylation. These results suggest that insulin contributes to fine-tuning of the β-cell response to GLP-1.