Molecular regulation of hormone secretion, growth and apoptosis of GLP-1-producing cells

Type 2 diabetes (T2D) spreads like an epidemic in today’s society, and there is a great need for new and improved treatments. T2D is characterized by hyperglycemia, resulting from impaired insulin production and insulin resistance in peripheral tissues. Incretin hormones, such as glucagon-like peptide-1 (GLP-1), secreted from L-cells dispersed along the intestinal tract, potentiate meal-stimulated insulin secretion in a glucose-dependent manner. Defective GLP-1 secretion has been indicated in T2D and administration of GLP-1 to T2D patients restores glucose-induced insulin secretion and normalizes glycemia, making stable analogs of GLP-1 among the best available treatments for T2D today. However, enhancing endogenous GLP-1 production/secretion by direct stimulation of GLP-1 secretion/promotion of growth and viability of L-cells may be a novel and more physiological option in incretin-based diabetes therapy. The aim of this work was to determine the effect of diabetic conditions and anti-diabetic agents on GLP-1-producing cells, in order to unravel some of the mechanisms regulating growth, survival and function of this cell type. Studies I-III were performed in vitro using the murine GLUTag cell line as a model. In study I, direct effects of metformin on apoptosis, and function of GLP-1-secreting cells were determined. Simulated diabetic hyperlipidemia resulted in increased caspase-3 activity and DNA fragmentation, indicating lipoapoptosis. Metformin treatment significantly decreased this lipoapoptosis in conjunction with increased phosphorylation of AMPK. In addition, metformin treatment stimulated GLP-1 secretion. In study II, we determined molecular mechanisms mediating lipotoxicity and metformininduced lipoprotection in GLP-1-secreting cells. Diabetic hyperlipidemia was simulated in this cell system by addition of the fatty acid palmitate. Palmitate increased ROS production in GLP- 1-secreting cells, and the lipotoxic effects of palmitate were abolished in the presence of the antioxidant Trolox. Further, palmitate phosphorylated p38 MAPK and inhibition of this enzyme significantly reduced lipoapoptosis. Pre-incubation with metformin further increased palmitate- induced ROS production, while significantly reducing the expression of p38 MAPK. Study III focused on direct effects of insulin and exendin-4/GLP-1 on lipoapoptosis and function of GLP-1-secreting cells. The GLP-1R was found to be expressed in the GLUTag cells, and diabetic lipotoxicity was partially inhibited by pre-incubation with insulin or the stable GLP-1 analog exendin-4. The lipoprotective effect of exendin-4 was GLP-1R-dependent, while independent of PKA activity. In addition, both insulin and exendin-4 significantly stimulated acute and long term GLP-1 secretion in the presence of glucose. In study IV, we investigated if a high fat diet (HFD) reduces the number of enteroendocrine GLP-1-secreting Lcells in C57/Bl6 mice. We also determined the effects of a HFD on GLP-1 plasma levels and possible effects on these parameters by metformin treatment. A HFD rapidly induced a diabetic phenotype with increased HbA1c levels, as well as fasting plasma insulin levels in conjunction with reduced oral glucose tolerance – indicating the manifestation of insulin resistance. A 14 day oral administration of metformin reduced HbA1c, fasting insulin and prandial FFA levels. The number of L-cells was significantly reduced after 12 weeks on a HFD, while -- in contrast - - there was a clear trend toward increased prandial plasma GLP-1 levels despite reduced food intake in HFD-fed mice. These findings may be of pathogenic significance not only in understanding mechanisms of the impaired incretin response characterizing T2D patients, but may also be harnessed to therapeutic advantage in efforts to enhance endogenous GLP-1 production. Such an approach has hitherto received little attention but may be superior to contemporary incretin-based antidiabetic therapy, which does not faithfully mimick physiologic GLP-1 release in for instance terms of secretory pattern (e.g. pulsatility) and actions on topographically adjacent hormone receptors (e.g. in the portal vein)

Glucagon-like peptide-1 receptor activation reduces ischaemic brain damage following stroke in Type 2 diabetic rats

Diabetes is a strong risk factor for premature and severe stroke. The GLP-1R (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic Goto–Kakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 μg/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 2–4 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-1R agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions

GLP-1 secretion by microglial cells and decreased CNS expression in obesity

Abstract Background Type 2 diabetes (T2D) is a strong risk factor for developing neurodegenerative pathologies. T2D patients have a deficiency in the intestinal incretin hormone GLP-1, which has been shown to exert neuroprotective and anti-inflammatory properties in the brain. Methods Here we investigate potential sources of GLP-1 in the CNS and the effect of diabetic conditions on the proglucagon mRNA expression in the CNS. The obese mouse model ob/ob, characterized by its high levels of free fatty acids, and the microglia cell line BV-2 were used as models. mRNA expression and protein secretion were analyzed by qPCR, immunofluorescence and ELISA. Results We show evidence for microglia as a central source of GLP-1 secretion. Furthermore, we observed that expression and secretion are stimulated by cAMP and dependent on microglial activation state. We also show that insulin-resistant conditions reduce the central mRNA expression of proglucagon. Conclusion The findings that microglial mRNA expression of proglucagon and GLP-1 protein expression are affected by high levels of free fatty acids and that both mRNA expression levels of proglucagon and secretion levels of GLP-1 are affected by inflammatory stimuli could be of pathogenic importance for the premature neurodegeneration and cognitive decline commonly seen in T2D patients, and they may also be harnessed to advantage in therapeutic efforts to prevent or treat such disorders.</p

Role of the AMP kinase in cytokine-induced human EndoC-beta H1 cell death

The aim of the present investigation was to delineate cytokine-induced signaling and death using the EndoC-beta H1 cells as a model for primary human beta-cells. The cytokines IL-1 beta and IFN-gamma induced a rapid and transient activation of NF-kappa B, STAT-1, ERK, JNK and eIF-2 alpha signaling. The EndoC-beta H1 cells died rapidly when exposed to IL-1 beta + IFN-gamma, and this occurred also in the presence of the actinomycin D. Inhibition of NF-kappa B and STAT-1 did not protect against cell death, nor did the cytokines activate iNOS expression. Instead, cytokines promoted a rapid decrease in EndoC-beta H1 cell respiration and ATP levels, and we observed protection by the AMPK activator AICAR against cytokine-induced cell death. It is concluded that EndoC-beta H1 cell death can be prevented by AMPK activation, which suggests a role for ATP depletion in cytokine-induced human beta-cell death