Monomeric G-protein, Rhes, is not an imidazoline-regulated protein in pancreatic beta-cells

The monomeric G-protein, Rhes, is a candidate imidazoline-regulated molecule involved in mediating the insulin secretory response to efaroxan [S.L. Chan, L.K. Monks, H. Gao, P. Deaville, N.G. Morgan, Identification of the monomeric G-protein, Rhes, as an efaroxan-regulated protein in the pancreatic beta-cell, Br. J. Pharmacol. 136 (1) (2002) 31-36]. This suggestion was based on observations regarding changes in Rhes mRNA expression in rat islets and pancreatic beta-cells after prolonged culture with efaroxan, leading to desensitization of the insulin response to the compound. To verify this report, we have evaluated the effects of the imidazoline compounds efaroxan and BL11282 on Rhes mRNA expression in isolated rat pancreatic islets maintained in conditions identical to those used by Chan et al. The results demonstrate that desensitization of the insulin response to efaroxan, or to another imidazoline, BL11282, does not change Rhes mRNA expression levels. Transfection of MIN6 cells with plasmids containing Rhes or Rhes-antisense also does not alter efaroxan- or BL11282-induced insulin secretion. Together, these data do not support the hypothesis that Rhes is an imidazoline-regulated protein.     

Pancreatic insulin content regulation by the estrogen receptor ER alpha

The function of pancreatic beta-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ER alpha and ER beta, are important molecules involved in glucose metabolism, yet their role in pancreatic beta-cell physiology is still greatly unknown. In this report we show that both ER alpha and ER beta are present in pancreatic beta-cells. Long term exposure to physiological concentrations of 17beta-estradiol (E2) increased beta-cell insulin content, insulin gene expression and insulin release, yet pancreatic beta-cell mass was unaltered. The up-regulation of pancreatic beta-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ER alpha and ER beta agonists as well as ER alphaKO and ER betaKO mice suggests that the estrogen receptor involved is ER alpha. The up-regulation of pancreatic insulin content by ER alpha activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.     

Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells)

Non-insulin-dependent diabetes mellitus is associated with, in addition to impaired insulin release, elevated levels of free fatty acids (FFA) in the blood. Insulin release is stimulated when beta-cells are acutely exposed to FFA, whereas chronic exposure may inhibit glucose-induced insulin secretion. In the present study we investigated the direct effects of long chain acyl-CoA (LC-CoA), the active intracellular form of FFA, on insulin exocytosis. Palmitoyl-CoA stimulated both insulin release from streptolysin-O-permeabilized HIT cells and fusion of secretory granules to the plasma membrane of mouse pancreatic beta-cells, as measured by cell capacitance. The LC-CoA effect was chain length-dependent, requiring chain lengths of at least 14 carbons. LC-CoA needed to be present to stimulate insulin release, and consequently there was no effect following its removal. The stimulatory effect was observed after inhibition of protein kinase activity and in the absence of ATP, even though both kinases and ATP, themselves, modulate exocytosis. The effect of LC-CoA was inhibited by cerulenin, which has been shown to block protein acylation. The data suggest that altered LC-CoA levels, resulting from FFA or glucose metabolism, may act directly on the exocytotic machinery to stimulate insulin release by a mechanism involving LC-CoA protein binding.     

GPR40 is expressed in glucagon producing cells and affects glucagon secretion

The free fatty acid receptor, GPR40, has been coupled with insulin secretion via its expression in pancreatic beta-cells. However, the role of GPR40 in the release of glucagon has not been studied and previous attempts to identify the receptor in alpha-cells have been unfruitful. Using double-staining for glucagon and GPR40 expression, we demonstrate that the two are expressed in the same cells in the periphery of mouse islets. In-R1-G9 hamster glucagonoma cells respond dose-dependently to linoleic acid stimulation by elevated phosphatidyl inositol hydrolysis and glucagon release and the cells become increasingly responsive to fatty acid stimulation when overexpressing GPR40. Isolated mouse islets also secrete glucagon in response to linoleic acid, a response that was abolished by antisense treatment against GPR40. This study demonstrates that GPR40 is present and active in pancreatic alpha-cells and puts further emphasis on the importance of this nutrient sensing receptor in islet function.     

IFN-gamma and tumor necrosis factor-alpha. Cytotoxicity to murine islets of Langerhans

We have previously reported that the cytokines IFN-gamma and TNF-alpha each upregulate the expression of class I MHC proteins and, in combination, induce the expression of class II MHC proteins on pancreatic islet cells. IFN-gamma and TNF-alpha are therefore implicated in the immunologic destruction of beta-cells in insulin-dependent diabetes mellitus. The objective of the present study was to define the effects of IFN-gamma and TNF-alpha on the function and viability of murine pancreatic islet beta-cells in vitro. Exposure of islets for 3 days to 200 U/ml of either IFN-gamma or TNF-alpha did not affect glucose-stimulated insulin release, but at higher concentrations (2000 U/ml) of either cytokine there was significant inhibition of glucose-stimulated insulin release. In combination, IFN-gamma and TNF-alpha each at 200 U/ml caused significant inhibition of glucose-stimulated insulin release; at 2000 U/ml glucose-stimulated insulin release was abolished. In time-course experiments, glucose-stimulated insulin release from islets exposed to IFN-gamma and TNF-alpha each at 1000 U/ml was significantly increased at 4-h (twofold increase compared with control islets), decreased back to control levels at 18 h, significantly inhibited by 24 h (threefold decrease compared with control islets), and completely abolished by 48 h. The progressive impairment of beta-cell function mediated by IFN-gamma plus TNF-alpha was associated with morphologic derangement of the islets that were almost totally disintegrated by day 6 of exposure to the cytokines. At day 6, insulin content of the islets was significantly reduced by exposure to TNF-alpha but not IFN-gamma. The combination of IFN-gamma and TNF-alpha resulted in a further dose-dependent depletion in insulin content compared with TNF-alpha alone. The synergistic functional and cytotoxic effects of IFN-gamma and TNF-alpha are consistent with a direct role for these cytokines in the destruction of beta-cells in insulin-dependent diabetes.     

3H-serotonin as a marker of oscillatory insulin secretion in clonal beta-cells (INS-1)

Serotonin release from preloaded pancreatic beta-cells has been used as a marker for insulin release in studying exocytosis from single cells using the amperometric technique. We found that single pancreatic beta-cells exhibited oscillations in exocytosis with a period of 1-1.5 min as measured amperometrically by serotonin release. We also show that 3H-serotonin can be used to monitor exocytosis from intact and streptolysin-O permeabilized clonal insulin-secreting cells preloaded with labeled serotonin and that serotonin release correlated with insulin secretion in the same cells. The use of 3H-serotonin provides a real-time indicator of exocytosis from populations of clonal insulin-secreting cells.     

Resistin induces insulin resistance in pancreatic islets to impair glucose-induced insulin release

An adipokine resistin, a small cysteine-rich protein, is one of the major risk factors of insulin resistance. In the present study, transiently resistin-expressing mice using adenovirus method showed an impaired glucose tolerance due to insulin resistance. We found that resistin-expressing mice exhibited impaired insulin secretory response to glucose. In addition, in vitro treatment with resistin for 1 day induced insulin resistance in pancreatic islets and impaired glucose-stimulated insulin secretion by elevating insulin release at basal glucose (2.8 mM) and suppressing insulin release at stimulatory glucose (8.3 mM). In addition, resistin inhibited insulin-induced phosphorylation of Akt in islets as well as other insulin target organs. Furthermore, resistin induced SOCS-3 expression in beta-cells. In conclusion, resistin induces insulin resistance in islet beta-cells at least partly via induction of SOCS-3 expression and reduction of Akt phosphorylation and impairs glucose-induced insulin secretion.     

Somatostatin inhibits insulin release via SSTR2 in hamster clonal beta-cells and pancreatic islets

Somatostatin (SST) inhibits pancreatic endocrine secretion. It is generally accepted that SSTR2 and SSTR5 mediate the inhibition of glucagon and insulin release, respectively. The present study was performed to test the hypothesis that SSTR2, but not SSTR5, mediates SST-induced inhibition of insulin release in hamster beta-cells. Both hamster clonal beta-cells HIT-T15 and pancreatic islets were used to test this hypothesis. Both SST and a nonpeptide SSTR2 agonist L-779,976 (1-100 nM) inhibited insulin release from HIT-T15 and islets in a concentration-dependent manner. In contrast, nonpeptide agonists for SSTR1, 3, 4 and 5 at the highest concentration studied (1 microM) failed to inhibit insulin release. PRL-2903, a peptide SSTR2 antagonist (0.1-1 muicroM), antagonized SST-induced inhibition of insulin release in a concentration-dependent manner. Taken together, we conclude that, in hamster beta-cells, SST inhibits insulin release via SSTR2 but not SSTR5.     

Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells

Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.     

Autoradiographic studies of the gamma-aminobutyric acid (GABA) system in the rat pancreas

The beta-cells of the pancreatic islets have been shown to contain gamma-aminobutyric acid (GABA) together with insulin. Autoradiographic analysis indicated that high affinity GABA binding sites (GABA receptors) are not present in the pancreas. High affinity GABA uptake sites are present, not in beta-cells, but in a few cells on the periphery of the islets. These observations cast doubt on the suggestion that GABA has a paracrine role in the pancreas.     

Regulation of the expression of components of the exocytotic machinery of insulin-secreting cells by microRNAs

Fine-tuning of insulin secretion from pancreatic beta-cells participates in blood glucose homeostasis. Defects in this process can lead to chronic hyperglycemia and diabetes mellitus. Several proteins controlling insulin exocytosis have been identified, but the mechanisms regulating their expression remain poorly understood. Here, we show that two non-coding microRNAs, miR124a and miR96, modulate the expression of proteins involved in insulin exocytosis and affect secretion of the beta-cell line MIN6B1. miR124a increases the levels of SNAP25, Rab3A and synapsin-1A and decreases those of Rab27A and Noc2. Inhibition of Rab27A expression is mediated by direct binding to the 3'-untranslated region of Rab27A mRNA. The effect on the other genes is indirect and linked to changes in mRNA levels. Over-expression of miR124a leads to exaggerated hormone release under basal conditions and a reduction in glucose-induced secretion. miR96 increases mRNA and protein levels of granuphilin, a negative modulator of insulin exocytosis, and decreases the expression of Noc2, resulting in lower capacity of MIN6B1 cells to respond to secretagogues. Our data identify miR124a and miR96 as novel regulators of the expression of proteins playing a critical role in insulin exocytosis and in the release of other hormones and neurotransmitters.     

Growth hormone is a growth factor for the differentiated pancreatic beta-cell

The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.