Islet amyloid polypeptide regulates multiple steps in stimulus-secretion coupling of beta cells in rat pancreatic islets

Islet amyloid polypeptide (IAPP) is produced in pancreatic beta cells. Intraislet function of IAPP is still uncertain. In the present study, we investigated effects of IAPP and somatostatin on stimulus-secretion coupling of beta cells in isolated rat pancreatic islets. Insulin secretion induced by 22.2 mM glucose was increased by an IAPP antiserum (0.1%) or an IAPP antagonist (IAPP8-37, 10 microM). Pretreatment of islets with pertussis toxin (PTX) abolished the stimulating effect of IAPP8-37 on glucose-induced insulin secretion. In contrast, IAPP antiserum and IAPP8-37 did not change insulin secretion induced by 30 mM KCl. Somatostatin (1 nM) inhibited insulin secretion induced by 22.2 mM glucose, 10 mM L-arginine, 25 microM forskolin, and 200 microM carbachol. IAPP (10 microM) enhanced the inhibitory effect of somatostatin on insulin secretion induced by L-arginine or forskolin. PTX pretreatment abolished the effects of somatostatin and IAPP on arginine-induced insulin secretion. In conclusion, IAPP regulates multiple steps in signal transductions of beta cells. The effects of IAPP on beta cells are mediated by PTX-sensitive regulatory G proteins.     

Human pancreatic acinar cells: studies of stimulus-secretion coupling

Elements of stimulus-secretion coupling were studied in human pancreatic acinar cells by using tissue samples obtained from cadaver organ donors. In pancreatic fragments, acetylcholine evoked amylase secretion as well as potassium release and increased the outflux of 45Ca and 86Rb from the prelabeled tissue. In patches of basolateral plasma membrane excised from acinar cell clusters, single-channel potassium currents were recorded. The inside of the plasma membrane faced the bath solution, allowing the effects of changes in the free ionized calcium concentration in contact with the membrane interior to be tested. Two types of calcium-activated potassium-selective channels were found with unit conductances of about 250 and 50 picosiemens (pS), respectively. In both cases channel opening was determined by the electrical potential difference across the plasma membrane and the free ionized calcium concentration in the bath solution. The probability of channel opening was markedly increased by elevation of the free ionized calcium concentration in contact with the membrane inside. The results suggest that the acetylcholine-evoked cellular potassium release occurs via selective membrane potassium channels opened by calcium released intracellularly after the action of the secretagogue.     

Peptidergic regulation of maturation of the stimulus-secretion coupling in fetal islet beta cells

The stimulus-secretion coupling of the insulin-producing pancreatic islet beta cell is subject to functional maturation during fetal life. We studied the maturation of a glucose-responsive insulin release from fetal rat islets and specifically investigated the impact of peptidergic regulation. To this end, islets were isolated from 21-day-old fetal rats and maintained for 7 days in tissue culture at 3.3 or 11.1 mM glucose and various supplements. In islets cultured in low glucose, acutely raising the ambient glucose concentration to 16.7 mM evoked a modest stimulation of short-term insulin release that was more pronounced in islets maintained in high glucose. Moreover, the insulin content was much higher in islets cultured in high than in low glucose. Culture with growth hormone (GH) markedly amplified both basal and stimulated short-term insulin secretion from islets maintained in either low or high glucose. Additionally, GH significantly elevated the insulin content in islets maintained in low glucose. Transforming growth factor alpha (TGF-alpha) increased basal, but not glucose-stimulated, insulin release and insulin content in islets cultured in low glucose. Gastrin, expressed in islets during fetal life, did not affect basal or glucose-stimulated insulin release, or insulin content, in islets maintained in either low or high glucose. The addition of gastrin to TGF-alpha did not affect the results obtained with the latter peptide. Gastrin-releasing peptide failed to influence basal or glucose-responsive insulin secretory rates, and insulin content, at either glucose concentration during culture. The somatostatin analog Sandostatin (octreotide acetate) neither influenced basal nor stimulated short-term insulin release at any glucose concentration present during culture, whereas the hormone significantly decreased the insulin content of islets cultured in high glucose. Pancreastatin, produced by porcine islet beta and delta cells, failed to influence basal or glucose-responsive insulin secretory rates, and islet insulin content, at either glucose concentration during culture. Culture with gastric inhibitory peptide (GIP) or glucagon-like peptide I (GLP-1), two proposed incretins, did not affect short-term insulin secretion in response to 3.3 or 16.7 mM glucose irrespective of the ambient glucose concentration during culture. To the contrary, GLP-1, but not GIP, increased the content of insulin in islets cultured in low glucose. We conclude that islet beta-cell differentiation and functional maturation of the stimulus-secretion coupling can be modulated in vitro in fetal rat pancreatic tissue by peptidergic regulation and glycemic stimulation. We suggest that GH and TGF-alpha stimulate, while somatostatin, through paracrine interaction, may inhibit, these processes. These effectors may be of regulatory significance in the in vivo development of glucose-sensitive beta cells, and defects in these mechanisms may result in glucose intolerance in adult subjects.     

Mathematical modelling of stimulus-secretion coupling in the pancreatic B-cell

Summary A mathematical model for cyclic AMP-Ca²⁺ interactions in pancreatic islets, defined in a previous study, was modified to include two new features. First, the cytosol and vacuolar Ca²⁺ pools were stratified in a cortical layer (representing 1 to 20% of the total cellular pool) and central core. Second, the changes in Ca²⁺ inflow and Ca²⁺ fractional outflow rate evoked by a rise in glucose concentration were discontinued during the 3rd and 4th min of stimulation. In this onion leaf sheet model, the biphasic pattern of glucose-induced insulin release could be simulated. This new model emphasizes the significance of both cytosol heterogeneity and signal discontinuity in the dynamics of insulin secretion.     

Transthyretin constitutes a functional component in pancreatic beta-cell stimulus-secretion coupling

Transthyretin (TTR) is a transport protein for thyroxine and, in association with retinol-binding protein, for retinol, mainly existing as a tetramer in vivo. We now demonstrate that TTR tetramer has a positive role in pancreatic beta-cell stimulus-secretion coupling. TTR promoted glucose-induced increases in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release. This resulted from a direct effect on glucose-induced electrical activity and voltage-gated Ca(2+) channels. TTR also protected against beta-cell apoptosis. The concentration of TTR tetramer was decreased, whereas that of a monomeric form was increased in sera from patients with type 1 diabetes. The monomer was without effect on glucose-induced insulin release and apoptosis. Thus, TTR tetramer constitutes a component in normal beta-cell function. Conversion of TTR tetramer to monomer may be involved in the development of beta-cell failure/destruction in type 1 diabetes.     

Over-expression of AMP-activated protein kinase impairs pancreatic {beta}-cell function in vivo

Treatment of type 1 diabetes by islet transplantation is currently limited by loss of functional beta-cell mass after transplantation. We investigated here whether adenovirus-mediated changes in AMP-activated protein kinase (AMPK) activity, previously shown to affect insulin secretion in vitro, might affect islet graft function in vivo. In isolated mouse and rat islets, insulin secretion stimulated by 17 (vs 3) mmol/l glucose was inhibited by 36.5% (P<0.01) and 43% (P<0.02) respectively after over-expression of constitutively-active AMPK- (AMPK CA) versus null (eGFP-expressing) viruses, and glucose oxidation was decreased by 38% (P<0.05) and 26.6% (P<0.05) respectively. Increases in apoptotic index (terminal deoxynucleotide transferase-mediated deoxyuridine trisphosphate biotin nick end-labelling) (TUNEL)) were also observed in AMPK CA- (22.8 +/- 3.6% TUNEL-positive cells, P<0.001), but not AMPK DN- (2.72 +/- 3.9%, positive cells, P=0.05) infected islets, versus null adenovirus-treated islets (0.68 +/- 0.36% positive cells). Correspondingly, transplantation of islets expressing AMPK CA into streptozotocin-diabetic C57 BL/6 mice improved glycaemic control less effectively than transplantation with either null (P<0.02) or AMPK-DN-infected (P<0.01) islets. We conclude that activation of AMPK inhibits beta-cell function in vivo and may represent a target for therapeutic intervention during islet transplantation.     

Alcohol impairs protein synthesis and degradation in cultured skeletal muscle cells

BACKGROUND: Acute and chronic alcohol intoxication decreases skeletal muscle protein synthesis under in vivo conditions. We investigated whether ethanol (EtOH) and its major metabolites, acetaldehyde and acetate, can directly modulate protein balance under in vitro conditions. METHODS: Human myocytes were incubated with different doses of EtOH for varying periods of time (i.e., 4-72 hr). Alternatively, cells were incubated with acetaldehyde, acetate, insulin, insulin-like growth factor-I (IGF-I), or with a combination of EtOH plus insulin or IGF-I. Rates of protein synthesis or degradation were determined by 35S-methionine/cysteine incorporation into or release from cellular protein. RESULTS: A significant, 15% to 20%, decrease in basal protein synthesis was observed after 24 hr, but not at earlier time points, in response to 80 mM EtOH. Incubation of myocytes for 72 hr decreased synthesis in cells incubated with EtOH ranging between 60 and 120 mM. The ability of IGF-I or insulin to stimulate protein synthesis was impaired by 30% and 60%, respectively, in cells incubated with 80 mM EtOH for 72 hr. Exposure of cells to 200 microM acetaldehyde or 5 mM Na-acetate also decreased basal protein synthesis. In contrast, neither EtOH, acetaldehyde, nor acetate altered the basal rate of protein degradation. However, EtOH completely impaired the ability of insulin and IGF-I to inhibit proteolysis. Finally, EtOH did not impair IGF-I receptor autophosphorylation, but inhibited the ability of insulin to phosphorylate its own receptor. EtOH also did not alter the number of insulin or IGF-I receptors or the formation of insulin/IGF-I hybrid receptors. CONCLUSIONS: We have demonstrated that EtOH can directly inhibit muscle protein synthesis under in vitro conditions. Neither EtOH nor its metabolites altered basal protein degradation, although EtOH did compromise the ability of both insulin and IGF-I to slow proteolysis. This impairment seems to be mediated by different defects in signal transduction.     

Translational control of protein synthesis in pancreatic acinar cells

Translational control of protein synthesis in the pancreas is important in regulating growth and the synthesis of digestive enzymes. Regulation of translation is primarily directed at the steps in initiation and involves reversible phosphorylation of initiation factors (eIFs) and ribosomal proteins. Major sites include the assembly of the eIF4F mRNA cap binding complex, the activity of guanine nucleotide exchange factor eIF2B, and the activity of ribosomal S6 kinase. All of these involve phosphorylation by different regulatory pathways. Stimulation of protein synthesis in acinar cells is primarily mediated by the phosphatidylinositol 3-kinase-mTOR pathway and involves both release of eIF4E (the limiting component of eIF4F) from its binding protein and phosphorylation of ribosomal S6 protein by S6K. eIF4E is itself phosphorylated by a distinct pathway. Inhibition of acinar protein synthesis can be mediated by inhibition of eIF2B following phosphorylation of eIF2α.     

Multiple signaling pathways control stimulus-secretion coupling in rat peritoneal mast cells.

Fura-2 and membrane capacitance measurements were performed to investigate intracellular Ca2+ concentration [( Ca2+]i) and secretory responses of rat peritoneal mast cells following secretagogue stimulation. Compound 48/80 and internally applied guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) induced transient rises in [Ca2+]i and caused membrane capacitance increases as secretion occurred. The 48/80-induced Ca2+ transients and secretory responses were blocked by guanosine 5'-[beta-thio]diphosphate and neomycin, indicating that inositolphospholipid breakdown mediated by guanine nucleotide-binding regulatory protein (G protein) plays an important role in stimulus-secretion coupling. However, pertussis toxin did not block Ca2+ transients induced by 48/80 or GTP[gamma-S], whereas secretory responses were either abolished (48/80) or developed only after a considerable delay (GTP[gamma-S]). Similar effects were obtained by perfusing cells with cAMP: (i) Ca2+ transients following stimulation with 48/80 remained unaffected by cAMP, but secretory responses were abolished; (ii) GTP[gamma-S] induced normal Ca2+ transients and degranulation in the presence of cAMP. Pretreatment of mast cells with phorbol 12-myristate 13-acetate (PMA) abolished 48/80- and GTP[gamma-S]-induced Ca2+ transients (but not inositol trisphosphate-induced Ca2+ transients), whereas secretion still occurred. At the same time, the Ca2+ requirement for secretion was reduced by PMA. These results indicate that secretion in mast cells is under control of an as yet unidentified signaling pathway that involves a G protein. This pathway is distinct from inositolphospholipid turnover and may provide the triggering mechanism for secretion, whereas the inositolphospholipid pathway serves to increase [Ca2+]i and renders the secretory process more sensitive to [Ca2+]i by activating protein kinase C. Persistent activation of protein kinase C through phorbol ester imposes negative feedback control on the inositolphospholipid pathway, whereas cAMP may inhibit the unidentified signaling pathway.     

The loss of Sirt1 in mouse pancreatic beta cells impairs insulin secretion by disrupting glucose sensing

Aims/hypothesis

Sirtuin 1 (SIRT1) has emerged as a key metabolic regulator of glucose homeostasis and insulin secretion. Enhanced SIRT1 activity has been shown to be protective against diabetes, although the mechanisms remain largely unknown. The aim of this study was to determine how SIRT1 regulates insulin secretion in the pancreatic beta cell.

Methods

Pancreatic beta cell-specific Sirt1 deletion was induced by tamoxifen injection in 9-week-old Pdx1CreER:floxSirt1 mice (Sirt1BKO). Controls were injected with vehicle. Mice were assessed metabolically via glucose challenge, insulin tolerance tests and physical variables. In parallel, Sirt1 short interfering RNA-treated MIN6 cells (SIRT1KD) and isolated Sirt1BKO islets were used to investigate the effect of SIRT1 inactivation on insulin secretion and gene expression.

Results

OGTTs showed impaired glucose disposal in Sirt1BKO mice due to insufficient insulin secretion. Isolated Sirt1BKO islets and SIRT1KD MIN6 cells also exhibited impaired glucose-stimulated insulin secretion. Subsequent analyses revealed impaired α-ketoisocaproic acid-induced insulin secretion and attenuated glucose-induced Ca²⁺ influx, but normal insulin granule exocytosis in Sirt1BKO beta cells. Microarray studies revealed a large cluster of mitochondria-related genes, the expression of which was dysregulated in SIRT1KD MIN6 cells. Upon further analysis, we demonstrated an explicit defect in mitochondrial function: the inability to couple nutrient metabolism to mitochondrial membrane hyperpolarisation and reduced oxygen consumption rates.

Conclusions/interpretation

Taken together, these findings indicate that in beta cells the deacetylase SIRT1 regulates the expression of specific mitochondria-related genes that control metabolic coupling, and that a decrease in beta cell Sirt1 expression impairs glucose sensing and insulin secretion.     

Aspects of novel sites of regulation of the insulin stimulus-secretion coupling in normal and diabetic pancreatic islets

Noninsulin-depenent diabetes mellitus (NIDDM), a major health care problem in the Western world, is a disease typified by a relative deficiency of insulin, leading to vast derangements in glucose and lipid homeostasis with disastrous vascular complications. Despite immense research efforts aimed at a clear understanding of the etiology of this complex disease, the molecular mechanisms causing the disorder still remain elusive. This article reviews extant data from recent publications implicating novel signal transduction pathways as important regulators of the insulin stimulus-secretion coupling in the pancreatic β-cell. The significance of nitric oxide and serine/threonine protein phosphatases, and their inactivation by insulin secretagogues, glucose metabolites, ATP, GTP, glutamate, and inositol hexaphosphate in this arena is scrutinized. Additionally, also presented is the growing concept that an important signal for insulin secretion may reside in the inextricable interplay between glucose and lipid metabolism, specifically the generation of malonyl-CoA, which inhibits carnitine palmitoyl-transferase 1 with the attendant accumulation of long-chain acyl CoA esters. Moreover, attention is directed towards novel intracellular actions of hypoglycemic sulfonylureas in the β-cell. Finally, the importance of “lipotoxicity” and aberrations in glucose uptake and metabolism in β-cell dysfunction is given consideration. Future research efforts should aim at further characterization of effects of second messengers on protein phosphorylation elements in β-cells. Additionally, long-term regulation by glucose and the diabetic state (e.g., fatty acids and ketones) on β-cell protein phosphatases, pyruvate dehydrogenase, and carnitine palmitoyltransferase 1 needs to be explored in greater depth. Clearly, the detrimental impact of diabetic hyperlipidemia on β-cell function has been a relatively neglected area, but future pharmacological approaches directed at preventing lipotoxicity may prove beneficial in the treatment of diabetes.     

Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.

The effects of putative transmethylation inhibitors were tested on stimulus-secretion coupling and neurotransmitter secretion at synapses between neuroblastoma X glioma hybrid cells and myotubes. 5'-Deoxy-5'-isobutylthio-3-deazaadenosine or 5'-deoxy-5'-isobutylthioadenosine inhibited CDP-choline synthesis catalyzed by cholinephosphate cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15) and thereby decreased the rate of phosphatidylcholine synthesis from CDP-choline, but did not affect the transmethylation pathway for phosphatidylcholine synthesis. These compounds also inhibited 45Ca2+ uptake by hybrid cells mediated by voltage-sensitive Ca2+ channels, acetylcholine secretion at synapses, and signal transduction through cell membranes mediated by myotube nicotinic acetylcholine receptors. In contrast, 3-deazaadenosine or adenosine inhibited the transmethylation pathway for phosphatidylcholine synthesis, but had no effect on Ca2+ action potentials, acetylcholine secretion, or signal transduction through cell membranes mediated by nicotinic acetylcholine receptors. These results show that the stimulus-secretion coupling and secretion reactions studied are not dependent on phospholipid methylation and suggest that the activity of action potential Ca2+ channels and the rate of neurotransmitter secretion are functionally coupled to the rate of phosphatidylcholine synthesis via the CDP-choline pathway.     

Lysosomes in normal pancreatic beta cells

Summary Lysosomes and their relationships with surrounding organelles were studied in pancreatic B-cells of normal Wistar rats by electron microscopy and cytochemical localization of acid phosphatase (AcPase) and arylsulphatase. Several forms of lysosomes were distinguished and shown to interact frequently with the secretory granules. Two different digestive processes were recognized. During crinophagy, lysosomes directly fuse with secretory granules while, during autophagy, a cytoplasmic area containing secretory granules and/or other organelles is circumscribed before the enzymatic digestion. In addition, lysosomes may transport arylsulphatase into secretory granules apparently not involved in a destructive process. Such a process could also account, at least in part, for the presence of AcPase in a certain number of mature and immature secretory granules.     

Insulin granule dynamics in pancreatic beta cells

Glucose-induced insulin secretion in response to a step increase in blood glucose concentrations follows a biphasic time course consisting of a rapid and transient first phase followed by a slowly developing and sustained second phase. Because Type 2 diabetes involves defects of insulin secretion, manifested as a loss of first phase and a reduction of second phase, it is important to understand the cellular mechanisms underlying biphasic insulin secretion. Insulin release involves the packaging of insulin in small (diameter 0.3 µm) secretory granules, the trafficking of these granules to the plasma membrane, the exocytotic fusion of the granules with the plasma membrane and eventually the retrieval of the secreted membranes by endocytosis. Until recently, studies on insulin secretion have been confined to the appearance of insulin in the extracellular space and the cellular events preceding exocytosis have been inaccessible to more detailed analysis. Evidence from a variety of secretory tissues, including pancreatic islet cells suggests, however, that the secretory granules can be functionally divided into distinct pools that are distinguished by their release competence and/or proximity to the plasma membrane. The introduction of fluorescent proteins that can be targeted to the secretory granules, in combination with the advent of new techniques that allow real-time imaging of granule trafficking in living cells (granule dynamics), has led to an explosion of our knowledge of the pre-exocytotic and post-exocytotic processes in the beta cell. Here we discuss these observations in relation to previous functional and ultra-structural data as well as the secretory defects of Type 2 diabetes.Abbreviations [Ca²⁺]_i Free cytoplasmic Ca²⁺-concentration - EGFP enhanced GFP - fF femtofarad (10^-15F) - GFP green fluorescent protein - K_ATP-channel ATP-regulated K⁺-channel - LDCV large dense-core vesicle - ms millisecond - mV millivolt - RRP readily releasable pool of granules - SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptors