Progression of Diet-Induced Diabetes in C57BL6J Mice Involves Functional Dissociation of Ca2+ Channels From Secretory Vesicles

OBJECTIVE: The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS: C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca(2+)](i)) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca(2+)](i). The functional data were complemented by analyses of histology and gene transcription. RESULTS: After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and beta-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20-50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca(2+)](i) signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca(2+) entry in HFD beta-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS: HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca(2+) entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion.

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived β-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human β cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D

Multivesicular exocytosis in rat pancreatic beta cells

AIMS/HYPOTHESIS: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. METHODS: Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. RESULTS: Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 μmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 μm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. CONCLUSIONS/INTERPRETATION: Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation

Function and expression of melatonin receptors on human pancreatic islets.

Melatonin is known to inhibit insulin secretion from rodent beta-cells through interactions with cell-surface MT1 and/or MT2 receptors, but the function of this hormone in human islets of Langerhans is not known. In the current study, melatonin receptor expression by human islets was examined by reverse transcription-polymerase chain reaction (RT-PCR) and the effects of exogenous melatonin on intracellular calcium ([Ca2+]i) levels and islet hormone secretion were determined by single cell microfluorimetry and radioimmunoassay, respectively. RT-PCR amplifications indicated that human islets express mRNAs coding for MT1 and MT2 melatonin receptors, although MT2 mRNA expression was very low. Analysis of MT1 receptor mRNA expression at the single cell level indicated that it was expressed by human islet alpha-cells, but not by beta-cells. Exogenous melatonin stimulated increases in intracellular calcium ([Ca2+]i) in dissociated human islet cells, and stimulated glucagon secretion from perifused human islets. It also stimulated insulin secretion and this was most probably a consequence of glucagon acting in a paracrine fashion to stimulate beta-cells as the MT1 receptor was absent in beta-cells. Melatonin did not decrease 3', 5'-cyclic adenosine monophosphate (cyclic AMP) levels in human islets, but it inhibited cyclic AMP in the mouse insulinoma (MIN6) beta-cell line and it also inhibited glucose-stimulated insulin secretion from MIN6 cells. These data suggest that melatonin has direct stimulatory effects at human islet alpha-cells and that it stimulates insulin secretion as a consequence of elevated glucagon release. This study also indicates that the effects of melatonin are species-specific with primarily an inhibitory role in rodent beta-cells and a stimulatory effect in human islets

Role of adenine nucleotides in insulin secretion from MIN6 pseudoislets

Insulin secretion from MIN6 cells configured as cell aggregates by culture on a gelatin substrate (pseudoislets) is enhanced compared to that of MIN6 cells grown as monolayers on tissue culture plastic, indicating the importance of β-cell-to-β-cell proximity for insulin release. In this study we have shown that glucose induced a biphasic release of insulin from pseudoislets, whereas the amplitude and duration of the responses of equivalent monolayer cells were much reduced. Purinergic aqonists have been implicated in intercellular communication between β-cells, so we investigated whether adenine nucleotides co-released with insulin are responsible for the enhanced secretory responses of pseudoislets. We have demonstrated that MIN6 cells express purinergic A1 and P2Y receptors, and that adenine nucleotides increased [Ca2+] with an efficacy of agonists being ATP>ADP>AMP. However, neither suramin nor the more selective A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine reduced glucose-induced insulin secretion from pseudoislets, and stimulation of monolayer cells with a range of adenine nucleotides did not enhance glucose-induced secretion. These results suggest that enhanced secretion from MIN6 pseudoislets is not due to increased paracrine/autocrine action of adenine nucleotides. © 2002 Elsevier Science Ireland Ltd. All rights reserved

Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca²⁺ Mobilization From Acidic Stores in Pancreatic α-Cells.

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of β-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca <sup>2+</sup> and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca <sup>2+</sup> ] <sub>i</sub> in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca <sup>2+</sup> but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca <sup>2+</sup> ] <sub>i</sub> in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca <sup>2+</sup> release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca <sup>2+</sup> ] <sub>i</sub> Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that β-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca <sup>2+</sup> ] <sub>i</sub> signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca <sup>2+</sup> release from the acidic stores and further amplified by Ca <sup>2+</sup> -induced Ca <sup>2+</sup> release from the sarco/endoplasmic reticulum