Albert Salehi

SUR1 Regulates PKA-independent cAMP-induced Granule Priming in Mouse Pancreatic B-cells

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS–insensitive) component correlated with a rapid increase in membrane capacitance of ∼80 fF that plateaued within ∼200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K d values were 6 and 29 μM for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1−/− mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1−/− mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl− into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane KATP-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca2+-induced exocytosis.

Overexpression of Alpha2A-Adrenergic Receptors Contributes to Type 2 Diabetes

Ratting Out a Diabetes Gene Inbred animals with inherited susceptibility to disease can be especially informative regarding pathogenetic mechanisms because they carry naturally occurring genetic variants of the same type that cause disease in humans. This principle is illustrated by Rosengren et al. (p. 217; published online 19 November), whose analysis of an inbred strain of rats prone to develop type 2 diabetes led to the discovery of a gene whose aberrant overexpression suppresses pancreatic insulin secretion in both rats and humans. The culprit gene, ADRA2A, encodes the alpha2A adrenergic receptor and is potentially a valuable lead for diabetes therapy because it can be targeted pharmacologically. Sequence variations in an adrenergic receptor gene cause reduced insulin secretion and contribute to type 2 diabetes. Several common genetic variations have been associated with type 2 diabetes, but the exact disease mechanisms are still poorly elucidated. Using congenic strains from the diabetic Goto-Kakizaki rat, we identified a 1.4-megabase genomic locus that was linked to impaired insulin granule docking at the plasma membrane and reduced β cell exocytosis. In this locus, Adra2a, encoding the alpha2A-adrenergic receptor [alpha(2A)AR], was significantly overexpressed. Alpha(2A)AR mediates adrenergic suppression of insulin secretion. Pharmacological receptor antagonism, silencing of receptor expression, or blockade of downstream effectors rescued insulin secretion in congenic islets. Furthermore, we identified a single-nucleotide polymorphism in the human ADRA2A gene for which risk allele carriers exhibited overexpression of alpha(2A)AR, reduced insulin secretion, and increased type 2 diabetes risk. Human pancreatic islets from risk allele carriers exhibited reduced granule docking and secreted less insulin in response to glucose; both effects were counteracted by pharmacological alpha(2A)AR antagonists.

A systems genetics approach identifies genes and pathways for type 2 diabetes in human islets.

Close to 50 genetic loci have been associated with type 2 diabetes (T2D), but they explain only 15% of the heritability. In an attempt to identify additional T2D genes, we analyzed global gene expression in human islets from 63 donors. Using 48 genes located near T2D risk variants, we identified gene coexpression and protein-protein interaction networks that were strongly associated with islet insulin secretion and HbA(1c). We integrated our data to form a rank list of putative T2D genes, of which CHL1, LRFN2, RASGRP1, and PPM1K were validated in INS-1 cells to influence insulin secretion, whereas GPR120 affected apoptosis in islets. Expression variation of the top 20 genes explained 24% of the variance in HbA(1c) with no claim of the direction. The data present a global map of genes associated with islet dysfunction and demonstrate the value of systems genetics for the identification of genes potentially involved in T2D.

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice.

Insulin is secreted from pancreatic β cells in response to an elevation of cytoplasmic Ca2+ resulting from enhanced Ca2+ influx through voltage‐gated Ca2+ channels. Mouse β cells express several types of Ca2+ channel (L‐, R‐ and possibly P/Q‐type). β cell‐selective ablation of the gene encoding the L‐type Ca2+ channel subtype Cav1.2 (βCav1.2−/− mouse) decreased the whole‐cell Ca2+ current by only ∼45%, but almost abolished first‐phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca2+ handling and glucose‐induced electrical activity. However, high‐resolution capacitance measurements of exocytosis in single β cells revealed that the loss of first‐phase insulin secretion in the βCav1.2−/− mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Cav1.2 channel. Thus, the conduit of Ca2+ entry determines the ability of the cation to elicit secretion.

GLP-1 Inhibits and Adrenaline Stimulates Glucagon Release by Differential Modulation of N- and L-Type Ca2+ Channel-Dependent Exocytosis

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).

Reduced Insulin Exocytosis in Human Pancreatic β-Cells With Gene Variants Linked to Type 2 Diabetes

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for β-cell dysfunction that includes aberrant granule docking, decreased Ca2+ sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in β-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.

Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas.

Proghrelin, the precursor of the orexigenic and adipogenic peptide hormone ghrelin, is synthetized in endocrine (A-like) cells in the gastric mucosa. During its cellular processing, proghrelin gives rise to the 28-amino acid peptide desacyl ghrelin, which after octanoylation becomes active acyl ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets of mice and rats. Surprisingly, acyl ghrelin and obestatin had almost identical effects in that they stimulated the secretion of glucagon and inhibited that of PP and somatostatin from both mouse and rat islets. Obestatin inhibited insulin secretion more effectively than acyl ghrelin. In mouse islets, acyl ghrelin inhibited insulin secretion at low doses and stimulated at high. In rat islets, acyl ghrelin inhibited insulin secretion in a dose-dependent manner but the IC(50) for the acyl ghrelin-induced inhibition of insulin release was 7.5 x 10(-8) M, while the EC(50) and IC(50) values, with respect to stimulation of glucagon release and to inhibition of PP and somatostatin release, were in the 3 x 10(-12)-15 x 10(-12) M range. The corresponding EC(50) and IC(50) values for obestatin ranged from 5 x 10(-12) to 20 x 10(-12) M. Desacyl ghrelin per se did not affect islet hormone secretion. However, at a ten times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant concentrations; with obestatin this was the case also for insulin secretion. The combination of obestatin, acyl ghrelin and desacyl ghrelin in concentrations and proportions similar to those found in plasma resulted in effects that were indistinguishable from those induced by obestatin alone. From the data it seems that the effects of endogenous, circulating acyl ghrelin may be overshadowed by obestatin or blunted by desacyl ghrelin.

Secreted Frizzled-Related Protein 4 Reduces Insulin Secretion and Is Overexpressed in Type 2 Diabetes

A plethora of candidate genes have been identified for complex polygenic disorders, but the underlying disease mechanisms remain largely unknown. We explored the pathophysiology of type 2 diabetes (T2D) by analyzing global gene expression in human pancreatic islets. A group of coexpressed genes (module), enriched for interleukin-1-related genes, was associated with T2D and reduced insulin secretion. One of the module genes that was highly overexpressed in islets from T2D patients is SFRP4, which encodes secreted frizzled-related protein 4. SFRP4 expression correlated with inflammatory markers, and its release from islets was stimulated by interleukin-1β. Elevated systemic SFRP4 caused reduced glucose tolerance through decreased islet expression of Ca(2+) channels and suppressed insulin exocytosis. SFRP4 thus provides a link between islet inflammation and impaired insulin secretion. Moreover, the protein was increased in serum from T2D patients several years before the diagnosis, suggesting that SFRP4 could be a potential biomarker for islet dysfunction in T2D.

Capacitance measurements of exocytosis in mouse pancreatic α-, β- and δ-cells within intact islets of Langerhans

Capacitance measurements of exocytosis were applied to functionally identified α‐, β‐ and δ‐cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in β‐cells during a depolarization to 0 mV was equivalent to 14 granules s−1, <5% of that observed in isolated β‐cells. β‐Cell secretion exhibited bell‐shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to ∼−20 mV) the maximum rate of release was ∼4 granules s−1. Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L‐type Ca2+ channel blocker nifedipine (25 μm) but only marginally (<20%) affected by the R‐type Ca2+ channel blocker SNX482 (100 nm). Exocytosis in the glucagon‐producing α‐cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s−1) and a sustained late component (30 granules s−1). Whereas addition of the N‐type Ca2+ channel blocker ω‐conotoxin GVIA (0.1 μm) inhibited glucagon secretion measured in the presence of 1 mm glucose to the same extent as an elevation of glucose to 20 mm, the L‐type Ca2+ channel blocker nifedipine (25 μm) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca2+‐influx through N‐type rather than L‐type Ca2+ channels. Exocytosis in the somatostatin‐secreting δ‐cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s−1) component followed by a sustained slower (60 granules s−1) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in β‐cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.

Somatostatin inhibits exocytosis in rat pancreatic α-cells by Gi2-dependent activation of calcineurin and depriming of secretory granules

1 Measurements of cell capacitance were used to investigate the molecular mechanisms by which somatostatin inhibits Ca2+‐induced exocytosis in single rat glucagon‐secreting pancreatic α‐cells. 2 Somatostatin decreased the exocytotic responses elicited by voltage‐clamp depolarisations by 80 % in the presence of cyclic AMP‐elevating agents such as isoprenaline and forskolin. Inhibition was time dependent and half‐maximal within 22 s. 3 The inhibitory action of somatostatin was concentration dependent with an IC50 of 68 nm and prevented by pretreatment of the cells with pertussis toxin. The latter effect was mimicked by intracellular dialysis with specific antibodies to Gi1/2 and by antisense oligonucleotides against G proteins of the subtype Gi2. 4 Somatostatin lacked inhibitory action when applied in the absence of forskolin or in the presence of the L‐type Ca2+ channel blocker nifedipine. The size of the ω‐conotoxin‐sensitive and forskolin‐independent component of exocytosis was limited to 60 fF. By contrast, somatostatin abolished L‐type Ca2+ channel‐dependent exocytosis in α‐cells exposed to forskolin. The magnitude of the latter pool amounted to 230 fF. 5 The inhibitory effect of somatostatin on exocytosis was mediated by activation of the serine/threonine protein phosphatase calcineurin and was prevented by pretreatment with cyclosporin A and deltamethrin or intracellularly applied calcineurin autoinhibitory peptide. Experiments using the stable ATP analogue AMP‐PCP indicate that somatostatin acts by depriming of granules. 6 We propose that somatostatin receptors associate with L‐type Ca2+ channels and couple to Gi2 proteins leading to a localised activation of calcineurin and depriming of secretory granules situated close to the L‐type Ca2+ channels.