Xingjun Jing

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.

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.

Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes.

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.

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.

Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic beta-cell exocytosis and release of insulin.

Cyclic AMP potentiates glucose-stimulated insulin release and mediates the stimulatory effects of hormones such as glucagon-like peptide 1 (GLP-1) on pancreatic β-cells. By inhibition of cAMP-degrading phosphodiesterase (PDE) and, in particular, selective inhibition of PDE3 activity, stimulatory effects on insulin secretion have been observed. Molecular and functional information on β-cell PDE3 is, however, scarce. To provide such information, we have studied the specific effects of the PDE3B isoform by adenovirus-mediated overexpression. In rat islets and rat insulinoma cells, approximate 10-fold overexpression of PDE3B was accompanied by a 6–8-fold increase in membrane-associated PDE3B activity. The cAMP concentration was significantly lowered in transduced cells (INS-1(832/13)), and insulin secretion in response to stimulation with high glucose (11.1 mm) was reduced by 40% (islets) and 50% (INS-1). Further, the ability of GLP-1 (100 nm) to augment glucose-stimulated insulin secretion was inhibited by ∼30% (islets) and 70% (INS-1). Accordingly, when stimulating with cAMP, a substantial decrease (65%) in exocytotic capacity was demonstrated in patch-clamped single β-cells. In untransduced insulinoma cells, application of the PDE3-selective inhibitor OPC3911 (10 μm) was shown to increase glucose-stimulated insulin release as well as cAMP-enhanced exocytosis. The findings suggest a significant role of PDE3B as an important regulator of insulin secretory processes.

Myosin 5a controls insulin granule recruitment during late-phase secretion.

We have examined the importance of the actin‐based molecular motor myosin 5a for insulin granule transport and insulin secretion. Expression of myosin 5a was downregulated in clonal INS‐1E cells using RNAinterference. Stimulated hormone secretion was reduced by 46% and single‐cell exocytosis, measured by capacitance recordings, was inhibited by 42% after silencing. Silencing of Slac‐2c/MYRIP, which links insulin granules to myosin 5a, resulted in similar inhibition of single‐cell exocytosis. Antibody inhibition of the myosin 5a–Slac‐2c/MYRIP interaction significantly reduced the recruitment of insulin granules for release. The pool of releasable granules independent of myosin 5a activity was estimated to approximately 550 granules. Total internal reflection microscopy was then applied to directly investigate granule recruitment to the plasma membrane. Silencing of myosin 5a inhibited granule recruitment during late phase of insulin secretion. In conclusion, we propose a model where insulin granules are transported through the actin network via both myosin 5a‐mediated transport and via passive diffusion, with the former playing the major role during stimulatory conditions.

Glutaredoxin-1 mediates NADPH-dependent stimulation of calcium-dependent insulin secretion.

Nicotinamide adenine dinucleotide phosphate (NADPH) enhances Ca(2+)-induced exocytosis in pancreatic beta-cells, an effect suggested to involve the cytosolic redox protein glutaredoxin-1 (GRX-1). We here detail the role of GRX-1 in NADPH-stimulated beta-cell exocytosis and glucose-stimulated insulin secretion. Silencing of GRX-1 by RNA interference reduced glucose-stimulated insulin secretion in both clonal INS-1 832/13 cells and primary rat islets. GRX-1 silencing did not affect cell viability or the intracellular redox environment, suggesting that GRX-1 regulates the exocytotic machinery by a local action. By contrast, knockdown of the related protein thioredoxin-1 (TRX-1) was ineffective. Confocal immunocytochemistry revealed that GRX-1 locates to the cell periphery, whereas TRX-1 expression is uniform. These data suggest that the distinct subcellular localizations of TRX-1 and GRX-1 result in differences in substrate specificities and actions on insulin secretion. Single-cell exocytosis was likewise suppressed by GRX-1 knockdown in both rat beta-cells and clonal 832/13 cells, whereas after overexpression exocytosis increased by approximately 40%. Intracellular addition of NADPH (0.1 mm) stimulated Ca(2+)-evoked exocytosis in both cell types. Interestingly, the stimulatory action of NADPH on the exocytotic machinery coincided with an approximately 30% inhibition in whole-cell Ca(2+) currents. After GRX-1 silencing, NADPH failed to amplify insulin release but still inhibited Ca(2+) currents in 832/13 cells. In conclusion, NADPH stimulates the exocytotic machinery in pancreatic beta-cells. This effect is mediated by the NADPH acceptor protein GRX-1 by a local redox reaction that accelerates beta-cell exocytosis and, in turn, insulin secretion.

Survival of pancreatic beta cells is partly controlled by a TCF7L2-p53-p53INP1-dependent pathway

The transcription factor T-cell factor 7-like 2 (TCF7L2) confers type 2 diabetes risk mainly through impaired insulin secretion, perturbed incretin effect and reduced beta-cell survival. The aim of this study was to identify the molecular mechanism through which TCF7L2 influences beta-cell survival. TCF7L2 target genes in INS-1 cells were identified using Chromatin Immunoprecipitation. Validation of targets was obtained by: siRNA silencing, real-time quantitative polymerase chain reaction, electrophoretic mobility shift assay, luciferase reporter assays and western blot. Apoptosis rate was measured by DNA degradation and caspase-3 content. Islet viability was estimated by measuring metabolic rate. TCF7L2 binds to 3646 gene promoters in INS-1 cells in high or low glucose, including Tp53, Pten, Uggt1, Adamts9 and Fto. SiRNA-mediated reduction in TCF7L2 activity resulted in increased apoptosis and increased expression of Tp53, which resulted in elevated p53 protein activity and an increased expression of the p53 target gene Tp53inp1 (encoding p53-induced-nuclear-protein 1). Reversing the increase in p53INP1 protein expression, seen after Tcf7l2 silencing, protected INS-1 cells from Tcf7l2 depletion-induced apoptosis. This result was replicated in primary rat islets. The risk T-allele of rs7903146 is associated with increased TCF7L2 mRNA expression and transcriptional activity. On the other hand, in vitro silencing of TCF7L2 lead to increased apoptosis. One possibility is that the risk T-allele increases expression of an inhibitory TCF7L2 isoform with lower transcriptional activity. These results identify the p53-p53INP1 pathway as a molecular mechanism through which TCF7L2 may affect beta-cell survival and established a molecular link between Tcf7l2 and two type 2 diabetes-associated genes, Tp53inp1 and Adamts9.

Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3.

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.