Functional genomics of the beta-cell: short-chain 3-hydroxyacyl-coenzyme A dehydrogenase regulates insulin secretion independent of K+ currents

Recent advances in functional genomics afford the opportunity to interrogate the expression profiles of thousands of genes simultaneously and examine the function of these genes in a high-throughput manner. In this study, we describe a rational and efficient approach to identifying novel regulators of insulin secretion by the pancreatic beta-cell. Computational analysis of expression profiles of several mouse and cellular models of impaired insulin secretion identified 373 candidate genes involved in regulation of insulin secretion. Using RNA interference, we assessed the requirements of 10 of these candidates and identified four genes (40%) as being essential for normal insulin secretion. Among the genes identified was Hadhsc, which encodes short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD), an enzyme of mitochondrial beta-oxidation of fatty acids whose mutation results in congenital hyperinsulinism. RNA interference-mediated gene suppression of Hadhsc in insulinoma cells and primary rodent islets revealed enhanced basal but normal glucose-stimulated insulin secretion. This increase in basal insulin secretion was not attenuated by the opening of the KATP channel with diazoxide, suggesting that SCHAD regulates insulin secretion through a KATP channel-independent mechanism. Our results suggest a molecular explanation for the hyperinsulinemia hypoglycemic seen in patients with SCHAD deficiency

Ubiquitin Fold Modifier 1 (UFM1) and Its Target UFBP1 Protect Pancreatic Beta Cells from ER Stress-Induced Apoptosis

UFM1 is a member of the ubiquitin like protein family. While the enzymatic cascade of UFM1 conjugation has been elucidated in recent years, the biological function remains largely unknown. In this report we demonstrate that the recently identified C20orf116 [1], which we name UFM1-binding protein 1 containing a PCI domain (UFBP1), andCDK5RAP3 interact with UFM1. Components of the UFM1 conjugation pathway (UFM1, UFBP1, UFL1 and CDK5RAP3) are highly expressed in pancreatic islets of Langerhans and some other secretory tissues. Co-localization of UFM1 with UFBP1 in the endoplasmic reticulum (ER)depends on UFBP1. We demonstrate that ER stress, which is common in secretory cells, induces expression of Ufm1, Ufbp1 and Ufl1 in the beta-cell line INS-1E.siRNA-mediated Ufm1 or Ufbp1knockdown enhances apoptosis upon ER stress.Silencing the E3 enzyme UFL1, results in similar outcomes, suggesting that UFM1-UFBP1 conjugation is required to prevent ER stress-induced apoptosis. Together, our data suggest that UFM1-UFBP1participate in preventing ER stress-induced apoptosis in protein secretory cells

Expression of the Transcription Factor STAT-1α in Insulinoma Cells Protects against Cytotoxic Effects of Multiple Cytokines

Destruction of pancreatic islet β-cells in type 1 diabetes appears to result from direct contact with infiltrating T-cells and macrophages and exposure to inflammatory cytokines such as interferon (IFN)-γ, interleukin (IL)-1β, and tumor necrosis factor TNF-α that such cells produce. We recently reported on a method for selection of insulinoma cells that are resistant to the cytotoxic effects of inflammatory cytokines (INS-1res), involving their growth in progressively increasing concentrations of IL-1β plus IFN-γ, and selection of surviving cells. In the current study, we have investigated the molecular mechanism of cytokine resistance in INS-1rescells. By focusing on the known components of the IFN-γ receptor signaling pathway, we have discovered that expression levels of signal transducer and activator of transcription (STAT)-1α are closely correlated with the cytokine-resistant and -sensitive phenotypes. That STAT-1α is directly involved in development of cytokine resistance is demonstrated by an increase of viability from 10 ± 2% in control cells to 50 ± 6% in cells with adenovirus-mediated overexpression of STAT-1α (p \u3c 0.001) after culture of both cell groups in the presence of 100 units/ml IFN-γ plus 10 ng/ml IL-1β for 48 h. The resistance to IL-1β plus IFN-γ in STAT-1α-expressing cells is due in part to interference with IL-1β-mediated stimulation of inducible nitric-oxide synthase expression and nitric oxide production. Furthermore, overexpression of STAT-1α does not impair robust glucose-stimulated insulin secretion in the INS-1-derived cell line 832/13. We conclude that expression of STAT-1α may be a means of protecting insulin-producing cell lines from cytokine damage, which, in conjunction with appropriate cell-impermeant macroencapsulation devices, may allow such cells to be used for insulin replacement in type 1 diabetes

Discrete and Complementary Mechanisms of Protection of β-Cells Against Cytokine-Induced and Oxidative Damage Achieved by bcl-2 Overexpression and a Cytokine Selection Strategy

We have been investigating the potential utility of engineered cell lines as surrogates for primary islet cells in treatment of type 1 diabetes. To this end, two strategies that have emerged for procuring cell lines with resistance to immune-mediated damage are 1) selection of cytokine-resistant cell lines by growth of INS-1 insulinoma cells in iteratively increasing concentrations of interleukin (IL)-1β + γ-interferon (IFN-γ), and 2) stable overexpression of the anti-apoptotic gene bcl-2 in INS-1 cells. Herein, we show that bcl-2−overexpressing cells are resistant to the cytotoxic effects of reactive oxygen and nitrogen species (ROS/RNS), but are only modestly protected against high concentrations of IL-1β + INF-γ, whereas the converse is true in cytokine selected cells. We also found that the combination of bcl-2 expression and cytokine selection confers a broader spectrum of resistance than either procedure alone, such that the resultant cells are highly resistant to cytokines and ROS/RNS, with no impairment in glucose-stimulated insulin secretion. INS-1−derived cells with combined bcl-2 expression and cytokine selection are also more resistant to damage induced by coculture with mitogen-activated peripheral blood mononuclear cells. Surprisingly, application of the cytokine selection procedure to bcl-2−overexpressing cells does not result in impairment of nuclear factor-κB translocation, iNOS expression, and NO production, as clearly occurs upon application of the selection procedure to cells without bcl-2 overexpression. Further investigation of the diverse pathways involved in the development of cytokine and ROS/RNS resistance may define simplified and specific strategies for preservation of β-cell mass

Caspase 3 activity is low in primary rat islets in response to ER stress and DNA damage.

<p>832/13 cells (<b>A</b>, <b>C</b>) and primary rat islets (<b>B</b>, <b>D</b>) were treated with DMSO (control), thapsigargin (100 nM or 1 μM, respectively), or etoposide (100 μM or 200 μM, respectively) for the indicated times. Clarified lysates were examined by immunoblot analysis (<b>A</b>, <b>B</b>) and caspase 3/7 colorimetric activity assay (<b>C</b>, <b>D</b>). Data represent the mean +S.E.M of 3 independent experiments. * p ≤ 0.05 as compared to DMSO treated cells.</p

Delayed apoptosis allows islet β-cells to implement an autophagic mechanism to promote cell survival

<div><p>Increased β-cell death coupled with the inability to replicate existing β-cells drives the decline in β-cell mass observed in the progression of both major forms of diabetes. Understanding endogenous mechanisms of islet cell survival could have considerable value for the development of novel strategies to limit β-cell loss and thereby promote β-cell recovery. Insulinoma cells have provided useful insight into β-cell death pathways but observations made in cell lines sometimes fail to translate to primary islets. Here, we report dramatic differences in the temporal regulation and engagement of the apoptotic program in primary rodent islets relative to the INS-1 derived 832/13 cell line. As expected, 832/13 cells rapidly induced cell stress markers in response to ER stress or DNA damage and were fully committed to apoptosis, resulting in >80% cell death within 24 h. In contrast, primary rat islets were largely refractory to cell death in response to ER stress and DNA damage, despite rapid induction of stress markers, such as XBP-1(s), CHOP, and PUMA. Gene expression profiling revealed a general suppression of pro-apoptotic machinery, such as Apaf-1 and caspase 3, and sustained levels of pro-survival factors, such as cIAP-1, cIAP-2, and XIAP, in rat islets. Furthermore, we observed sustained induction of autophagy following chronic ER stress and found that inhibition of autophagy rendered islet β-cells highly vulnerable to ER stress-induced cell death. We propose that islet β-cells dampen the apoptotic response to delay the onset of cell death, providing a temporal window in which autophagy can be activated to limit cellular damage and promote survival.</p></div

Early and late apoptotic marker analysis demonstrates low levels of apoptosis in primary rat islets.

<p>832/13 cells (<b>A</b>, <b>C</b>) and primary rat islets (<b>B</b>, <b>D</b>) were treated with DMSO (control), thapsigargin (100 nM or 1 μM, respectively), or etoposide (100 μM or 200 μM, respectively) for the indicated times. (<b>A</b>, <b>B</b>) Cells were stained with Annexin V and counted by flow cytometry. (<b>C</b>, <b>D</b>) Cells were dispersed onto coverslips and stained with TUNEL and counterstained with DAPI. Positive cells were counted using Fiji. Data represent the mean +S.E.M. of 3 independent experiments. * p ≤ 0.05 as compared to DMSO treated cells.</p