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NADPH : The Missing Link Between Glucose Metabolism and Insulin Secretion?
MONTREAL 2008 ABSTRACTS
| 337
POSTER PRESENTATIONS 135 Subcellular Distribution and Function of Bcl-2 and Bcl-xL in the ȕ-cell DAN S. LUCIANI*, SCOTT WIDENMAIER, JACKY YEUNG, CHRISTOPHER McINTOSH, JAMES D. JOHNSON. Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC.
136#171 ABSTRACT C&SS ISLET BIOLOGY
Bcl-2 family proteins control ȕ-cell apoptosis and emerging evidence also suggests important roles in ȕ-cell physiology. Their function is regulated by the interaction of pro- and antiapoptotic family members at several intracellular organelles. Bcl2 and Bcl-xL are both believed to be important for ȕ-cell survival, but their relative abundance and localization within the ȕ-cell are not well characterized. Here we examined the subcellular distribution of endogenous and overexpressed Bcl-2 and Bcl-xL in ȕ-cells to clarify their organelle-specific functions. In both cultured mouse E-cells and pancreatic sections, immunofluorescent staining with monoclonal and polyclonal antibodies to Bcl-2 revealed clear nuclear localization with no detectable cytosolic signal. Overexpressed Bcl-2:GFP on the other hand was observed exclusively in the cytoplasm with prominent mitochondrial co-localization. Endogenous Bcl-xL was found by both immunostaining and cell fractionation to be primarily mitochondrial and non-nuclear, and this distribution was also seen in cells overexpressing Bcl-xL:YFP. Small molecule inhibitors of Bcl-xL/Bcl-2 caused immediate mitochondrial hyperpolarization, KATP-channel-dependent cytosolic Ca2+ signals, and had immediate effects on glucose-stimulated insulin secretion. In conclusion, endogenous Bcl-2 and Bcl-xL differ markedly in their localization within the pancreatic ȕ-cell. Taken together with the prominent mitochondrial effects of Bcl-2/Bcl-xL inhibitors, these findings suggest that Bcl-xL specifically, may act as an important regulator of ȕ-cell mitochondrial apoptosis and physiology.
ABSTRACT #93
137
The Glucagon-Like Peptide-1 Analogue Exenatide Reduces Beta-Cell Apoptosis and Enhances Survival of Cultured Human Islets: Implications for Clinical Islet Transplantation. ZILIANG AO, MARK MELOCHE, TIMOTHY KIEFFER, GARTH WARNOCK, LUCY MARZBAN. University of British Columbia, BC, Canada. Human islet transplantation as a means of beta-cell replacement is a promising approach for treatment of type 1 diabetes but is currently limited by loss of islets during isolation procedure, pre-transplant culture, and following transplantation. Any approach that enhances survival and/or function of human islets during pre-transplant culture period will therefore be of great importance in clinical islet transplantation. Exenatide is a long acting analogue of glucagon-like peptide-1 that has been shown to stimulate insulin secretion and increase beta-cell mass and was recently approved as an anti-diabetic drug for treatment of patients with type 2 diabetes. In the present study, we sought to determine whether exenatide treatment could reduce loss of beta-cells and improve survival of human islets during pre-transplant culture period as a potential approach to increase the yield of islets isolated from pancreatic donors. Human islets isolated from cadaveric donors were cultured in CMRL (5 mM glucose, 10% fetal bovine serum, 37qC) in the presence or absence of exenatide (1 nM) at different time points for up to 7 days. Beta-cell apoptosis was assessed by insulin/TUNEL/DAPI staining performed on paraffinembedded islet sections as well as measurement of cleaved caspase-3 by enzyme-linked immunosurbent assay (ELISA). As expected, culture of human islets resulted in loss of islet beta-cells in a timedependent manner leading to a decrease in the islet beta/alpha cell ratio during culture period (d0: 1.8 ± 0.3 vs. d7: 1.3 ± 0.2, P<0.05) and an increase in the proportion of apoptotic (TUNEL-positive) islet cells (d0: 3.4 ± 1.2% vs. d7: 12.1 ± 2.4%, P<0.05). Treatment with exenatide for 3, 5, or 7 days resulted in an increase in the islet beta/alpha cell ratio (+Ex: 1.6 ± 0.2%, P<0.05), intensity of insulin immunostaining and a decrease in the proportion of TUNEL-positive islet cells (+Ex: 7.5 ± 1.5%, P<0.05). Double insulin/TUNEL staining revealed that the majority of TUNEL-positive cells were beta cells. This decrease in the number of TUNEL-positive beta-cells in exenatide-treated islets was associated with a marked decrease in the activation of caspase-3 (34%). These findings suggest that short-term treatment with exenatide reduces beta-cell apoptosis in isolated human islets and decreases caspase-3 activation by a direct and/or indirect mechanism leading to an increase in the yield of islet betacells. Treatment of human islets with exenatide during pre-transplant culture period may have therapeutic value in clinical islet transplantation.
138
C&SS
NADPH : The Missing Link Between Glucose Metabolism and Insulin Secretion? ISLET BIOLOGY CLAUDIANE GUAY*, ÉRIK JOLY, MURTHY S. MADIRAJU, MARC PRENTKI, Montreal Diabetes Research Centre, Université de Montréal, Montréal, QC Patients with type 2 diabetes have inappropriate regulation of insulin secretion in response to glucose and other nutrients in pancreatic E-cells. To better understand this dysregulation, intensive work has been done to uncover factors coupling glucose metabolism to insulin secretion. Generation of metabolic coupling factors (MCF), such as ATP, malonyl-CoA and NADPH, by glucose metabolism have been suggested to regulate insulin secretion. In the present study, we hypothesized that malic enzyme (ME) and isocitrate dehydrogenase (IDH), the two main enzymes producing NADPH in E-cells, are implicated in the regulation of glucose-induced insulin secretion (GIIS). To test this hypothesis, we used RNAi-knockdown of ME and IDH. Transfection of INS 832/13 cells with shRNA directed against ME reduced ME activity by 62 % which was associated with a decrease in insulin secretion (33 % at 5 mM and 38 % at 10 mM glucose). siRNA directed against IDH decreased IDH mRNA level by 80 % and IDH activity by 50 % in INS 832/13 transfected cells. Experiments are currently performed to examine the effect of reducing IDH expression on GIIS. NADPH levels will also be measured in those conditions, to verify if ME and IDH are implicated in the regulation of GIIS via the production of NADPH. Those experiments are important not only to determine if ME and IDH are implicated in the regulation of GIIS, but also to underscore the role of MCF generated from glucose metabolism in the regulation of insulin secretion by the E-cells.
ABSTRACT #163 C&SS ISLET BIOLOGY
| 337
POSTER PRESENTATIONS 135 Subcellular Distribution and Function of Bcl-2 and Bcl-xL in the ȕ-cell DAN S. LUCIANI*, SCOTT WIDENMAIER, JACKY YEUNG, CHRISTOPHER McINTOSH, JAMES D. JOHNSON. Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC.
136#171 ABSTRACT C&SS ISLET BIOLOGY
Bcl-2 family proteins control ȕ-cell apoptosis and emerging evidence also suggests important roles in ȕ-cell physiology. Their function is regulated by the interaction of pro- and antiapoptotic family members at several intracellular organelles. Bcl2 and Bcl-xL are both believed to be important for ȕ-cell survival, but their relative abundance and localization within the ȕ-cell are not well characterized. Here we examined the subcellular distribution of endogenous and overexpressed Bcl-2 and Bcl-xL in ȕ-cells to clarify their organelle-specific functions. In both cultured mouse E-cells and pancreatic sections, immunofluorescent staining with monoclonal and polyclonal antibodies to Bcl-2 revealed clear nuclear localization with no detectable cytosolic signal. Overexpressed Bcl-2:GFP on the other hand was observed exclusively in the cytoplasm with prominent mitochondrial co-localization. Endogenous Bcl-xL was found by both immunostaining and cell fractionation to be primarily mitochondrial and non-nuclear, and this distribution was also seen in cells overexpressing Bcl-xL:YFP. Small molecule inhibitors of Bcl-xL/Bcl-2 caused immediate mitochondrial hyperpolarization, KATP-channel-dependent cytosolic Ca2+ signals, and had immediate effects on glucose-stimulated insulin secretion. In conclusion, endogenous Bcl-2 and Bcl-xL differ markedly in their localization within the pancreatic ȕ-cell. Taken together with the prominent mitochondrial effects of Bcl-2/Bcl-xL inhibitors, these findings suggest that Bcl-xL specifically, may act as an important regulator of ȕ-cell mitochondrial apoptosis and physiology.
ABSTRACT #93
137
The Glucagon-Like Peptide-1 Analogue Exenatide Reduces Beta-Cell Apoptosis and Enhances Survival of Cultured Human Islets: Implications for Clinical Islet Transplantation. ZILIANG AO, MARK MELOCHE, TIMOTHY KIEFFER, GARTH WARNOCK, LUCY MARZBAN. University of British Columbia, BC, Canada. Human islet transplantation as a means of beta-cell replacement is a promising approach for treatment of type 1 diabetes but is currently limited by loss of islets during isolation procedure, pre-transplant culture, and following transplantation. Any approach that enhances survival and/or function of human islets during pre-transplant culture period will therefore be of great importance in clinical islet transplantation. Exenatide is a long acting analogue of glucagon-like peptide-1 that has been shown to stimulate insulin secretion and increase beta-cell mass and was recently approved as an anti-diabetic drug for treatment of patients with type 2 diabetes. In the present study, we sought to determine whether exenatide treatment could reduce loss of beta-cells and improve survival of human islets during pre-transplant culture period as a potential approach to increase the yield of islets isolated from pancreatic donors. Human islets isolated from cadaveric donors were cultured in CMRL (5 mM glucose, 10% fetal bovine serum, 37qC) in the presence or absence of exenatide (1 nM) at different time points for up to 7 days. Beta-cell apoptosis was assessed by insulin/TUNEL/DAPI staining performed on paraffinembedded islet sections as well as measurement of cleaved caspase-3 by enzyme-linked immunosurbent assay (ELISA). As expected, culture of human islets resulted in loss of islet beta-cells in a timedependent manner leading to a decrease in the islet beta/alpha cell ratio during culture period (d0: 1.8 ± 0.3 vs. d7: 1.3 ± 0.2, P<0.05) and an increase in the proportion of apoptotic (TUNEL-positive) islet cells (d0: 3.4 ± 1.2% vs. d7: 12.1 ± 2.4%, P<0.05). Treatment with exenatide for 3, 5, or 7 days resulted in an increase in the islet beta/alpha cell ratio (+Ex: 1.6 ± 0.2%, P<0.05), intensity of insulin immunostaining and a decrease in the proportion of TUNEL-positive islet cells (+Ex: 7.5 ± 1.5%, P<0.05). Double insulin/TUNEL staining revealed that the majority of TUNEL-positive cells were beta cells. This decrease in the number of TUNEL-positive beta-cells in exenatide-treated islets was associated with a marked decrease in the activation of caspase-3 (34%). These findings suggest that short-term treatment with exenatide reduces beta-cell apoptosis in isolated human islets and decreases caspase-3 activation by a direct and/or indirect mechanism leading to an increase in the yield of islet betacells. Treatment of human islets with exenatide during pre-transplant culture period may have therapeutic value in clinical islet transplantation.
138
C&SS
NADPH : The Missing Link Between Glucose Metabolism and Insulin Secretion? ISLET BIOLOGY CLAUDIANE GUAY*, ÉRIK JOLY, MURTHY S. MADIRAJU, MARC PRENTKI, Montreal Diabetes Research Centre, Université de Montréal, Montréal, QC Patients with type 2 diabetes have inappropriate regulation of insulin secretion in response to glucose and other nutrients in pancreatic E-cells. To better understand this dysregulation, intensive work has been done to uncover factors coupling glucose metabolism to insulin secretion. Generation of metabolic coupling factors (MCF), such as ATP, malonyl-CoA and NADPH, by glucose metabolism have been suggested to regulate insulin secretion. In the present study, we hypothesized that malic enzyme (ME) and isocitrate dehydrogenase (IDH), the two main enzymes producing NADPH in E-cells, are implicated in the regulation of glucose-induced insulin secretion (GIIS). To test this hypothesis, we used RNAi-knockdown of ME and IDH. Transfection of INS 832/13 cells with shRNA directed against ME reduced ME activity by 62 % which was associated with a decrease in insulin secretion (33 % at 5 mM and 38 % at 10 mM glucose). siRNA directed against IDH decreased IDH mRNA level by 80 % and IDH activity by 50 % in INS 832/13 transfected cells. Experiments are currently performed to examine the effect of reducing IDH expression on GIIS. NADPH levels will also be measured in those conditions, to verify if ME and IDH are implicated in the regulation of GIIS via the production of NADPH. Those experiments are important not only to determine if ME and IDH are implicated in the regulation of GIIS, but also to underscore the role of MCF generated from glucose metabolism in the regulation of insulin secretion by the E-cells.
ABSTRACT #163 C&SS ISLET BIOLOGY