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Differential mechanisms of glucose and saturated fatty acids in augmentation of insulin secretion in mouse pancreatic islets
S146
Poster Session I
expressed and purified NBDl from E. coli. Purified NBDl was found to exist as a tetramer indicating strong homomeric attractions and a possible role of NBDl in SURl assembly.
P588 Differential Mechanisms of Glucose and Saturated Fatty Acids in Augmentation of Insulin Secretion in Mouse Pancreatic Islets PETER THAMS, Kirsten Capito. Department of Medical Biochemistry & Generics, University of Copenhagen,
Copenhagen, Denmark
PSf37 IAPP and Insulin Release from Human Pancwtic Islets - Indication of KAw-Independent IAPP Secretion ELLA KARLSSON, Stellan Sandier. Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
Background and aims: The amyloidogenic segment of human Islet amyloid polypeptide (IAPP) and its secretion, are prerequiesites for formation of pancreatic amyloid. It is unknown whether the secretion pattern of IAPP from human islets differs from that of rodent islets. The aim of this study was to examine the release of IAPP and insulin from human islets in re sponse to different agents. We also wanted to identify possible steps, at an early and late stage, during islet hormone release, at which, a dissociation of IAPP and insulin release might occur. Material and methods: Human and rat islets were cultured in medium RPMI 1640 (5.6 mM glucose) and RPM1 (11.1 mM glucose), respectively + 10% fetal calf serum, for 5 days prior to the experiment. Based on findings in rodent islets, incubation conditions known to either stimulate or inhibit secretion were chosen. The islets were exposed to 5 mM theophylline, 3 uM adrenaline, 10 mM mannoheptulose at 17 mM glucose, or 5 mM adrenaline at 5.6 mM glucose, or 10 nM leptin at 11 mM glucose, for 1 h. IAPP and insulin were measured by RIA. In other experiments, mannoheptulose of 5-50 mM was further studied to evaluate the importance of glucose phosphorylation for IAPP and insulin release, from human and rat islets. Islet peptide release via the KArr-channel independent pathway, was studied by opening of the KAm-channels with diazoxide while restoring the Ca*+ influx with a high extracellular K+ concentration. Results: The human islet response regarding IAPP and insulin release, was similar to some agents, e.g. the release ratio IAPP/insulin were unaltered after exposure to 5 mM theophylline and 3 uM adrenaline, (theophylline: 1.1% i 0.2%, adrenaline: 1.5% f 0.4% vs. control: 1.5% f 0.3%). n=9, whereas a dissociated release was seen after incubation with 5 mM arginine, as illustrated by an increased release ratio IAPP/insulin (arginine: 2.7% zt 0.6% vs. control: 1.5% + 0.3%). n=9. A decreased stimulated IAPP release ratio at 17/1.7 mM glucose, was seen in the presence of 10 nM leptin (leptin: 2.1% f 0.5% vs. control: 2.5%f 0.6%) n=4. A maximal 75%- inhibition, of IAPP release, was seen in rat islets at 5 mM mannoheptulose, whilst the human islets showed a maximal 40%-inhibition at 20 mM. The decrease in insulin release from human islets was of the same magnitude as that of IAPP, whereas rat islets showed a more marked inhibition (90%) of the insulin release after mannoheptulose treatment. Islet IAPP was shown to be released from human islets via both KATp-dependent and KATp-independent pathways, in response to 17 mM glucose, as illustrated by an abolished inhibition by diazoxide, of the glucose-stimulated islet IAPP release, when the extracellular K+ concentration was raised from 6 to 30 mM K+ (diazoxide at 6 mM K+: 175.0 f 33.3 fmoVug DNA/h; d&oxide at 30 mM K+: 668.6 f 55.7 fmol/ug DNA/h vs. control at 6 mM K+: 621.0 f 28.0 fmol/ug DNA/h), n=6. Rat islets behaved in a similar way. The present study demonstrates species differences in the regulation of IAPP and insulin secretion. In general, human islets were more resistant to the test substances than the rat islets. In rat islets, insulin release seemed to be more firmly associated to glucose phosphorylation than was the release of IAPP Islet IAPP was shown to be released from human beta cells via both K+-ATp-dependent and K+-A~-independent pathways, in response to high glucose.
Saturated fatty acids like pahnitate and myristate have been suggested to constitute the coupling factors in glucose amplification of K+*T~ channel-independent insulin secretion. In the present study, this proposal has been challenged by a comparison of the stimulatory potential of saturated fatty acids and glucose under different experimental conditions. In normal Krebs-Ringer medium, albumin-bound palmitate (165 WM total; 1.2 I.LMfree unbound) failed to induce insulin secretion at 3.3 mM glucose, but potentiated insulin secretion at 10 or 16.7 mM glucose. In the presence of K+ (20 mM) and diazoxide (250 bM), which stimulates Ca2+ influx and opens K+AT~channels, respectively, palmitate (165 NM total; 1.2 PM free) potentiated insulin secretion at 3.3, 10 and 16.7 mM glucose, whereas glucose (10; 16.7 mM) stimulation of insulin secretion was minimal. In the presence of K+ (60 mM) and d&oxide (250 PM), glucose (10; 16.7 mM) stimulation of K+ATpchannel-independent insulin secretion was amplified, whereas palmitate (165 PM total; 1.2 FM free) augmentation of insulin secretion at 3.3, 10 and 16.7 mM glucose was abrogated. In this way, palmitate mimicked the stimulatoty pattern of the protein kinase C activator 12-0-tetradecanoylphorbol13-acetate (0.16 PM), which also failed to potentiate insulin secretion at a maximum depolarizing concentration of K+ (60 mM). In accordance, the protein kinase C inhibitor, calphostin C (1 PM) led to a total elimination of both palmitate (165 PM total; 1.2 WM free) and myristate (165 PM total; 2.4 PM free) potentiation of glucose (16.7 mM)-induced insulin secretion. Calphostin C (1 PM), however, failed to affect insulin secretion induced by glucose (16.7 ml@. In conclusion, these data demonstrate that glucose augmentation of insulin secretion may occur independently of saturated fatty acids like palmitate and my&ate, which appear to potentiate glucose-induced insulin secretion by activation of protein kinase C.
P589 Lipotoxicity of the Pancreatic Beta-Cell Is Associated with a Glucose-Dependent, Substrate-Driven Increase in Esterilication of Fatty Acids into Neutral Lipids ISABELLE BRIAUD, Jamie S. Harmon, Cynthia L. Kelpe, Vengadesh B. Segu, Vincent Poitout. Pacific Nothwesf Research Institute, Seattle, WA, United States of America
Prolonged exposure of isolated islets to palmitate inhibits insulin gene expression only in the presence of elevated glucose concentrations. The aim of this study was to determine whether or not this was associated with increased esterification of fatty acids into neutral lipids. Expression of glycerol-3-phosphate-acyltransferase, diacylglycerol acyltransferase, and hormone-sensitive-lipase, 3 key enzymes in neutral lipid metabolism, was demonstrated in isolated rat islets by fluorescence-based RT-PCR. A 72-h exposure to 16.7 mM glucose, 0.5 mM palmitate, or both, did not affect mRNA levels for any of the 3 enzymes. Isolated rat islets were cultured for 72h with trace amounts of labeled palmitate with or without 05mM unlabeled palmitate at 2.8 or 16.7 mM glucose. In the presence of 16.7 mM glucose, the total number of counts incorporated from labeled palmitate into complex lipids (phospholipids (PL) + diacylglycerols (DAG) + triacylglycerols (TAG)) was increased 2.71 f 0.57 fold (p&01, n=14. In the presence of 0.5 mM unlabeled palmitate, the percentage of counts incorporated into DAG and TAG was increased at the expense of the counts incorporated into PL. This was associated with an increase in TAG content in the presence of 16.7 mM glucose (303 f 58 vs. 171 f 31 ng/islet, n=6, p=O.O3), as determined by mass assay. In HIT-T15 cells cultured for 72h in 11.1 mM glucose, the proportion of counts incorporated into TAG, negligible in the absence of palmitate (3.8 i. 0.7 % of the total, n=6), was significantly increased to 10.7 3t 0.9%
Poster Session I
expressed and purified NBDl from E. coli. Purified NBDl was found to exist as a tetramer indicating strong homomeric attractions and a possible role of NBDl in SURl assembly.
P588 Differential Mechanisms of Glucose and Saturated Fatty Acids in Augmentation of Insulin Secretion in Mouse Pancreatic Islets PETER THAMS, Kirsten Capito. Department of Medical Biochemistry & Generics, University of Copenhagen,
Copenhagen, Denmark
PSf37 IAPP and Insulin Release from Human Pancwtic Islets - Indication of KAw-Independent IAPP Secretion ELLA KARLSSON, Stellan Sandier. Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
Background and aims: The amyloidogenic segment of human Islet amyloid polypeptide (IAPP) and its secretion, are prerequiesites for formation of pancreatic amyloid. It is unknown whether the secretion pattern of IAPP from human islets differs from that of rodent islets. The aim of this study was to examine the release of IAPP and insulin from human islets in re sponse to different agents. We also wanted to identify possible steps, at an early and late stage, during islet hormone release, at which, a dissociation of IAPP and insulin release might occur. Material and methods: Human and rat islets were cultured in medium RPMI 1640 (5.6 mM glucose) and RPM1 (11.1 mM glucose), respectively + 10% fetal calf serum, for 5 days prior to the experiment. Based on findings in rodent islets, incubation conditions known to either stimulate or inhibit secretion were chosen. The islets were exposed to 5 mM theophylline, 3 uM adrenaline, 10 mM mannoheptulose at 17 mM glucose, or 5 mM adrenaline at 5.6 mM glucose, or 10 nM leptin at 11 mM glucose, for 1 h. IAPP and insulin were measured by RIA. In other experiments, mannoheptulose of 5-50 mM was further studied to evaluate the importance of glucose phosphorylation for IAPP and insulin release, from human and rat islets. Islet peptide release via the KArr-channel independent pathway, was studied by opening of the KAm-channels with diazoxide while restoring the Ca*+ influx with a high extracellular K+ concentration. Results: The human islet response regarding IAPP and insulin release, was similar to some agents, e.g. the release ratio IAPP/insulin were unaltered after exposure to 5 mM theophylline and 3 uM adrenaline, (theophylline: 1.1% i 0.2%, adrenaline: 1.5% f 0.4% vs. control: 1.5% f 0.3%). n=9, whereas a dissociated release was seen after incubation with 5 mM arginine, as illustrated by an increased release ratio IAPP/insulin (arginine: 2.7% zt 0.6% vs. control: 1.5% + 0.3%). n=9. A decreased stimulated IAPP release ratio at 17/1.7 mM glucose, was seen in the presence of 10 nM leptin (leptin: 2.1% f 0.5% vs. control: 2.5%f 0.6%) n=4. A maximal 75%- inhibition, of IAPP release, was seen in rat islets at 5 mM mannoheptulose, whilst the human islets showed a maximal 40%-inhibition at 20 mM. The decrease in insulin release from human islets was of the same magnitude as that of IAPP, whereas rat islets showed a more marked inhibition (90%) of the insulin release after mannoheptulose treatment. Islet IAPP was shown to be released from human islets via both KATp-dependent and KATp-independent pathways, in response to 17 mM glucose, as illustrated by an abolished inhibition by diazoxide, of the glucose-stimulated islet IAPP release, when the extracellular K+ concentration was raised from 6 to 30 mM K+ (diazoxide at 6 mM K+: 175.0 f 33.3 fmoVug DNA/h; d&oxide at 30 mM K+: 668.6 f 55.7 fmol/ug DNA/h vs. control at 6 mM K+: 621.0 f 28.0 fmol/ug DNA/h), n=6. Rat islets behaved in a similar way. The present study demonstrates species differences in the regulation of IAPP and insulin secretion. In general, human islets were more resistant to the test substances than the rat islets. In rat islets, insulin release seemed to be more firmly associated to glucose phosphorylation than was the release of IAPP Islet IAPP was shown to be released from human beta cells via both K+-ATp-dependent and K+-A~-independent pathways, in response to high glucose.
Saturated fatty acids like pahnitate and myristate have been suggested to constitute the coupling factors in glucose amplification of K+*T~ channel-independent insulin secretion. In the present study, this proposal has been challenged by a comparison of the stimulatory potential of saturated fatty acids and glucose under different experimental conditions. In normal Krebs-Ringer medium, albumin-bound palmitate (165 WM total; 1.2 I.LMfree unbound) failed to induce insulin secretion at 3.3 mM glucose, but potentiated insulin secretion at 10 or 16.7 mM glucose. In the presence of K+ (20 mM) and diazoxide (250 bM), which stimulates Ca2+ influx and opens K+AT~channels, respectively, palmitate (165 NM total; 1.2 PM free) potentiated insulin secretion at 3.3, 10 and 16.7 mM glucose, whereas glucose (10; 16.7 mM) stimulation of insulin secretion was minimal. In the presence of K+ (60 mM) and d&oxide (250 PM), glucose (10; 16.7 mM) stimulation of K+ATpchannel-independent insulin secretion was amplified, whereas palmitate (165 PM total; 1.2 FM free) augmentation of insulin secretion at 3.3, 10 and 16.7 mM glucose was abrogated. In this way, palmitate mimicked the stimulatoty pattern of the protein kinase C activator 12-0-tetradecanoylphorbol13-acetate (0.16 PM), which also failed to potentiate insulin secretion at a maximum depolarizing concentration of K+ (60 mM). In accordance, the protein kinase C inhibitor, calphostin C (1 PM) led to a total elimination of both palmitate (165 PM total; 1.2 WM free) and myristate (165 PM total; 2.4 PM free) potentiation of glucose (16.7 mM)-induced insulin secretion. Calphostin C (1 PM), however, failed to affect insulin secretion induced by glucose (16.7 ml@. In conclusion, these data demonstrate that glucose augmentation of insulin secretion may occur independently of saturated fatty acids like palmitate and my&ate, which appear to potentiate glucose-induced insulin secretion by activation of protein kinase C.
P589 Lipotoxicity of the Pancreatic Beta-Cell Is Associated with a Glucose-Dependent, Substrate-Driven Increase in Esterilication of Fatty Acids into Neutral Lipids ISABELLE BRIAUD, Jamie S. Harmon, Cynthia L. Kelpe, Vengadesh B. Segu, Vincent Poitout. Pacific Nothwesf Research Institute, Seattle, WA, United States of America
Prolonged exposure of isolated islets to palmitate inhibits insulin gene expression only in the presence of elevated glucose concentrations. The aim of this study was to determine whether or not this was associated with increased esterification of fatty acids into neutral lipids. Expression of glycerol-3-phosphate-acyltransferase, diacylglycerol acyltransferase, and hormone-sensitive-lipase, 3 key enzymes in neutral lipid metabolism, was demonstrated in isolated rat islets by fluorescence-based RT-PCR. A 72-h exposure to 16.7 mM glucose, 0.5 mM palmitate, or both, did not affect mRNA levels for any of the 3 enzymes. Isolated rat islets were cultured for 72h with trace amounts of labeled palmitate with or without 05mM unlabeled palmitate at 2.8 or 16.7 mM glucose. In the presence of 16.7 mM glucose, the total number of counts incorporated from labeled palmitate into complex lipids (phospholipids (PL) + diacylglycerols (DAG) + triacylglycerols (TAG)) was increased 2.71 f 0.57 fold (p&01, n=14. In the presence of 0.5 mM unlabeled palmitate, the percentage of counts incorporated into DAG and TAG was increased at the expense of the counts incorporated into PL. This was associated with an increase in TAG content in the presence of 16.7 mM glucose (303 f 58 vs. 171 f 31 ng/islet, n=6, p=O.O3), as determined by mass assay. In HIT-T15 cells cultured for 72h in 11.1 mM glucose, the proportion of counts incorporated into TAG, negligible in the absence of palmitate (3.8 i. 0.7 % of the total, n=6), was significantly increased to 10.7 3t 0.9%