Reactive Oxygen Species Stimulate Insulin Secretion in Rat Pancreatic Islets: Studies Using Mono-Oleoyl-Glycerol

Chronic exposure (24–72 hrs) of pancreatic islets to elevated glucose and fatty acid leads to glucolipoxicity characterized by basal insulin hypersecretion and impaired glucose-stimulated insulin secretion (GSIS). Our aim was to determine the mechanism for basal hypersecretion of insulin. We used mono-oleoyl-glycerol (MOG) as a tool to rapidly increase lipids in isolated rat pancreatic ß-cells and in the clonal pancreatic ß-cell line INS-1 832/13. MOG (25–400 µM) stimulated basal insulin secretion from ß-cells in a concentration dependent manner without increasing intracellular Ca2+ or O2 consumption. Like GSIS, MOG increased NAD(P)H and reactive oxygen species (ROS). The mitochondrial reductant ß-hydroxybutyrate (ß-OHB) also increased the redox state and ROS production, while ROS scavengers abrogated secretion. Diazoxide (0.4 mM) did not prevent the stimulatory effect of MOG, confirming that the effect was independent of the KATP-dependent pathway of secretion. MOG was metabolized to glycerol and long-chain acyl-CoA (LC-CoA), whereas, acute oleate did not similarly increase LC-CoA. Inhibition of diacylglycerol kinase (DGK) did not mimic the effect of MOG on insulin secretion, indicating that MOG did not act primarily by inhibiting DGK. Inhibition of acyl-CoA synthetase (ACS) reduced the stimulatory effect of MOG on basal insulin secretion by 30% indicating a role for LC-CoA. These data suggest that basal insulin secretion is stimulated by increased ROS production, due to an increase in the mitochondrial redox state independent of the established components of GSIS

Reactive Oxygen Species Stimulate Insulin Secretion in Rat Pancreatic Islets: Studies Using Mono-Oleoyl-Glycerol

Chronic exposure (24–72 hrs) of pancreatic islets to elevated glucose and fatty acid leads to glucolipoxicity characterized by basal insulin hypersecretion and impaired glucose-stimulated insulin secretion (GSIS). Our aim was to determine the mechanism for basal hypersecretion of insulin. We used mono-oleoyl-glycerol (MOG) as a tool to rapidly increase lipids in isolated rat pancreatic ß-cells and in the clonal pancreatic ß-cell line INS-1 832/13. MOG (25–400 µM) stimulated basal insulin secretion from ß-cells in a concentration dependent manner without increasing intracellular Ca2+ or O2 consumption. Like GSIS, MOG increased NAD(P)H and reactive oxygen species (ROS). The mitochondrial reductant ß-hydroxybutyrate (ß-OHB) also increased the redox state and ROS production, while ROS scavengers abrogated secretion. Diazoxide (0.4 mM) did not prevent the stimulatory effect of MOG, confirming that the effect was independent of the KATP-dependent pathway of secretion. MOG was metabolized to glycerol and long-chain acyl-CoA (LC-CoA), whereas, acute oleate did not similarly increase LC-CoA. Inhibition of diacylglycerol kinase (DGK) did not mimic the effect of MOG on insulin secretion, indicating that MOG did not act primarily by inhibiting DGK. Inhibition of acyl-CoA synthetase (ACS) reduced the stimulatory effect of MOG on basal insulin secretion by 30% indicating a role for LC-CoA. These data suggest that basal insulin secretion is stimulated by increased ROS production, due to an increase in the mitochondrial redox state independent of the established components of GSIS

Type 1 Diabetes Alters Lipid Handling and Metabolism in Human Fibroblasts and Peripheral Blood Mononuclear Cells

Triggers of the autoimmune response that leads to type 1 diabetes (T1D) remain poorly understood. A possibility is that parallel changes in both T cells and target cells provoke autoimmune attack. We previously documented greater Ca2+ transients in fibroblasts from T1D subjects than non-T1D after exposure to fatty acids (FA) and tumor necrosis factor α (TNFα). These data indicate that metabolic and signal transduction defects present in T1D can be elicited ex vivo in isolated cells. Changes that precede T1D, including inflammation, may activate atypical responses in people that are genetically predisposed to T1D. To identify such cellular differences in T1D, we quantified a panel of metabolic responses in fibroblasts and peripheral blood cells (PBMCs) from age-matched T1D and non-T1D subjects, as models for non-immune and immune cells, respectively. Fibroblasts from T1D subjects accumulated more lipid, had higher LC-CoA levels and converted more FA to CO2, with less mitochondrial proton leak in response to oleate alone or with TNFα, using the latter as a model of inflammation. T1D-PBMCs contained and also accumulated more lipid following FA exposure. In addition, they formed more peroxidized lipid than controls following FA exposure. We conclude that both immune and non-immune cells in T1D subjects differ from controls in terms of responses to FA and TNFα. Our results suggest a differential sensitivity to inflammatory insults and FA that may precede and contribute to T1D by priming both immune cells and their targets for autoimmune reactions

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 mu M) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action

Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation

Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes

Metabolic signals in the regulation of insulin release

In the present thesis, an attempt was made to elucidate the role of some of the glucose-induced intracellular metabolic effector signals in insulin secretion and to develop new applications that will enable further investigations of oscillatory signalling and secretion from pancreatic ß-cells. The sequence of early metabolic and ionic events was examined in clonal pancreatic insulin secreting cells (HIT) in response to glucose stimulation. It was determined that the earliest metabolic changes that occur in response to glucose were in pyridine and flavin nucleotides as well as in the ATP/ADP ratio. These changes were followed sequentially by increases in oxygen consumption, intracellular pH and cytoplasnidc free Ca2+ concentration ([Ca2+]i). The results support the hypothesis that metabolism is the driving force for changes in [Ca2+]i and not the reverse. The time sequence above does not include insulin secretion due to the difficulty in measuring this parameter in a cuvette system. Perifusion methods for studying oscillations in insulin secretion also prevent linking the secretory and metabolic events to each other, due to the fact that the cells are inaccessible in a chamber or column. By making use of multiwell culture plates, we have measured oscillations in secretion and the ATP/ADP ratio and have been able to demonstrate that the patterns of change in the ATP/ADP ratio and insulin release are similar. A comparison of the clonal pancreatic cell lines HIT and INS-1 showed a period of 4 min and 1.5 min, respectively, for oscillations in both secretion and the ATP/ADP ratio. The period of oscillations in [Ca2+]i was 5 min in HIT cells and 1.4 min in INS-1 cells. These data support the role of oscillations in the ATP/ADP ratio in pulsatile insulin release. Measurements of exocytosis can be accomplished by monitoring the release of serotonin from preloaded single ß-cells with carbon fibers using the amperometric technique. We show that preloading INS-1 cells with 3H-serotonin allows the detection of exocytosis from populations of both intact and permeabilized cells. Oscillations in insulin release correlated with oscillations in 3H-serotonin release from INS-1 cells. Insulin and 3H-serotonin were measured from the same cells, validating the use of serotonin as an indicator of insulin release. This technique allows for rapid on-line detemiination of exocytosis from cells loaded with the tritiated label. Permeabilized HIT cells were used to study the effects of long-chain acyl-CoA (LC-CoA) on Ca2+ handling and exocytosis. Acyl-CoA was shown to reduce the Ca2+ set point maintained by saponin permeabilized cells, exhibiting both dose and chain length dependencies. The effect was shown to be mediated by the endoplasmic reticulum (ER) Ca2+-ATPase, using inhibitors of mitochondlial Ca2+ uptake and the specific Ca2+-ATPase inhibitor, thapsigargin. In addition, the acyl- CoA effect was influenced by the ATP/ADP ratio, which is a regulator of the ER Ca2+-ATPase in these cells. This interaction with the ATP/ADP ratio suggested that the CoA moiety of the LC-CoA, which resembles ADP, acted to alleviate the inhibition of the ATPase by ADP, thereby increasing Ca2+ sequestration into the ER. We have shown that LC-CoA can acutely stimulate exocytosis from streptolysin-O permeabilized HIT cells. This effect is also dose and chain length dependent, with longer chain lengths being most effective. The effect is not mediated by classical protein kinase C (cPKC) isoforms and seems not to be dependent on the free Ca2+ concentration. LC-CoA simulates exocytosis in the absence of ATP, suggesting that the effect on insulin release comes after ATP dependent docking of secretory granules to the plasma membrane. cPKC activation has been shown to stimulate glucose-induced insulin release. The translocation of PKC to the plasma membrane has been shown, by some but not others, to be stimulated by glucose. The fact that some investigators have failed to observe such a glucose-induced translocation of CPKC seems to lie in the extraction procedures for assessing translocation of the enzyme. We show that upon cell disruption, cPKC translocation is Ca2+-dependent and reversible. Diacylglycerol and phorbol 12-myristate 13-acetate (TPA,) activators of cPKC, stimulate glucose-induced insulin release with different time courses. The activation of glucose-induced insulin release continued for hours after removal of TPA from the media, suggesting the existence of a long lasting phosphorylation event. A dissociation between the changes in [Ca2+]i and insulin secretion was shown subsequent to stimulation with TPA. Under these conditions, the amplitude of the oscillations in secretion were stimulated two-fold with no major effect on [Ca2+]i. It is well accepted that glucose-induced insulin release is coupled to signals generated by glucose metabolism. In many cases the effects of these signals are not completely understood. The studies presented here were designed to increase our knowledge of how some of these signals affect the insulin secretory process

Ca2+, NAD(P)H and membrane potential changes in pancreatic beta-cells by methyl succinate: comparison with glucose

The present study was undertaken to determine the main metabolic secretory signals generated by the mitochondrial substrate MeS (methyl succinate) compared with glucose in mouse and rat islets and to understand the differences. Glycolysis and mitochondrial metabolism both have key roles in the stimulation of insulin secretion by glucose. Both fuels elicited comparable oscillatory patterns of Ca2+ and changes in plasma and mitochondrial membrane potential in rat islet cells and clonal pancreatic beta-cells (INS-1). Saturation of the Ca2+ signal occurred between 5 and 6 mM MeS, while secretion reached its maximum at 15 mM, suggesting operation of a K(ATP)-channel-independent pathway. Additional responses to MeS and glucose included elevated NAD(P)H autofluorescence in INS-1 cells and islets and increases in assayed NADH and NADPH and the ATP/ADP ratio. Increased NADPH and ATP/ADP ratios occurred more rapidly with MeS, although similar levels were reached after 5 min of exposure to each fuel, whereas NADH increased more with MeS than with glucose. Reversal of MeS-induced cell depolarization by Methylene Blue completely inhibited MeS-stimulated secretion, whereas basal secretion and KCl-induced changes in these parameters were not affected. MeS had no effect on secretion or signals in the mouse islets, in contrast with glucose, possibly due to a lack of malic enzyme. The data are consistent with the common intermediates being pyruvate, cytosolic NADPH or both, and suggest that cytosolic NADPH production could account for the more rapid onset of MeS-induced secretion compared with glucose stimulatio

Direct Stimulation of Islet Insulin Secretion by Glycolytic and Mitochondrial Metabolites in KCl-Depolarized Islets.

We have previously demonstrated that islet depolarization with 70 mM KCl opens Cx36 hemichannels and allows diffusion of small metabolites and cofactors through the β-cell plasma membrane. We have investigated in this islet "permeabilized" model whether glycolytic and citric acid cycle intermediates stimulate insulin secretion and how it correlates with ATP production (islet content plus extracellular nucleotide accumulation). Glycolytic intermediates (10 mM) stimulated insulin secretion and ATP production similarly. However, they showed differential sensitivities to respiratory chain or enzyme inhibitors. Pyruvate showed a lower secretory capacity and less ATP production than phosphoenolpyruvate, implicating an important role for glycolytic generation of ATP. ATP production by glucose-6-phosphate was not sensitive to a pyruvate kinase inhibitor that effectively suppressed the phosphoenolpyruvate-induced secretory response and islet ATP rise. Strong suppression of both insulin secretion and ATP production induced by glucose-6-phosphate was caused by 10 μM antimycin A, implicating an important role for the glycerophosphate shuttle in transferring reducing equivalents to the mitochondria. Five citric acid cycle intermediates were investigated for their secretory and ATP production capacity (succinate, fumarate, malate, isocitrate and α-ketoglutarate at 5 mM, together with ADP and/or NADP+ to feed the NADPH re-oxidation cycles). The magnitude of the secretory response was very similar among the different mitochondrial metabolites but α-ketoglutarate showed a more sustained second phase of secretion. Gabaculine (1 mM, a GABA-transaminase inhibitor) suppressed the second phase of secretion and the ATP-production stimulated by α-ketoglutarate, supporting a role for the GABA shuttle in the control of glucose-induced insulin secretion. None of the other citric acid intermediates essayed showed any suppression of both insulin secretion or ATP-production by the presence of gabaculine. We propose that endogenous GABA metabolism in the "GABA-shunt" facilitates ATP production in the citric acid cycle for an optimal insulin secretion

Inborn Errors of Long-Chain Fatty Acid β-Oxidation Link Neural Stem Cell Self-Renewal to Autism

Summary: Inborn errors of metabolism (IEMs) occur with high incidence in human populations. Especially prevalent among these are inborn deficiencies in fatty acid β-oxidation (FAO), which are clinically associated with developmental neuropsychiatric disorders, including autism. We now report that neural stem cell (NSC)-autonomous insufficiencies in the activity of TMLHE (an autism risk factor that supports long-chain FAO by catalyzing carnitine biosynthesis), of CPT1A (an enzyme required for long-chain FAO transport into mitochondria), or of fatty acid mobilization from lipid droplets reduced NSC pools in the mouse embryonic neocortex. Lineage tracing experiments demonstrated that reduced flux through the FAO pathway potentiated NSC symmetric differentiating divisions at the expense of self-renewing stem cell division modes. The collective data reveal a key role for FAO in controlling NSC-to-IPC transition in the mammalian embryonic brain and suggest NSC self renewal as a cellular mechanism underlying the association between IEMs and autism. : The mechanisms underlying the association between inborn errors of fatty acid metabolism and developmental brain disorders such as autism remain unclear. Xie et al. find that TMLHE, a carnitine biosynthesis enzyme, and carnitine-dependent long-chain fatty acid β-oxidation control the neural stem cell pool during neocortical development by maintaining self-renewing divisions

3H-serotonin as a marker of oscillatory insulin secretion in clonal β-cells (INS-1)

Serotonin release from preloaded pancreatic β-cells has been used as a marker for insulin release in studying exocytosis from single cells using the amperometric technique. We found that single pancreatic β-cells exhibited oscillations in exocytosis with a period of 1–1.5 min as measured amperometrically by serotonin release. We also show that 3H-serotonin can be used to monitor exocytosis from intact and streptolysin-O permeabilized clonal insulin-secreting cells preloaded with labeled serotonin and that serotonin release correlated with insulin secretion in the same cells. The use of 3H-serotonin provides a real-time indicator of exocytosis from populations of clonal insulin-secreting cells