Glucagon-Like Peptide-1 Induced Signaling and Insulin Secretion Do Not Drive Fuel and Energy Metabolism in Primary Rodent Pancreatic β-Cells

Background

Glucagon like peptide-1 (GLP-1) and its analogue exendin-4 (Ex-4) enhance glucose stimulated insulin secretion (GSIS) and activate various signaling pathways in pancreatic β-cells, in particular cAMP, Ca²⁺ and protein kinase-B (PKB/Akt). In many cells these signals activate intermediary metabolism. However, it is not clear whether the acute amplification of GSIS by GLP-1 involves in part metabolic alterations and the production of metabolic coupling factors.

Methodology/Prinicipal Findings

GLP-1 or Ex-4 at high glucose caused release (∼20%) of the total rat islet insulin content over 1 h. While both GLP-1 and Ex-4 markedly potentiated GSIS in isolated rat and mouse islets, neither had an effect on β-cell fuel and energy metabolism over a 5 min to 3 h time period. GLP-1 activated PKB without changing glucose usage and oxidation, fatty acid oxidation, lipolysis or esterification into various lipids in rat islets. Ex-4 caused a rise in [Ca²⁺]_i and cAMP but did not enhance energy utilization, as neither oxygen consumption nor mitochondrial ATP levels were altered.

Conclusions/Significance

The results indicate that GLP-1 barely affects β-cell intermediary metabolism and that metabolic signaling does not significantly contribute to GLP-1 potentiation of GSIS. The data also indicate that insulin secretion is a minor energy consuming process in the β-cell, and that the β-cell is different from most cell types in that its metabolic activation appears to be primarily governed by a “push” (fuel substrate driven) process, rather than a “pull” mechanism secondary to enhanced insulin release as well as to Ca²⁺, cAMP and PKB signaling.     

Inhibition of Monoacylglycerol Lipase Activity Decreases Glucose-Stimulated Insulin Secretion in INS-1 (832/13) Cells and Rat Islets

Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca²⁺ to glucose and depolarization but to a lesser degree suggesting that altered Ca²⁺ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.     

Glucose Stimulation Induces Dynamic Change of Mitochondrial Morphology to Promote Insulin Secretion in the Insulinoma Cell Line INS-1E

Fission and fusion of mitochondrial tubules are the major processes regulating mitochondrial morphology. However, the physiological significance of mitochondrial shape change is poorly understood. Glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells requires mitochondrial ATP production which evokes Ca²⁺ influx through plasma membrane depolarization, triggering insulin vesicle exocytosis. Therefore, GSIS reflects mitochondrial function and can be used for evaluating functional changes associated with morphological alterations of mitochondria. Using the insulin-secreting cell line INS-1E, we found that glucose stimulation induced rapid mitochondrial shortening and recovery. Inhibition of mitochondrial fission through expression of the dominant-negative mutant DLP1-K38A eliminated this dynamic mitochondrial shape change and, importantly, blocked GSIS. We found that abolishing mitochondrial morphology change in glucose stimulation increased the mitochondrial inner membrane proton leak, and thus significantly diminished the mitochondrial ATP producing capacity in response to glucose stimulation. These results demonstrate that dynamic change of mitochondrial morphology is a previously unrecognized component for metabolism-secretion coupling of pancreatic β-cells by participating in efficient ATP production in response to elevated glucose levels.     

Mitochondrial GTP regulates glucose-stimulated insulin secretion

Nucleotide-specific isoforms of the tricarboxylic acid (TCA) cycle enzyme succinyl-CoA synthetase (SCS) catalyze substrate-level synthesis of mitochondrial GTP (mtGTP) and ATP (mtATP). While mtATP yield from glucose metabolism is coupled with oxidative phosphorylation and can vary, each molecule of glucose metabolized within pancreatic beta cells produces approximately one mtGTP, making mtGTP a potentially important fuel signal. In INS-1 832/13 cells and cultured rat islets, siRNA suppression of the GTP-producing pathway (DeltaSCS-GTP) reduced glucose-stimulated insulin secretion (GSIS) by 50%, while suppression of the ATP-producing isoform (DeltaSCS-ATP) increased GSIS 2-fold. Insulin secretion correlated with increases in cytosolic calcium, but not with changes in NAD(P)H or the ATP/ADP ratio. These data suggest a role for mtGTP in controlling pancreatic GSIS through modulation of mitochondrial metabolism, possibly involving mitochondrial calcium. Furthermore, in light of its tight coupling to TCA oxidation rates, mtGTP production may serve as an important molecular signal of TCA-cycle activity.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.     

Chronic suppression of acetyl-CoA carboxylase 1 in beta-cells impairs insulin secretion via inhibition of glucose rather than lipid metabolism

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.     

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic islet β-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic α-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic β-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP⁺ ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.     

The mitochondrial citrate/isocitrate carrier plays a regulatory role in glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control 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 K_ATP-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.     

SAD-A Potentiates Glucose-Stimulated Insulin Secretion as a Mediator of Glucagon-Like Peptide 1 Response in Pancreatic β Cells

Type 2 diabetes is characterized by defective glucose-stimulated insulin secretion (GSIS) from pancreatic β cells, which can be restored by glucagon-like peptide 1 (GLP-1), an incretin hormone commonly used for the treatment of type 2 diabetes. However, molecular mechanisms by which GLP-1 affects glucose responsiveness in islet β cells remain poorly understood. Here we investigated a role of SAD-A, an AMP-activated protein kinase (AMPK)-related kinase, in regulating GSIS in mice with conditional SAD-A deletion. We show that selective deletion of SAD-A in pancreas impaired incretin''s effect on GSIS, leading to glucose intolerance. Conversely, overexpression of SAD-A significantly enhanced GSIS and further potentiated GLP-1''s effect on GSIS from isolated mouse islets. In support of SAD-A as a mediator of incretin response, SAD-A is expressed exclusively in pancreas and brain, the primary targeting tissues of GLP-1 action. Additionally, SAD-A kinase is activated in response to stimulation by GLP-1 through cyclic AMP (cAMP)/Ca²⁺-dependent signaling pathways in islet β cells. Furthermore, we identified Thr443 as a key autoinhibitory phosphorylation site which mediates SAD-A''s effect on incretin response in islet β cells. Consequently, ablation of Thr443 significantly enhanced GLP-1''s effect on GSIS from isolated mouse islets. Together, these findings identified SAD-A kinase as a pancreas-specific mediator of incretin response in islet β cells.     

Mitochondrial Metabolism of Pyruvate Is Essential for Regulating Glucose-stimulated Insulin Secretion

It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (K_ATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP⁺ ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.     

Glucose Regulates Steady-state Levels of PDX1 via the Reciprocal Actions of GSK3 and AKT Kinases

The pancreatic beta cell is sensitive to even small changes in PDX1 protein levels; consequently, Pdx1 haploinsufficiency can inhibit beta cell growth and decrease insulin biosynthesis and gene expression, leading to compromised glucose-stimulated insulin secretion. Using metabolic labeling of primary islets and a cultured β cell line, we show that glucose levels modulate PDX1 protein phosphorylation at a novel C-terminal GSK3 consensus that maps to serines 268 and 272. A decrease in glucose levels triggers increased turnover of the PDX1 protein in a GSK3-dependent manner, such that PDX1 phosphomutants are refractory to the destabilizing effect of low glucose. Glucose-stimulated activation of AKT and inhibition of GSK3 decrease PDX1 phosphorylation and delay degradation. Furthermore, direct pharmacologic inhibition of AKT destabilizes, and inhibition of GSK3 increases PDX1 protein stability. These studies define a novel functional role for the PDX1 C terminus in mediating the effects of glucose and demonstrate that glucose modulates PDX1 stability via the AKT-GSK3 axis.     

Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects

The tumor suppressor liver kinase B1 (LKB1) is an important regulator of pancreatic β cell biology. LKB1-dependent phosphorylation of distinct AMPK (adenosine monophosphate-activated protein kinase) family members determines proper β cell polarity and restricts β cell size, total β cell mass, and glucose-stimulated insulin secretion (GSIS). However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood. We report here that in the absence of LKB1 in β cells, GSIS is dramatically and persistently improved. The enhancement is seen both in vivo and in vitro and cannot be explained by altered cell polarity, increased β cell number, or increased insulin content. Increased secretion does require membrane depolarization and calcium influx but appears to rely mostly on a distal step in the secretion pathway. Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway. Thus LKB1 is essential for mitochondrial homeostasis in β cells and in parallel is a powerful negative regulator of insulin secretion. This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.     

Mathematical Model of Metabolism and Electrophysiology of Amino Acid and Glucose Stimulated Insulin Secretion: In Vitro Validation Using a β-Cell Line

We integrated biological experimental data with mathematical modelling to gain insights into the role played by L-alanine in amino acid-stimulated insulin secretion (AASIS) and in D-glucose-stimulated insulin secretion (GSIS), details important to the understanding of complex β-cell metabolic coupling relationships. We present an ordinary differential equations (ODEs) based simplified kinetic model of core metabolic processes leading to ATP production (glycolysis, TCA cycle, L-alanine-specific reactions, respiratory chain, ATPase and proton leak) and Ca²⁺ handling (essential channels and pumps in the plasma membrane) in pancreatic β-cells and relate these to insulin secretion. Experimental work was performed using a clonal rat insulin-secreting cell line (BRIN-BD11) to measure the consumption or production of a range of important biochemical parameters (D-glucose, L-alanine, ATP, insulin secretion) and Ca²⁺ levels. These measurements were then used to validate the theoretical model and fine-tune the parameters. Mathematical modelling was used to predict L-lactate and L-glutamate concentrations following D-glucose and/or L-alanine challenge and Ca²⁺ levels upon stimulation with a non metabolizable L-alanine analogue. Experimental data and mathematical model simulations combined suggest that L-alanine produces a potent insulinotropic effect via both a stimulatory impact on β-cell metabolism and as a direct result of the membrane depolarization due to Ca²⁺ influx triggered by L-alanine/Na⁺ co-transport. Our simulations indicate that both high intracellular ATP and Ca²⁺ concentrations are required in order to develop full insulin secretory responses. The model confirmed that K⁺ _ATP channel independent mechanisms of stimulation of intracellular Ca²⁺ levels, via generation of mitochondrial coupling messengers, are essential for promotion of the full and sustained insulin secretion response in β-cells.     

Essential Role of Mitochondrial Ca2+ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells

In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca²⁺ influx, which triggers insulin exocytosis. The mitochondrial Ca²⁺ uniporter (MCU) mediates Ca²⁺ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca²⁺ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca²⁺. Suppression of the putative Ca²⁺/H⁺ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca²⁺-induced matrix acidification. These results demonstrate that MCU-mediated Ca²⁺ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.     

Extracellular ATP and zinc are co-secreted with insulin and activate multiple P2X purinergic receptor channels expressed by islet beta-cells to potentiate insulin secretion

It is well established that ATP is co-secreted with insulin and zinc from pancreatic beta-cells (β-cells) in response to elevations in extracellular glucose concentration. Despite this knowledge, the physiological roles of extracellular secreted ATP and zinc are ill-defined. We hypothesized that secreted ATP and zinc are autocrine purinergic signaling molecules that activate P2X purinergic receptor (P2XR) channels expressed by β-cells to enhance glucose-stimulated insulin secretion (GSIS). To test this postulate, we performed ELISA assays for secreted insulin at fixed time points within a “real-time” assay and confirmed that the physiological insulin secretagogue glucose stimulates secretion of ATP and zinc into the extracellular milieu along with insulin from primary rat islets. Exogenous ATP and zinc alone or together also induced insulin secretion in this model system. Most importantly, the presence of an extracellular ATP scavenger, a zinc chelator, and P2 receptor antagonists attenuated GSIS. Furthermore, mRNA and protein were expressed in immortalized β-cells and primary islets for a unique subset of P2XR channel subtypes, P2X₂, P2X₃, P2X₄, and P2X₆, which are each gated by extracellular ATP and modulated positively by extracellular zinc. On the basis of these results, we propose that, within endocrine pancreatic islets, secreted ATP and zinc have profound autocrine regulatory influence on insulin secretion via ATP-gated and zinc-modulated P2XR channels.