Compensatory responses to pyruvate carboxylase suppression in islet beta-cells. Preservation of glucose-stimulated insulin secretion

We have previously reported that glucose-stimulated insulin secretion (GSIS) is tightly correlated with pyruvate carboxylase (PC)-catalyzed anaplerotic flux into the tricarboxylic acid cycle and stimulation of pyruvate cycling activity. To further evaluate the role of PC in beta-cell function, we constructed a recombinant adenovirus containing a small interfering RNA (siRNA) specific to PC (Ad-siPC). Ad-siPC reduced PC mRNA levels by 83 and 64% and PC protein by 56 and 35% in INS-1-derived 832/13 cells and primary rat islets, respectively. Surprisingly, this manipulation did not impair GSIS in rat islets. In Ad-siPC-treated 832/13 cells, GSIS was slightly increased, whereas glycolytic rate and glucose oxidation were unaffected. Flux through PC at high glucose was decreased by only 20%, suggesting an increase in PC-specific activity. Acetyl carnitine, a surrogate for acetyl-CoA, an allosteric activator of PC, was increased by 36% in Ad-siPC-treated cells, suggesting a mechanism by which PC enzymatic activity is maintained with suppressed PC protein levels. In addition, the NADPH:NADP ratio, a proposed coupling factor for GSIS, was unaffected in Ad-siPC-treated cells. We conclude that beta-cells activate compensatory mechanisms in response to suppression of PC expression that prevent impairment of anaplerosis, pyruvate cycling, NAPDH production, and GSIS.     

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.     

Biochemical mechanism of lipid-induced impairment of glucose-stimulated insulin secretion and reversal with a malate analogue

Hyperlipidemia appears to play an integral role in loss of glucose-stimulated insulin secretion (GSIS) in type 2 diabetes. This impairment can be simulated in vitro by chronic culture of 832/13 insulinoma cells with high concentrations of free fatty acids, or by study of lipid-laden islets from Zucker diabetic fatty rats. Here we show that impaired GSIS is not a simple result of saturation of lipid storage pathways, as adenovirus-mediated overexpression of a cytosolically localized variant of malonyl-CoA decarboxylase in either cellular model results in dramatic lowering of cellular triglyceride stores but no improvement in GSIS. Instead, the glucose-induced increment in "pyruvate cycling" activity (pyruvate exchange with tricarboxylic acid cycle intermediates measured by (13)C NMR), previously shown to play an important role in GSIS, is completely ablated in concert with profound suppression of GSIS in lipid-cultured 832/13 cells, whereas glucose oxidation is unaffected. Moreover, GSIS is partially restored in both lipid-cultured 832/13 cells and islets from Zucker diabetic fatty rats by addition of a membrane permeant ester of a pyruvate cycling intermediate (dimethyl malate). We conclude that chronic exposure of islet beta-cells to fatty acids grossly alters a mitochondrial pathway of pyruvate metabolism that is important for normal GSIS.     

Stevioside improves pancreatic beta-cell function during glucotoxicity via regulation of acetyl-CoA carboxylase

Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity influences ACC and whether this action can be counteracted by SVS are not known. To investigate this, we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose, respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid (OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5-Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in beta-cells via a unique mechanism of action.     

Normal flux through ATP-citrate lyase or fatty acid synthase is not required for glucose-stimulated insulin secretion

It has been proposed that de novo synthesis of long-chain acyl-CoA (LC-CoA) is a signal for glucose-stimulated insulin secretion (GSIS). Key enzymes involved in synthesis of fatty acids from glucose include ATP-citrate lyase (CL) and fatty acid synthase (FAS). An inhibitor of CL, hydroxycitrate (HC), has been reported to inhibit insulin secretion in some laboratories but not in others. Here we show that high concentrations of NaCl created during preparation of HC by standard methods explain the inhibition of GSIS, and that removal of the excess NaCl prevents the effect. To further investigate the role of CL, two small interfering RNA adenoviruses (Ad-siCL2 and Ad-siCL3) were generated. Ad-siCL3 reduced CL mRNA levels by 92 +/- 6% and CL protein levels by 75 +/- 4% but did not affect GSIS in 832/13 cells compared with cells treated with a control adenovirus (Ad-siControl). Similar results were obtained with Ad-siCL2. Ad-siCL3-treated cells also exhibited a 52 +/- 7% reduction in cytosolic oxaloacetate, an 83 +/- 4% reduction in malonyl-CoA levels, and inhibition of [U-(14)C]glucose incorporation into lipid by 43 +/- 4%, all expected metabolic out-comes of CL suppression. Similarly, treatment of 832/13 cells with a recombinant adenovirus specific to FAS (Ad-siFAS) reduced FAS mRNA levels by 81 +/- 2% in 832/13 cells, resulting in a 59 +/- 4% decrease in [U-(14)C]glucose incorporation into lipid, without affecting GSIS. Finally, treatment of primary rat islets with Ad-siCL3 or Ad-siFAS reduced CL and FAS mRNA levels by 65 +/- 4% and 52 +/- 3%, respectively, but had no effect on GSIS relative to Ad-siControl-treated islets. These findings demonstrate that a normal rate of flux of glucose carbons through CL and FAS is not required for GSIS in insulinoma cell lines or rat islets.     

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.     

The role of rapid lipogenesis in insulin secretion: Insulin secretagogues acutely alter lipid composition of INS-1 832/13 cells

Pancreatic beta cell mitochondria convert insulin secretagogues into products that support insulin exocytosis. We explored the idea that lipids are some of these products formed from acyl group transfer out of mitochondria to the cytosol, the site of lipid synthesis. There are two isoforms of acetyl-CoA carboxylase, the enzyme that forms malonyl-CoA from which C₂ units for lipid synthesis are formed. We found that ACC1, the isoform seen in lipogenic tissues, is the only isoform present in human and rat pancreatic islets and INS-1 832/13 cells. Inhibitors of ACC and fatty acid synthase inhibited insulin release in islets and INS-1 cells. Carbon from glucose and pyruvate were rapidly incorporated into many lipid classes in INS-1 cells. Glucose and other insulin secretagogues acutely increased many lipids with C₁₄-C₂₄ chains including individual cholesterol esters, phospholipids and fatty acids. Many phosphatidylcholines and phosphatidylserines were increased and many phosphatidylinositols and several phosphatidylethanolamines were decreased. The results suggest that lipid remodeling and rapid lipogenesis from secretagogue carbon support insulin secretion.     

Diminished Acyl-CoA Synthetase Isoform 4 Activity in INS 832/13 Cells Reduces Cellular Epoxyeicosatrienoic Acid Levels and Results in Impaired Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells is potentiated by fatty acids (FA). The initial step in the metabolism of intracellular FA is the conversion to acyl-CoA by long chain acyl-CoA synthetases (Acsls). Because the predominantly expressed Acsl isoforms in INS 832/13 cells are Acsl4 and -5, we characterized the role of these Acsls in beta-cell function by using siRNA to knock down Acsl4 or Acsl5. Compared with control cells, an 80% suppression of Acsl4 decreased GSIS and FA-potentiated GSIS by 32 and 54%, respectively. Knockdown of Acsl5 did not alter GSIS. Acsl4 knockdown did not alter FA oxidation or long chain acyl-CoA levels. With Acsl4 knockdown, incubation with 17 mm glucose increased media epoxyeicosatrienoic acids (EETs) and reduced cell membrane levels of EETs. Further, exogenous EETs reduced GSIS in INS 832/13 cells, and in Acsl4 knockdown cells, an EET receptor antagonist partially rescued GSIS. These results strongly suggest that Acsl4 activates EETs to form EET-CoAs that are incorporated into glycerophospholipids, thereby sequestering EETs. Exposing INS 832/13 cells to arachidonate or linoleate reduced Acsl4 mRNA and protein expression and reduced GSIS. These data indicate that Acsl4 modulates GSIS by regulating the levels of unesterified EETs and that arachidonate controls the expression of its activator Acsl4.     

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.     

Inhibition of fatty acid translocase cluster determinant 36 (CD36), stimulated by hyperglycemia, prevents glucotoxicity in INS-1 cells

The purpose of the present study was to determine whether exposure of pancreatic islets to glucotoxic conditions changes fatty acid translocase cluster determinant 36 (CD36) and examine the role of CD36 on the induction of glucotoxicity. We measured the changes of CD36 and insulin secretion in high glucose (30 mM) exposed INS-1 cells and CD36 suppressed INS-1 cells by transfection of CD36 siRNA. The intracellular peroxide level of INS-1 cells increased in the high glucose media compared to normal glucose (5.6mM) media. The mRNA levels of insulin and PDX-1, as well as glucose stimulated insulin secretion (GSIS) were decreased in INS-1 cells exposed to high glucose media compared to normal glucose media, while CD36 and palmitate uptake were significantly elevated with exposure to high glucose media for 12h. The inhibition of CD36 reversed the decreased GSIS and intracellular peroxide level in INS-1 cells. These results suggest that high glucose may exacerbate glucotoxicity via increasing fatty acid influx by elevation of CD36 expression, and that CD36 may be a possible target molecule for preventing glucotoxicity in pancreatic beta-cells.     

Phosphoenolpyruvate Cycling via Mitochondrial Phosphoenolpyruvate Carboxykinase Links Anaplerosis and Mitochondrial GTP with Insulin Secretion

Pancreatic β-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical K_ATP-dependent glucose-stimulated insulin secretion (GSIS), there are important K_ATP-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel ¹³C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling.β-Cells in pancreatic islets of Langerhans make and release insulin in response to changes in blood glucose levels. The mechanisms by which high concentrations of glucose stimulate insulin release from islets remain unclear. The canonical explanation for GSIS² is that glucose metabolism increases mitochondrial ATP production, thereby raising the cytosolic ATP:ADP ratio that triggers the closure of ATP-sensitive K⁺ channels. This, in turn, depolarizes the membrane and stimulates the opening of voltage-dependent Ca²⁺ channels with increased Ca²⁺ influx promoting the exocytosis of insulin. Although K_ATP channels certainly have an important role in β-cells, K_ATP-independent signals are implicated to play a fundamental role in GSIS. In particular, β-cells are known to have notably elevated rates of anaplerotic flux of the carbon from glucose into the mitochondria and back out to pyruvate (pyruvate cycling) that is tightly correlated with insulin secretion (1–4).Recently, mtGTP synthesis was identified as a novel K_ATP-independent mitochondrial signal for insulin secretion (5). mtGTP is synthesized as a product of glucose metabolism by the GTP-specific isoform of the matrix enzyme SCS. mtGTP synthetic rates are determined by the rate of TCA cycle flux as well as by the ratio of activities of the ATP-specific and GTP-specific isoforms of SCS. The mtGTP signal is trapped within the matrix of the mitochondria, suggesting that another GTPase in the matrix transmits the mtGTP signal to the cytosol. Because both mtGTP synthesis and anaplerotic flux correlate with insulin secretion, we investigated whether the GTP-dependent mitochondrial isoform of PEPCK, an enzyme that lies at the intersection of anaplerosis and mtGTP metabolism (see Fig. 1A), is important for GSIS.Open in a separate windowFIGURE 1.PEP cycle. PEP is produced during glycolysis and is further metabolized to pyruvate by PK. Pyruvate that enters the TCA cycle by pyruvate dehydrogenase will generate GTP via direct synthesis by SCS-GTP. Anaplerotic pyruvate entry by PC will generate oxaloacetate. PEPCK-M will then consume oxaloacetate and GTP to produce PEP, GDP, and CO₂. PEP is then transported out of the mitochondrial matrix by an anion transporter (Ex) in exchange for another metabolite depending on the transporter. Mitochondrial PEP, thus, contributes to the PEP pool that is determined by the rate of appearance (ν_PEPCK-M+1+ ν_Glyc+1) of PEP minus the rate of disappearance (ν_PK+1). One turn of the PEP cycle will result in the net exchange of one ion into the mitochondrial matrix. Unidirectional fluxes are indicated by ν followed by the enzyme with the forward direction being +1 and the reverse −1. GDP in turn can be reused by SCS-GTP. PDH, pyruvate dehydrogenase.     

A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in beta-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.     

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.     

Overexpression of a modified human malonyl-CoA decarboxylase blocks the glucose-induced increase in malonyl-CoA level but has no impact on insulin secretion in INS-1-derived (832/13) beta-cells

The long-chain acyl-CoA (LC-CoA) model of glucose-stimulated insulin secretion (GSIS) holds that secretion is linked to a glucose-induced increase in malonyl-CoA level and accumulation of LC-CoA in the cytosol. We have previously tested the validity of this proposal by overexpressing goose malonyl-CoA decarboxylase (MCD) in INS-1 cells, but these studies have been criticized due to: 1) the small insulin secretion response (2-4-fold) of the INS-1 cells used; 2) unknown contribution of the ATP-sensitive K(+) (K(ATP)) channel-independent pathway of GSIS in INS-1 cells, which has been implicated as the site at which lipids regulate insulin granule exocytosis; and 3) deletion of the N-terminal mitochondrial targeting sequence, but not the C-terminal peroxisomal targeting sequence in the goose MCD construct, raising the possibility that a significant fraction of the overexpressed enzyme was localized to peroxisomes. To address these outstanding concerns, INS-1-derived 832/13 cells, which exhibit robust K(ATP) channel-dependent and -independent pathways of GSIS, were treated with a new adenovirus encoding human MCD lacking both its mitochondrial and peroxisomal targeting sequences (AdCMV-MCD Delta 5), resulting in large increases in cytosolic MCD activity. Treatment of 832/13 cells with AdCMV-MCD Delta 5 completely blocked the glucose-induced rise in malonyl-CoA and attenuated the inhibitory effect of glucose on fatty acid oxidation. However, MCD overexpression had no effect on K(ATP) channel-dependent or -independent GSIS in 832/13 cells. Furthermore, combined treatment of 832/13 cells with AdCMV-MCD Delta 5 and triacsin C, an inhibitor of long chain acyl-CoA synthetase that reduces LC-CoA levels, did not impair GSIS. These findings extend our previous observations and are not consistent with the LC-CoA hypothesis as originally set forth.     

Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.     

In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi

Pancreatic β-cells secrete insulin, which controls blood glucose levels, and defects in insulin secretion are responsible for diabetes mellitus. The actin cytoskeleton and some myosins support insulin granule trafficking and release, although a role for the class I myosin Myo1b, an actin- and membrane-associated load-sensitive motor, in insulin biology is unknown. We found by immunohistochemistry that Myo1b is expressed in islet cells of the rat pancreas. In cultured rat insulinoma 832/13 cells, Myo1b localized near actin patches, the trans-Golgi network (TGN) marker TGN38, and insulin granules in the perinuclear region. Myo1b depletion by small interfering RNA in 832/13 cells reduced intracellular proinsulin and insulin content and glucose-stimulated insulin secretion (GSIS) and led to the accumulation of (pro)insulin secretory granules (SGs) at the TGN. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, Myo1b depletion in insulinoma cells reduced the number of (pro)insulin-containing SGs budding from the TGN. The studies indicate for the first time that in pancreatic β-cells Myo1b controls GSIS at least in part by mediating an early stage in insulin granule trafficking from the TGN.     

Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

Aims/Hypothesis

Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.

Methods

Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca²⁺ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.

Results

Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.

Conclusions/Interpretation

Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca²⁺ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.     

Selected plant species from the Cree pharmacopoeia of northern Quebec possess anti-diabetic potential

Type II diabetes is a major health problem worldwide. Some populations, such as aboriginal peoples, are particularly at risk for this disease. In the Cree Nation of Quebec, Canada, prevalence in adults is approaching 20%, and the consequences are compounded by low compliance with modern medicine. In 2003, we conducted an ethnobotanical study of Cree medicinal plants used for the treatment of symptoms of diabetes. This served as the basis for a project designed to identify efficacious complementary treatment options more readily accepted by this population. The present study assesses the in vitro anti-diabetic potential of extracts from the 8 most promising plants to emerge from the ethnobotanical study. Cell-based bioassays were employed to screen for (i) potentiation of glucose uptake by skeletal muscle cells (C2C12) and adipocytes (3T3-L1); (ii) potentiation of glucose-stimulated insulin secretion (GSIS) and insulin production by pancreatic beta cells (INS 832/13); (iii) potentiation of triglyceride accumulation in differentiating 3T3-L1 cells; (iv) protection against glucose toxicity and glucose deprivation in pre-sympathetic neurons (PC12-AC). Additionally, anti-oxidant activity was measured biochemically by the diphenylpicrylhydrazyl (DPPH) reduction assay. All plant extracts potentiated basal or insulin-stimulated glucose uptake to some degree in muscle cells or adipocytes. Adipocyte differentiation was accelerated by 4 extracts. Five extracts conferred protection in PC12 cells. Three extracts displayed free radical scavenging activity similar to known anti-oxidants. None of the plant extracts enhanced GSIS or insulin content in INS 832/13 beta cells. It is concluded that the Cree pharmacopoeia contains several plants with significant anti-diabetic potential.     

The CB1 Antagonist Rimonabant Decreases Insulin Hypersecretion in Rat Pancreatic Islets

Type 2 diabetes and obesity are characterized by elevated nocturnal circulating free fatty acids, elevated basal insulin secretion, and blunted glucose‐stimulated insulin secretion (GSIS). The CB1 receptor antagonist, Rimonabant, has been shown to improve glucose tolerance and insulin sensitivity in vivo but its direct effect on islets has been unclear. Islets from lean littermates and obese Zucker (ZF) and Zucker Diabetic Fatty (ZDF) rats were incubated for 24 h in vitro and exposed to 11 mmol/l glucose and 0.3 mmol/l palmitate (GL) with or without Rimonabant. Insulin secretion was determined at basal (3 mmol/l) or stimulatory (15 mmol/l) glucose concentrations. As expected, basal secretion was significantly elevated in islets from obese or GL‐treated lean rats whereas the fold increase in GSIS was diminished. Rimonabant decreased basal hypersecretion in islets from obese rats and GL‐treated lean rats without decreasing the fold increase in GSIS. However, it decreased GSIS in islets from lean rats without affecting basal secretion. These findings indicate that Rimonabant has direct effects on islets to reduce insulin secretion when secretion is elevated above normal levels by diet or in obesity. In contrast, it appears to decrease stimulated secretion in islets from lean animals but not in obese or GL‐exposed islets.