Glucose-stimulated Insulin Secretion In INS-1 Cells Research Articles

Constitutive Pleiotrophin Deletion Results in a Phenotype with an Altered Pancreatic Morphology and Function in Old Mice.

Pleiotrophin (PTN) is crucial for embryonic development and pancreas organogenesis as it regulates metainflammation, metabolic homeostasis, thermogenesis, and glucose tolerance. Pleiotrophin deletion is associated with a lipodystrophic phenotype in which adipose tissue plasticity is altered in late life. This study explored the impact of pleiotrophin deletion on pancreatic morphology and function in later life. We analyzed glucose tolerance and circulating parameters on female wild-type (Ptn+/+) and knock-out (Ptn-/-) mice. At 9 and 15 months, we conducted morphometric analyses of pancreatic islets and evaluated the levels of insulin, glucagon, somatostatin, glucose transporter 2 (GLUT2), vesicle-associated membrane protein 2 (VAMP2), and synaptosome-associated protein 25 (SNAP25) via immunofluorescence. The effect of PTN on glucose-stimulated insulin secretion (GSIS) was evaluated in INS1E cells and isolated islets. Ptn-/- mice showed hyperinsulinemia, impaired glucose tolerance, and increased homeostatic model assessment for insulin resistance (HOMA-IR) with age. While Ptn+/+ islets enlarge with age, in Ptn-/- mice, the median size decreased, and insulin content increased. Vesicle transport and exocytosis proteins were significantly increased in 9-month-old Ptn-/- islets. Islets from Ptn-/- mice showed impaired GSIS and decreased cell membrane localization of GLUT2 whereas, PTN increased GSIS in INS1E cells. Ptn deletion accelerated age-related changes in the endocrine pancreas, affecting islet number and size, and altering VAMP2 and SNAP25 levels and GLUT2 localization leading to impaired GSIS and insulin accumulation in islets.

Preliminary Study on the Protective Effects and Molecular Mechanism of Procyanidins against PFOS-Induced Glucose-Stimulated Insulin Secretion Impairment in INS-1 Cells.

This study aimed to investigate the effects of perfluorooctanesulfonic acid (PFOS) exposure on glucose-stimulated insulin secretion (GSIS) of rat insulinoma (INS-1) cells and the potential protective effects of procyanidins (PC). The effects of PFOS and/or PC on GSIS of INS-1 cells were investigated after 48 h of exposure (protein level: insulin; gene level: glucose transporter 2 (Glut2), glucokinase (Gck), and insulin). Subsequently, the effects of exposure on the intracellular reactive oxygen species (ROS) activity were measured. Compared to the control group, PFOS exposure (12.5, 25, and 50 μM) for 48 h had no significant effect on the viability of INS-1 cells. PFOS exposure (50 μM) could reduce the level of insulin secretion and reduce the relative mRNA expression levels of Glut2, Gck, and insulin. It is worth noting that PC could partially reverse the damaging effect caused by PFOS. Significantly, there was an increase in ROS after exposure to PFOS and a decline after PC intervention. PFOS could affect the normal physiological function of GSIS in INS-1 cells. PC, a plant natural product, could effectively alleviate the damage caused by PFOS by inhibiting ROS activity.

Myokine Regulation of Insulin Secretion: Impact of Inflammation and Type 2 Diabetes

Skeletal muscle (SkM) secretes protein factors (myokines) that can exert multiple actions. To study the control of myokine regulation of β-cell function, SkM biopsies were taken from non-diabetic (ND) and Type 2 diabetic (T2D) subjects and satellite cells cultured to myotubes (MT). MT were also treated with lipopolysaccharide (infectious inflammation – II) or a combination of glucose (10 mM), insulin (120 pM), and palmitate (0.4 mM) (metabolic inflammation – MI) to model the inflammatory and metabolic conditions seen in vivo with T2D. Conditioned media (CM) was collected from MT after 24 h and used to treat INS-1 cells for 24 h. Cell viability, total insulin content, glucose-stimulated insulin secretion (GSIS) and maximal (IBMX-stimulated) IS (ISmax) were monitored. Under baseline conditions, CM from ND and T2D MT had no effects on INS-1 cell viability, insulin content, GSIS, or ISmax. After exposure to II, CM from ND-MT augmented GSIS in INS-1 cells by 100 ± 25% over control (p < 0.05); T2D-CM had no effect. After exposure to MI, T2D-CM suppressed GSIS by 35 ± 5% (p < 0.05); ND-CM was without effect. Under either of these conditions cell viability, total insulin content and ISmax were unaffected. Effects of CM on GSIS were lost after CM was boiled. Both augmentation of GSIS by ND-CM from II-treated MT, and suppression by T2D-CM from MI-treated MT, were inhibited by wortmannin, Ro 31-8220, and SB203580. In summary: (1) ND-MT are able to augment GSIS when stressed, (2) T2D-MT responding to a diabetic-like environment secrete myokines that suppress GSIS, (3) Unknown protein factors exert effects specifically on GSIS, possibly through PI-3K, PKC, and/or p38 MAPK. In T2D, both insulin resistance and a suppression of adaptive increased insulin secretion are intrinsic properties of SkM that can contribute to the full T2D phenotype.

Palmitic acid-induced lipotoxicity promotes a novel interplay between Akt-mTOR, IRS-1, and FFAR1 signaling in pancreatic \u03b2-cells

BackgroundFree fatty acid receptor 1 (FFAR1) is G-protein coupled receptor predominantly expressed in pancreatic β-cells that is activated by a variety of free fatty acids (FFAs). Once activated, it promotes glucose-stimulated insulin secretion (GSIS). However, increased levels of FFAs lead to lipotoxicity, inducing loss of β-cell function. FFAR1 plays a key role in the development of type 2 diabetes (T2D), and previous studies have indicated the importance of developing anti-diabetic therapies against FFAR1, although its role in the regulation of β-cell function remains unclear. The present study investigated the role of FFAR1 under lipotoxic conditions using palmitic acid (PA). The rat insulinoma 1 clone 832/13 (INS-1 832/13) cell line was used as a model as it physiologically resembles native pancreatic β-cells. Key players of the insulin signaling pathway, such as mTOR, Akt, IRS-1, and the insulin receptor (INSR1β), were selected as candidates to be analyzed under lipotoxic conditions.ResultsWe revealed that PA-induced lipotoxicity affected GSIS in INS-1 cells and negatively modulated the activity of both IRS-1 and Akt. Reduced phosphorylation of both IRS-1 S636/639 and Akt S473 was observed, in addition to decreased expression of both INSR1β and FFAR1. Moreover, transient knockdown of FFAR1 led to a reduction in IRS-1 mRNA expression and an increase in INSR1β mRNA. Finally, PA affected localization of FFAR1 from the cytoplasm to the perinucleus.ConclusionsIn conclusion, our study suggests a novel regulatory involvement of FFAR1 in crosstalk with mTOR–Akt and IRS-1 signaling in β-cells under lipotoxic conditions.

Pro-inflammatory cytokines attenuate glucose-stimulated insulin secretion from INS-1E insulinoma cells by restricting mitochondrial pyruvate oxidation capacity - Novel mechanistic insight from real-time analysis of oxidative phosphorylation.

Pro-inflammatory cytokines cause pancreatic beta cell failure during the development of type 2 diabetes. This beta cell failure associates with mitochondrial dysfunction, but the precise effects of cytokines on mitochondrial respiration remain unclear. To test the hypothesis that pro-inflammatory cytokines impair glucose-stimulated insulin secretion (GSIS) by inhibiting oxidative ATP synthesis, we probed insulin release and real-time mitochondrial respiration in rat INS-1E insulinoma cells that were exposed to a combination of 2 ng/mL interleukin-1-beta and 50 ng/mL interferon-gamma. We show that 24-h exposure to these cytokines dampens both glucose- and pyruvate-stimulated insulin secretion (P < 0.0001 and P < 0.05, respectively), but does not affect KCl-induced insulin release. Mirroring secretory defects, glucose- and pyruvate-stimulated mitochondrial respiration are lowered after cytokine exposure (P < 0.01). Further analysis confirms that cytokine-induced mitochondrial respiratory defects occur irrespective of whether fuel oxidation is coupled to, or uncoupled from, ATP synthesis. These observations demonstrate that pro-inflammatory cytokines attenuate GSIS by restricting mitochondrial pyruvate oxidation capacity. Interleukin-1-beta and interferon-gamma also increase mitochondrial superoxide levels (P < 0.05), which may reinforce the inhibition of pyruvate oxidation, and cause a modest (20%) but significant (P < 0.01) loss of INS-1E cells. Cytokine-induced INS-1E cell failure is insensitive to palmitoleate and linoleate, which is at odds with the cytoprotection offered by unsaturated fatty acids against harm caused by nutrient excess. Our data disclose a mitochondrial mechanism for cytokine-impaired GSIS in INS-1E cells, and suggest that inflammatory and nutrient-related beta cell failure emerge, at least partly, through distinct paths.

Epicatechin potentiation of glucose-stimulated insulin secretion in INS-1 cells is not dependent on its antioxidant activity.

Epicatechin (EC) is a monomeric flavan-3-ol. We have previously demonstrated that glucose-intolerant rats fed flavan-3-ols exhibit improved pancreatic islet function corresponding with an increase in circulating EC-derived metabolites. Thus, we speculate that EC may act as a cellular signaling molecule in vivo to modulate insulin secretion. In this study we further examined the effects of different concentrations of EC on H2O2 or hyperglycemia-induced ROS production, as well as on saturated fatty acid (SFA)-impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cell line in vitro. We showed that EC at a high concentration (30 μmol/L), but not a low concentration (0.3 μmol/L), significantly decreased H2O2 or hyperglycemia-induced ROS production in INS-1 cells. However, EC (0.3 μmol/L) significantly enhanced SFA-impaired GSIS in INS-1 cells. Addition of KN-93, a CaMKII inhibitor, blocked the effect of EC on insulin secretion and decreased CaMKII phosphorylation. Addition of GW1100, a GPR40 antagonist, significantly attenuated EC-enhanced GSIS, but only marginally affected CaMKII phosphorylation. These results demonstrate that EC at a physiological concentration promotes GSIS in SFA-impaired β-cells via activation of the CaMKII pathway and is consistent with its function as a GPR40 ligand. The findings support a role for EC as a cellular signaling molecule in vivo and further delineate the signaling pathways of EC in β-cells.

Interleukin-22 restored mitochondrial damage and impaired glucose-stimulated insulin secretion through down-regulation of uncoupling protein-2 in INS-1 cells.

Defective glucose-stimulated insulin secretion (GSIS) induced by chronic exposure to fatty acids is a hallmark of type 2 diabetes (T2D). Interleukin-22 (IL-22) has been shown to exert beneficial effects on insulin secretion and to protect pancreatic β-cells from stress. Moreover, uncoupling protein-2 (UCP-2) plays a central role in the regulation of GSIS and β-cell dysfunction, whereas the role of UCP-2 in IL-22-enhanced glycemic control under conditions of lipotoxicity remains unclear. In this present study, we investigated the effects of IL-22 on rat insulin-secreting cells (INS-1 cells) and the mechanisms that underlie IL-22 and lipotoxicity-impaired GSIS in vitro. Chronic palmitate (PA) treatment impaired insulin secretion and activated UCP-2 expression in INS-1 cells. Furthermore, in INS-1 cells, both reduced mitochondrial membrane potential (ΔΨm) and impaired GSIS induced by PA treatment were effectively reversed by an inhibitor of UCP-2 (genipin). Additionally, compared with the PA-treated group, INS-1 cells treated with IL-22 down-regulated UCP-2 expression, increased mitochondrial membrane potential, and restored GSIS. Together, our findings indicate that chronic exposure to PA could activate UCP-2, resulting in mitochondrial damage and impaired GSIS in INS-1 cells. We also suggest that IL-22 plays a protective role in this process via the down-regulation of UCP-2.

Possible Role of AMPK/SIRT1 Signaling on Energy Balance in Geniposide- Treated INS-1 Cells

Our previous work showed that in pancreatic INS-1 cells, geniposide exerted a contrary role on both glucosestimulated insulin secretion (GSIS) and glucose uptake and metabolism in the presence of low and high glucose. But the molecular mechanisms are presently not well understood. In the present study, we design to probe the role of AMP-activated protein kinase (AMPK) and NAD+- dependent deacetylase sirtuin-1 (SIRT1) on geniposide regulating GSIS, and analyze the interaction between AMPK and SIRT1 in pancreatic β cells. Our results indicate that geniposide induce the phosphorylation of AMPK and enhance the expression of SIRT1 in the presence of low concentration of glucose, but in the presence of high concentrations of glucose, geniposide played a contrary role on those. Furthermore, Compound C (an AMPK inhibitor) and Ex527 (a potent and selective inhibitor of SIRT1) prevent the effects of geniposide on glucose uptake, ATP production and GSIS in INS-1 cells. Taken together, our findings suggest that AMPK/SIRT1 are associated with the role of geniposide on energy balance in INS-1 cells.

PRMT1 promotes glucose toxicity-induced β cell dysfunction by regulating the nucleo-cytoplasmic trafficking of PDX-1 in a FOXO1-dependent manner in INS-1 cells.

Protein N-arginine methyltransferase-1 (PRMT1), the major asymmetric arginine methyltransferase, plays important roles in various cellular processes. Previous reports have demonstrated that levels and activities of PRMT1 can vary in animals with type 2 diabetes mellitus. The aim of this study was to assess the expression and mechanism of action of PRMT1 during glucose toxicity-induced β cell dysfunction. Liposome-mediated gene transfection was used to transfect INS-1 cells with siPRMT1, which inhibits PRMT1 expression, and pALTER-FOXO1, which overexpresses forkhead box protein O1 (FOXO1). The cells were then cultured in media containing 5.6 or 25 mmol/L glucose with or without the small molecule PRMT1 inhibitor AMI-1 for 48 h. The protein levels of PRMT1, the arginine methylated protein α-metR, FOXO1, Phospho-FOXO1, pancreas duodenum homeobox-1 (PDX-1), and the intracellular localization of PDX-1 and FOXO1 were then measured by western blotting. FOXO1 methylation was detected by immunoprecipitated with anti-PRMT1 antibody and were immunoblotted with α-metR. The levels of insulin mRNA were measured by real-time fluorescence quantitative PCR. Glucose-stimulated insulin secretion (GSIS) and intracellular insulin content were measured using radioimmunoassays. Intracellular Ca(2+) ([Ca(2+)]i) was detected using Fura-2 AM. Intracellular cAMP levels were measured using ELISA. Chronic exposure to high glucose impaired insulin secretion, decreased insulin mRNA levels and insulin content, increased intracellular [Ca(2+)]i and cAMP levels, and abolishes their responses to glucose. Inhibiting PRMT1 expression improved insulin secretion, increased mRNA levels and insulin content by regulating the intracellular translocation of PDX-1 and FOXO1, decreasing the methylation of FOXO1, and reducing intracellular [Ca(2+)]i and cAMP concentrations. Transient overexpression of constitutively active FOXO1 in nuclear reversed the AMI-1-induced improvement of β cell function without changing arginine methylation. It is concluded therefore that PRMT1 regulates GSIS in INS-1 cells, through enhanced methylation-induced nuclear localization of FOXO1, which subsequently suppresses the nuclear localization of PDX-1. Our results suggest a novel mechanism that might contribute to the deficient insulin secretion observed under conditions of chronically hyperglycemia.

Geniposide Regulates Glucose-Stimulated Insulin Secretion Possibly through Controlling Glucose Metabolism in INS-1 Cells

Glucose-stimulated insulin secretion (GSIS) is essential to the control of metabolic fuel homeostasis. The impairment of GSIS is a key element of β-cell failure and one of causes of type 2 diabetes mellitus (T2DM). Although the KATP channel-dependent mechanism of GSIS has been broadly accepted for several decades, it does not fully describe the effects of glucose on insulin secretion. Emerging evidence has suggested that other mechanisms are involved. The present study demonstrated that geniposide enhanced GSIS in response to the stimulation of low or moderately high concentrations of glucose, and promoted glucose uptake and intracellular ATP levels in INS-1 cells. However, in the presence of a high concentration of glucose, geniposide exerted a contrary role on both GSIS and glucose uptake and metabolism. Furthermore, geniposide improved the impairment of GSIS in INS-1 cells challenged with a high concentration of glucose. Further experiments showed that geniposide modulated pyruvate carboxylase expression and the production of intermediates of glucose metabolism. The data collectively suggest that geniposide has potential to prevent or improve the impairment of insulin secretion in β-cells challenged with high concentrations of glucose, likely through pyruvate carboxylase mediated glucose metabolism in β-cells.

The ETS-Domain Transcription Factor Elk-1 Regulates COX-2 Gene Expression and Inhibits Glucose-Stimulated Insulin Secretion in the Pancreaticβ-Cell Line INS-1

Cyclooxygenase-2 (COX-2) expression is associated with many aspects of physiological and pathological conditions, including pancreatic β-cell dysfunction. Prostaglandin E2 (PGE2) production, as a consequence of COX-2 gene induction, has been reported to impair β-cell function. The molecular mechanisms involved in the regulation of COX-2 gene expression are not fully understood. We previously demonstrated that transcription factor Elk-1 significantly upregulated COX-2 gene promoter activity. In this report, we used pancreatic β-cell line (INS-1) to explore the relationships between Elk-1 and COX-2. We first investigated the effects of Elk-1 on COX-2 transcriptional regulation and expression in INS-1 cells. We thus undertook to study the binding of Elk-1 to its putative binding sites in the COX-2 promoter. We also analysed glucose-stimulated insulin secretion (GSIS) in INS-1 cells that overexpressed Elk-1. Our results demonstrate that Elk-1 efficiently upregulates COX-2 expression at least partly through directly binding to the −82/−69 region of COX-2 promoter. Overexpression of Elk-1 inhibits GSIS in INS-1 cells. These findings will be helpful for better understanding the transcriptional regulation of COX-2 in pancreatic β-cell. Moreover, Elk-1, the transcriptional regulator of COX-2 expression, will be a potential target for the prevention of β-cell dysfunction mediated by PGE2.

Cav1.2 and Cav1.3 are differentially coupled to glucagon-like peptide-1 potentiation of glucose-stimulated insulin secretion in the pancreatic beta-cell line INS-1.

The incretin peptides, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), potentiate glucose-stimulated insulin secretion (GSIS) and beta-cell proliferation and differentiation. Ca(2+) influx via voltage-gated L-type Ca(2+) channels is required for GLP-1 and GIP potentiation of GSIS. We investigated the role of the L-type Ca(2+) channels Ca(v)1.2 and Ca(v)1.3 in mediating GLP-1- and GIP-stimulated events in INS-1 cells and INS-1 cell lines expressing dihydropyridine-insensitive (DHPi) mutants of either Ca(v)1.2 or Ca(v)1.3. Ca(v)1.3/DHPi channels supported full potentiation of GSIS by GLP-1 (50 nM) compared with untransfected INS-1 cells. However, GLP-1-potentiated GSIS mediated by Ca(v)1.2/DHPi channels was markedly reduced compared with untransfected INS-1 cells. In contrast, GIP (10 nM) potentiation of GSIS mediated by both Ca(v)1.2/DHPi and Ca(v)1.3/DHPi channels was similar to that observed in untransfected INS-1 cells. Disruption of intracellular Ca(2+) release with thapsigargin, ryanodine, or 2-aminoethyldiphenylborate and inhibition of protein kinase A (PKA) or protein kinase C (PKC) significantly reduced GLP-1 potentiation of GSIS by Ca(v)1.3/DHPi channels and by endogenous L-type channels in INS-1 cells, but not by Ca(v)1.2/DHPi channels. Inhibition of glucose-stimulated phospholipase C activity with 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not inhibit potentiation of GSIS by GLP-1 in INS-1 cells. In contrast, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase, both markedly inhibited GLP-1 potentiation of GSIS by endogenous channels in INS-1 cells and Ca(v)1.3/DHPi channels, but not by Ca(v)1.2/DHPi channels. Thus, Ca(v)1.3 is preferentially coupled to GLP-1 potentiation of GSIS in INS-1 cells via a mechanism that requires intact intracellular Ca(2+) stores, PKA and PKC activity, and activation of ERK1/2.

Chronic palmitate exposure inhibits AMPKα and decreases glucose-stimulated insulin secretion from β-cells: modulation by fenofibrate

Adenosine monophosphate-activated protein kinase (AMPK), a vital regulator of glucose metabolism, may affect insulin secretion in beta-cells. However, the role of AMPK in beta-cell lipotoxicity remains unclear. Fenofibrate has been reported to regulate lipid homeostasis and is involved in insulin secretion in pancreatic beta-cells. In the present study, we aimed to investigate the effect of palmitate on AMPK expression and glucose-stimulated insulin secretion (GSIS) in rat islets and INS-1 beta-cell, as well as the effect of fenofibrate on AMPK and GSIS in INS-1 cells treated with palmitate. Isolated rat islets and INS-1 beta-cells were treated with and without palmitate or fenofibrate for 48 h. The mRNA levels of the AMPK alpha isoforms were measured by real-time PCR. Western blotting was used to detect the protein expression of total AMPKalpha (TAMPKalpha), phosphorylated AMPKalpha (P-AMPKalpha), and phosphorylated acetyl coenzyme A carboxylase (P-ACC). Insulin secretion was detected by radioimmunoassay induced by 20 mmol/L glucose as GSIS. The results showed that chronic exposure of beta-cells to palmitate for 48 h inhibited the expression of AMPK alpha1 mRNA and T-AMPK alpha protein levels, as well as P-AMPK alpha and PACC protein expressions in a dose-dependent manner. Accordingly, GSIS was inhibited by palmitate. Compared with the palmitate-treated cells, fenofibrate ameliorated these changes impaired by palmitate and exhibited a significant elevation in the expression of AMPK alpha and GSIS. Our findings suggest a role of AMPK alpha reduction in beta-cell lipotoxicity and a novel role of fenofibrate in improving GSIS associated with the AMPK alpha activation in beta-cells chronically exposed to palmitate.

Troglitazone ameliorates lipotoxicity in the beta cell line INS-1 expressing PPAR gamma

To elucidate the mechanisms by which troglitazone, which is a direct ligand for peroxisome proliferator-activated receptor (PPAR) gamma, ameliorates insulin resistance, we have demonstrated that PPAR gamma is expressed in a pancreatic beta cell line, INS-1, using reverse transcription-polymerase chain reaction (RT-PCR). We incubated the cells with 5 μmol/l troglitazone and 1 mmol/l of each major free fatty acid (FFA; palmitic acid, oleic acid, and linoleic acid), alone or in combination, for 48 h. After that, we evaluated glucose-stimulated insulin secretion (GSIS) and 25 mmol/l KCl-induced insulin secretion in the presence of diazoxide, which clamps membrane potential. Our results showed: (1) treatment with troglitazone for 48 h caused enhancement of GSIS, although troglitazone significantly suppressed cell viability assessed by MTT assay. (2) In cells co-treated with troglitazone and FFA, troglitazone ameliorated lipotoxicity due to FFA. (3) In the presence of 300 μmol/l diazoxide and 25 mmol/l KCl, troglitazone did not affect the recovery of GSIS in INS-1 cells. These results suggest that insulin secretion from the rat insulinoma cell line, INS-1, is modulated by troglitazone, acting somewhere in the ATP-sensitive K + channel pathway, possibly through PPAR gamma.

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) β-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.