Single-Cell RNA Sequencing Reveals a Role for Reactive Oxygen Species and Peroxiredoxins in Fatty Acid-Induced Rat ß-Cell Proliferation.

The functional mass of insulin-secreting pancreatic ß-cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing ß-cells. Type 2 diabetes appears when these compensatory mechanisms fail. Nutrients including glucose and fatty acids are important contributors to the ß-cell compensatory response, but their underlying mechanisms of action remain poorly understood. We investigated the transcriptional mechanisms of ß-cell proliferation in response to fatty acids. Isolated rat islets were exposed to 16.7 mmol/L glucose with or without 0.5 mmol/L oleate (C18:1) or palmitate (C16:0) for 48 h. The islet transcriptome was assessed by single-cell RNA sequencing. ß-Cell proliferation was measured by flow cytometry. Unsupervised clustering of pooled ß-cells identified different subclusters, including proliferating ß-cells. ß-Cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved in energy metabolism and mitochondrial activity. Comparison of proliferating versus nonproliferating ß-cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, N-acetyl cysteine and the peroxiredoxin inhibitor conoidin A both blocked oleate-induced ß-cell proliferation. Our study reveals a key role for ROS signaling through peroxiredoxin activation in oleate-induced ß-cell proliferation.

HB-EGF Signaling Is Required for Glucose-Induced Pancreatic ß-Cell Proliferation in Rats.

The molecular mechanisms of ß-cell compensation to metabolic stress are poorly understood. We previously observed that nutrient-induced ß-cell proliferation in rats is dependent on epidermal growth factor receptor (EGFR) signaling. The aim of this study was to determine the role of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) in the ß-cell proliferative response to glucose, a ß-cell mitogen and key regulator of ß-cell mass in response to increased insulin demand. We show that exposure of isolated rat and human islets to HB-EGF stimulates ß-cell proliferation. In rat islets, inhibition of EGFR or HB-EGF blocks the proliferative response not only to HB-EGF but also to glucose. Furthermore, knockdown of HB-EGF in rat islets blocks ß-cell proliferation in response to glucose ex vivo and in vivo in transplanted glucose-infused rats. Mechanistically, we demonstrate that HB-EGF mRNA levels are increased in ß-cells in response to glucose in a carbohydrate-response element-binding protein (ChREBP)-dependent manner. In addition, chromatin immunoprecipitation studies identified ChREBP binding sites in proximity to the HB-EGF gene. Finally, inhibition of Src family kinases, known to be involved in HB-EGF processing, abrogated glucose-induced ß-cell proliferation. Our findings identify a novel glucose/HB-EGF/EGFR axis implicated in ß-cell compensation to increased metabolic demand.

The regulator of G-protein signaling RGS16 promotes insulin secretion and ß-cell proliferation in rodent and human islets.

OBJECTIVE: G protein-coupled receptor (GPCR) signaling regulates insulin secretion and pancreatic ß cell-proliferation. While much knowledge has been gained regarding how GPCRs are activated in ß cells, less is known about the mechanisms controlling their deactivation. In many cell types, termination of GPCR signaling is controlled by the family of Regulators of G-protein Signaling (RGS). RGS proteins are expressed in most eukaryotic cells and ensure a timely return to the GPCR inactive state upon removal of the stimulus. The aims of this study were i) to determine if RGS16, the most highly enriched RGS protein in ß cells, regulates insulin secretion and ß-cell proliferation and, if so, ii) to elucidate the mechanisms underlying such effects. METHODS: Mouse and human islets were infected with recombinant adenoviruses expressing shRNA or cDNA sequences to knock-down or overexpress RGS16, respectively. 60 h post-infection, insulin secretion and cAMP levels were measured in static incubations in the presence of glucose and various secretagogues. ß-cell proliferation was measured in infected islets after 72 h in the presence of 16.7 mM glucose ± somatostatin and various inhibitors. RESULTS: RGS16 mRNA levels are strongly up-regulated in islets of Langerhans under hyperglycemic conditions in vivo and ex vivo. RGS16 overexpression stimulated glucose-induced insulin secretion in isolated mouse and human islets while, conversely, insulin secretion was impaired following RGS16 knock-down. Insulin secretion was no longer affected by RGS16 knock-down when islets were pre-treated with pertussis toxin to inactivate Gαi/o proteins, or in the presence of a somatostatin receptor antagonist. RGS16 overexpression increased intracellular cAMP levels, and its effects were blocked by an adenylyl cyclase inhibitor. Finally, RGS16 overexpression prevented the inhibitory effect of somatostatin on insulin secretion and ß-cell proliferation. CONCLUSIONS: Our results identify RGS16 as a novel regulator of ß-cell function that coordinately controls insulin secretion and proliferation by limiting the tonic inhibitory signal exerted by δ-cell-derived somatostatin in islets.

The ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Is Associated With Progressive Insulin Resistance and Decreased Functional ß-Cell Mass in Mice.

Cystic fibrosis (CF) is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF-related diabetes affects 50% of adult CF patients. How CFTR deficiency predisposes to diabetes is unknown. Herein, we examined the impact of the most frequent cftr mutation in humans, deletion of phenylalanine at position 508 (ΔF508), on glucose homeostasis in mice. We compared ΔF508 mutant mice with wild-type (WT) littermates. Twelve-week-old male ΔF508 mutants had lower body weight, improved oral glucose tolerance, and a trend toward higher insulin tolerance. Glucose-induced insulin secretion was slightly diminished in ΔF508 mutant islets, due to reduced insulin content, but ΔF508 mutant islets were not more sensitive to proinflammatory cytokines than WT islets. Hyperglycemic clamps confirmed an increase in insulin sensitivity with normal ß-cell function in 12- and 18-week-old ΔF508 mutants. In contrast, 24-week-old ΔF508 mutants exhibited insulin resistance and reduced ß-cell function. ß-Cell mass was unaffected at 11 weeks of age but was significantly lower in ΔF508 mutants versus controls at 24 weeks. This was not associated with gross pancreatic pathology. We conclude that the ΔF508 CFTR mutation does not lead to an intrinsic ß-cell secretory defect but is associated with insulin resistance and a ß-cell mass deficit in aging mutants.

ß-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1.

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic ß cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via ß-arrestins. Further, G protein- and ß-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses ("biased agonism"). Whether GPR40 engages ß-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-ß-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting ß-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of ß-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a ß-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.

The islet estrogen receptor-α is induced by hyperglycemia and protects against oxidative stress-induced insulin-deficient diabetes.

The female steroid, 17ß-estradiol (E2), is important for pancreatic ß-cell function and acts via at least three estrogen receptors (ER), ERα, ERß, and the G-protein coupled ER (GPER). Using a pancreas-specific ERα knockout mouse generated using the Cre-lox-P system and a Pdx1-Cre transgenic line (PERαKO ⁻/⁻), we previously reported that islet ERα suppresses islet glucolipotoxicity and prevents ß-cell dysfunction induced by high fat feeding. We also showed that E2 acts via ERα to prevent ß-cell apoptosis in vivo. However, the contribution of the islet ERα to ß-cell survival in vivo, without the contribution of ERα in other tissues is still unclear. Using the PERαKO ⁻/⁻ mouse, we show that ERα mRNA expression is only decreased by 20% in the arcuate nucleus of the hypothalamus, without a parallel decrease in the VMH, making it a reliable model of pancreas-specific ERα elimination. Following exposure to alloxan-induced oxidative stress in vivo, female and male PERαKO ⁻/⁻ mice exhibited a predisposition to ß-cell destruction and insulin deficient diabetes. In male PERαKO ⁻/⁻ mice, exposure to E2 partially prevented alloxan-induced ß-cell destruction and diabetes. ERα mRNA expression was induced by hyperglycemia in vivo in islets from young mice as well as in cultured rat islets. The induction of ERα mRNA by hyperglycemia was retained in insulin receptor-deficient ß-cells, demonstrating independence from direct insulin regulation. These findings suggest that induction of ERα expression acts to naturally protect ß-cells against oxidative injury.

Epidermal growth factor receptor signaling promotes pancreatic ß-cell proliferation in response to nutrient excess in rats through mTOR and FOXM1.

The cellular and molecular mechanisms underpinning the compensatory increase in ß-cell mass in response to insulin resistance are essentially unknown. We previously reported that a 72-h coinfusion of glucose and Intralipid (GLU+IL) induces insulin resistance and a marked increase in ß-cell proliferation in 6-month-old, but not in 2-month-old, Wistar rats. The aim of the current study was to identify the mechanisms underlying nutrient-induced ß-cell proliferation in this model. A transcriptomic analysis identified a central role for the forkhead transcription factor FOXM1 and its targets, and for heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a ligand of the EGF receptor (EGFR), in nutrient-induced ß-cell proliferation. Phosphorylation of ribosomal S6 kinase, a mammalian target of rapamycin (mTOR) target, was increased in islets from GLU+IL-infused 6-month-old rats. HB-EGF induced proliferation of insulin-secreting MIN6 cells and isolated rat islets, and this effect was blocked in MIN6 cells by the EGFR inhibitor AG1478 or the mTOR inhibitor rapamycin. Coinfusion of either AG1478 or rapamycin blocked the increase in FOXM1 signaling, ß-cell proliferation, and ß-cell mass and size in response to GLU+IL infusion in 6-month-old rats. We conclude that chronic nutrient excess promotes ß-cell mass expansion via a pathway that involves EGFR signaling, mTOR activation, and FOXM1-mediated cell proliferation.

Per-Arnt-Sim kinase regulates pancreatic duodenal homeobox-1 protein stability via phosphorylation of glycogen synthase kinase 3ß in pancreatic ß-cells.

In pancreatic ß-cells, glucose induces the binding of the transcription factor pancreatic duodenal homeobox-1 (PDX-1) to the insulin gene promoter to activate insulin gene transcription. At low glucose levels, glycogen synthase kinase 3ß (GSK3ß) is known to phosphorylate PDX-1 on C-terminal serine residues, which triggers PDX-1 proteasomal degradation. We previously showed that the serine/threonine Per-Arnt-Sim domain-containing kinase (PASK) regulates insulin gene transcription via PDX-1. However, the mechanisms underlying this regulation are unknown. In this study, we aimed to identify the role of PASK in the regulation of PDX-1 phosphorylation, protein expression, and stability in insulin-secreting cells and isolated rodent islets of Langerhans. We observed that glucose induces a decrease in overall PDX-1 serine phosphorylation and that overexpression of WT PASK mimics this effect. In vitro, PASK directly phosphorylates GSK3ß on its inactivating phosphorylation site Ser(9). Overexpression of a kinase-dead (KD), dominant negative version of PASK blocks glucose-induced Ser(9) phosphorylation of GSK3ß. Accordingly, GSK3ß Ser(9) phosphorylation is reduced in islets from pask-null mice. Overexpression of WT PASK or KD GSK3ß protects PDX-1 from degradation and results in increased PDX-1 protein abundance. Conversely, overexpression of KD PASK blocks glucose-induction of PDX-1 protein. We conclude that PASK phosphorylates and inactivates GSK3ß, thereby preventing PDX-1 serine phosphorylation and alleviating GSK3ß-mediated PDX-1 protein degradation in pancreatic ß-cells.

The free fatty acid receptor G protein-coupled receptor 40 (GPR40) protects from bone loss through inhibition of osteoclast differentiation.

The mechanisms linking fat intake to bone loss remain unclear. By demonstrating the expression of the free fatty acid receptor G-coupled protein receptor 40 (GPR40) in bone cells, we hypothesized that this receptor may play a role in mediating the effects of fatty acids on bone remodeling. Using micro-CT analysis, we showed that GPR40(-/-) mice exhibit osteoporotic features suggesting a positive role of GPR40 on bone density. In primary cultures of bone marrow, we showed that GW9508, a GRP40 agonist, abolished bone-resorbing cell differentiation. This alteration of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation occurred via the inhibition of the nuclear factor κB (NF-κB) signaling pathway as demonstrated by decrease in gene reporter activity, inhibitor of κB kinase (IKKα/ß) activation, inhibitor of κB (IkBα) phosphorylation, and nuclear factor of activated T cells 1 (NFATc1) expression. The GPR40-dependent effect of GW9508 was confirmed using shRNA interference in osteoclast precursors and GPR40(-/-) primary cell cultures. In addition, in vivo administration of GW9508 counteracted ovariectomy-induced bone loss in wild-type but not GPR40(-/-) mice, enlightening the obligatory role of the GPR40 receptor. Then, in a context of growing prevalence of metabolic and age-related bone disorders, our results demonstrate for the first time in translational approaches that GPR40 is a relevant target for the design of new nutritional and therapeutic strategies to counter bone complications.

Discovery of novel glucose-regulated proteins in isolated human pancreatic islets using LC-MS/MS-based proteomics.

The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is characterized by insulin resistance and insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent regulator of beta-cell function under physiological conditions, identification of the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of human beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins (∼p < 0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (pleiotropic regulator 1), processing (retinoblastoma binding protein 6), and function (nuclear RNA export factor 1), in addition to neuron navigator 1 and plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and synaptotagmin-17. Up-regulation of dicer 1 and SLC27A2 and down-regulation of phospholipase Cß4 were confirmed by Western blots. Many proteins found to be differentially abundant after high glucose stimulation are annotated as uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.

Human mutation within Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) causes basal insulin hypersecretion.

PAS kinase (PASK) is a glucose-regulated protein kinase involved in the control of pancreatic islet hormone release and insulin sensitivity. We aimed here to identify mutations in the PASK gene that may be associated with young-onset diabetes in humans. We screened 18 diabetic probands with unelucidated maturity-onset diabetes of the young (MODY). We identified two rare nonsynonymous mutations in the PASK gene (p.L1051V and p.G1117E), each of which was found in a single MODY family. Wild type or mutant PASKs were expressed in HEK 293 cells. Kinase activity of the affinity-purified proteins was assayed as autophosphorylation at amino acid Thr307 or against an Ugp1p-derived peptide. Whereas the PASK p.G1117E mutant displayed a ∼25% increase with respect to wild type PASK in the extent of autophosphorylation, and a ∼2-fold increase in kinase activity toward exogenous substrates, the activity of the p.L1051V mutant was unchanged. Amino acid Gly1117 is located in an α helical region opposing the active site of PASK and may elicit either: (a) a conformational change that increases catalytic efficiency or (b) a diminished inhibitory interaction with the PAS domain. Mouse islets were therefore infected with adenoviruses expressing wild type or mutant PASK and the regulation of insulin secretion was examined. PASK p.G1117E-infected islets displayed a 4-fold decrease in glucose-stimulated (16.7 versus 3 mM) insulin secretion, chiefly reflecting a 4.5-fold increase in insulin release at low glucose. In summary, we have characterized a rare mutation (p.G1117E) in the PASK gene from a young-onset diabetes family, which modulates glucose-stimulated insulin secretion.

Lack of preservation of insulin gene expression by a glucagon-like peptide 1 agonist or a dipeptidyl peptidase 4 inhibitor in an in vivo model of glucolipotoxicity.

UNLABELLED: Prolonged exposure of pancreatic beta-cells to elevated levels of glucose and fatty acids adversely affects insulin secretion and gene expression. AIM: To examine whether the GLP-1 agonist exenatide or the inhibitor of the GLP-1-degrading enzyme dipeptidyl peptidase 4 (DPP-4) sitagliptin rescue insulin gene expression in rats infused for 72h with glucose+Intralipid, independently from their glucose-lowering action. METHODS: Wistar rats were infused alternatively with glucose or Intralipid for cycles of 4h each for a total of 72h. The animals received exenatide (5microg/kg/day IV) or sitagliptin (5mg/kg/day IV) continuously starting 4 days prior to and continuing throughout the 3-day infusion period. RESULTS: Plasma glucose, fatty acids, insulin and C-peptide levels were unaffected by exenatide or sitagliptin treatment during the infusion period. Insulin mRNA levels increased in response to the glucose infusion, but this increase was abolished in islets from rats receiving glucose+Intralipid. Neither exenatide nor sitagliptin administration rescued insulin mRNA in glucose+Intralipid infused rats. CONCLUSIONS: Neither a GLP-1 agonist nor a DPP-4 inhibitor, at doses that do not alter blood glucose levels, prevented the inhibition of insulin gene expression in this in vivo model of glucolipotoxicity.

Cystic fibrosis-related diabetes: from CFTR dysfunction to oxidative stress.

Cystic fibrosis (CF) represents the most common lethal autosomal recessive disorder in the Caucasian population. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in abnormal Na(+) and Cl(-) transport in several tissues. Its main clinical manifestations include bronchopulmonary infections along with gastrointestinal and nutritional disorders. Intense and recurrent inflammation ultimately leads to an overabundance of activated neutrophils and macrophages that contribute to free radical generation. Furthermore, CFTR defects directly affect glutathione transport and homeostasis, while intestinal fat malabsorption limits uptake of endogenous antioxidant vitamins. Collectively, these abnormal events disturb the balance between pro- and anti-oxidants and promote oxidative stress, which may play a significant role in CF-related diabetes (CFRD), a severe complication associated with a drastic increase of morbidity and mortality. This review will focus on the involvement of oxidative stress in CF pathology, especially its role in the occurrence of CFRD. The multiple abnormalities in the oxidant/antioxidant balance could be a potential target for a new therapeutic approach.

Involvement of Per-Arnt-Sim Kinase and extracellular-regulated kinases-1/2 in palmitate inhibition of insulin gene expression in pancreatic beta-cells.

OBJECTIVE: Prolonged exposure of pancreatic beta-cells to simultaneously elevated levels of fatty acids and glucose (glucolipotoxicity) impairs insulin gene transcription. However, the intracellular signaling pathways mediating these effects are mostly unknown. This study aimed to ascertain the role of extracellular-regulated kinases (ERKs)1/2, protein kinase B (PKB), and Per-Arnt-Sim kinase (PASK) in palmitate inhibition of insulin gene expression in pancreatic beta-cells. RESEARCH DESIGN AND METHODS: MIN6 cells and isolated rat islets were cultured in the presence of elevated glucose, with or without palmitate or ceramide. ERK1/2 phosphorylation, PKB phosphorylation, and PASK expression were examined by immunoblotting and real-time PCR. The role of these kinases in insulin gene expression was assessed using pharmacological and molecular approaches. RESULTS: Exposure of MIN6 cells and islets to elevated glucose induced ERK1/2 and PKB phosphorylation, which was further enhanced by palmitate. Inhibition of ERK1/2, but not of PKB, partially prevented the inhibition of insulin gene expression in the presence of palmitate or ceramide. Glucose-induced expression of PASK mRNA and protein levels was reduced in the presence of palmitate. Overexpression of wild-type PASK increased insulin and pancreatic duodenal homeobox-1 gene expression in MIN6 cells and rat islets incubated with glucose and palmitate, whereas overexpression of a kinase-dead PASK mutant in rat islets decreased expression of insulin and pancreatic duodenal homeobox-1 and increased C/EBPbeta expression. CONCLUSIONS: Both the PASK and ERK1/2 signaling pathways mediate palmitate inhibition of insulin gene expression. These findings identify PASK as a novel mediator of glucolipotoxicity on the insulin gene in pancreatic beta-cells.

Pioglitazone acutely reduces insulin secretion and causes metabolic deceleration of the pancreatic beta-cell at submaximal glucose concentrations.

Thiazolidinediones (TZDs) have beneficial effects on glucose homeostasis via enhancement of insulin sensitivity and preservation of beta-cell function. How TZDs preserve beta-cells is uncertain, but it might involve direct effects via both peroxisome proliferator-activated receptor-gamma-dependent and -independent pathways. To gain insight into the independent pathway(s), we assessed the effects of short-term (

Characterization of the human pancreatic islet proteome by two-dimensional LC/MS/MS.

The pancreatic beta-cell plays a central role in the maintenance of glucose homeostasis and in the pathogenesis of both type 1 and type 2 diabetes mellitus. Elucidation of the insulin secretory defects observed in diabetes first requires a better understanding of the complex mechanisms regulating insulin secretion, which are only partly understood. While there have been reports detailing proteomic analyses of islet cell lines or isolated rodent islets, the information gained is not always applicable to humans. Therefore, definition of the human islet proteome could contribute to a better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the confident identification of 29,021 different tryptic peptides covering 3365 proteins (> or =2 unique peptide identifications per protein). As expected, the three major islet hormones (insulin, glucagon, and somatostatin) were detected, as well as various beta-cell enriched secretory products, ion channels, and transcription factors. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was observed, including the integrin signaling cascade and the MAP kinase, NF-kappa beta, and JAK/STAT pathways. The resulting peptide reference library provides a resource for future higher throughput and quantitative studies of islet biology.

Palmitate inhibits insulin gene expression by altering PDX-1 nuclear localization and reducing MafA expression in isolated rat islets of Langerhans.

Abnormalities in lipid metabolism have been proposed as contributing factors to both defective insulin secretion from the pancreatic beta cell and peripheral insulin resistance in type 2 diabetes. Previously, we have shown that prolonged exposure of isolated rat islets of Langerhans to excessive fatty acid levels impairs insulin gene transcription. This study was designed to assess whether palmitate alters the expression and binding activity of the key regulatory factors pancreas-duodenum homeobox-1 (PDX-1), MafA, and Beta2, which respectively bind to the A3, C1, and E1 elements in the proximal region of the insulin promoter. Nuclear extracts of isolated rat islets cultured with 0.5 mm palmitate exhibited reduced binding activity to the A3 and C1 elements but not the E1 element. Palmitate did not affect the overall expression of PDX-1 but reduced its nuclear localization. In contrast, palmitate blocked the stimulation of MafA mRNA and protein expression by glucose. Combined adenovirus-mediated overexpression of PDX-1 and MafA in islets completely prevented the inhibition of insulin gene expression by palmitate. These results demonstrate that prolonged exposure of islets to palmitate inhibits insulin gene transcription by impairing nuclear localization of PDX-1 and cellular expression of MafA.

The islet beta cell-enriched MafA activator is a key regulator of insulin gene transcription.

The islet-enriched MafA, PDX-1, and BETA2 activators contribute to both beta cell-specific and glucose-responsive insulin gene transcription. To investigate how these factors impart activation, their combined impact upon insulin enhancer-driven expression was first examined in non-beta cell line transfection assays. Individual expression of PDX-1 and BETA2 led to little or no activation, whereas MafA alone did so modestly. MafA together with PDX-1 or BETA2 produced synergistic activation, with even higher insulin promoter activity found when all three proteins were present. Stimulation was attenuated upon compromising either MafA transactivation or DNA-binding activity. MafA interacted with endogenous PDX-1 and BETA2 in coimmunoprecipitation and in vitro GST pull-down assays, suggesting that regulation involved direct binding. Dominant-negative acting and small interfering RNAs of MafA also profoundly reduced insulin promoter activity in beta cell lines. In addition, MafA was induced in parallel with insulin mRNA expression in glucose-stimulated rat islets. Insulin mRNA levels were also elevated in rat islets by adenoviral-mediated expression of MafA. Collectively, these results suggest that MafA plays a key role in coordinating and controlling the level of insulin gene expression in islet beta cells.

A role for the malonyl-CoA/long-chain acyl-CoA pathway of lipid signaling in the regulation of insulin secretion in response to both fuel and nonfuel stimuli.

The malonyl-CoA/long-chain acyl-CoA (LC-CoA) model of glucose-induced insulin secretion (GIIS) predicts that malonyl-CoA derived from glucose metabolism inhibits fatty acid oxidation, thereby increasing the availability of LC-CoA for lipid signaling to cellular processes involved in exocytosis. For directly testing the model, INSr3 cell clones overexpressing malonyl-CoA decarboxylase in the cytosol (MCDc) in a tetracycline regulatable manner were generated, and INS(832/13) and rat islets were infected with MCDc-expressing adenoviruses. MCD activity was increased more than fivefold, and the malonyl-CoA content was markedly diminished. This was associated with enhanced fat oxidation at high glucose, a suppression of the glucose-induced increase in cellular free fatty acid (FFA) content, and reduced partitioning at elevated glucose of exogenous palmitate into lipid esterification products. MCDc overexpression, in the presence of exogenous FFAs but not in their absence, reduced GIIS in all beta-cell lines and in rat islets. It also markedly curtailed the stimulation of insulin secretion by other fuel and nonfuel secretagogues. In the absence of MCDc overexpression, the secretory responses to all types of secretagogues were amplified by the provision of exogenous fatty acids. In the presence of exogenous FFAs, the fatty acyl-CoA synthetase inhibitor triacsin C reduced secretion in response to glucose and nonfuel stimuli. The data show the existence of important links between the metabolic coupling factor malonyl-CoA, the partitioning of fatty acids, and the stimulation of insulin secretion to both fuel and nonfuel stimuli.

Palmitate inhibition of insulin gene expression is mediated at the transcriptional level via ceramide synthesis.

Chronic exposure to elevated levels of fatty acids impairs pancreatic beta cell function, a phenomenon thought to contribute to the progressive deterioration of insulin secretion in type 2 diabetes. We have previously demonstrated that prolonged exposure of isolated islets to elevated levels of palmitate inhibits preproinsulin mRNA levels in the presence of high glucose concentrations. However, whether this occurs via transcriptional or post-transcriptional mechanisms has not been determined. In addition, the nature of the lipid metabolites involved in palmitate inhibition of insulin gene expression is unknown. In this study, we show that palmitate decreases glucose-stimulated preproinsulin mRNA levels in isolated rat islets, an effect that is not mediated by changes in preproinsulin mRNA stability, but is associated with inhibition of glucose-stimulated insulin promoter activity. Prolonged culture of isolated islets with palmitate is associated with increased levels of intracellular ceramide. Palmitate-induced ceramide generation is prevented by inhibitors of de novo ceramide synthesis. Further, exogenous ceramide inhibits insulin mRNA levels, whereas blockade of de novo ceramide synthesis prevents palmitate inhibition of insulin gene expression. We conclude that prolonged exposure to elevated levels of palmitate affects glucose-stimulated insulin gene expression via transcriptional mechanisms and ceramide synthesis.