Biomedical subjects

M Prentki

Publications and source records attributed to M Prentki.

At least 19 recordsLinked to original sources

PubMed2006

Beta cell compensation for insulin resistance in Zucker fatty rats: increased lipolysis and fatty acid signalling.

AIMS/HYPOTHESIS: The aim of this study was to determine the role of fatty acid signalling in islet beta cell compensation for insulin resistance in the Zucker fatty fa/fa (ZF) rat, a genetic model of severe obesity, hyperlipidaemia and insulin resistance that does not develop diabetes. MATERIALS AND METHODS: NEFA augmentation of insulin secretion and fatty acid metabolism were studied in isolated islets from ZF and Zucker lean (ZL) control rats. RESULTS: Exogenous palmitate markedly potentiated glucose-stimulated insulin secretion (GSIS) in ZF islets, allowing robust secretion at physiological glucose levels (5-8 mmol/l). Exogenous palmitate also synergised with glucagon-like peptide-1 and the cyclic AMP-raising agent forskolin to enhance GSIS in ZF islets only. In assessing islet fatty acid metabolism, we found increased glucose-responsive palmitate esterification and lipolysis processes in ZF islets, suggestive of enhanced triglyceride-fatty acid cycling. Interruption of glucose-stimulated lipolysis by the lipase inhibitor Orlistat (tetrahydrolipstatin) blunted palmitate-augmented GSIS in ZF islets. Fatty acid oxidation was also higher at intermediate glucose levels in ZF islets and steatotic triglyceride accumulation was absent. CONCLUSIONS/INTERPRETATION: The results highlight the potential importance of NEFA and glucoincretin enhancement of insulin secretion in beta cell compensation for insulin resistance. We propose that coordinated glucose-responsive fatty acid esterification and lipolysis processes, suggestive of triglyceride-fatty acid cycling, play a role in the coupling mechanisms of glucose-induced insulin secretion as well as in beta cell compensation and the hypersecretion of insulin in obesity.

Animals↗

PubMed2004

Glucagon-like peptide-1 prevents beta cell glucolipotoxicity.

AIMS/HYPOTHESIS: We have provided evidence that glucagon-like peptide-1, a potential therapeutic agent in the treatment of diabetes, activates phosphatidylinositol-3 kinase/protein kinase B signalling in the pancreatic beta cell. Since this pathway promotes cell survival in a variety of systems, we tested whether glucagon-like peptide-1 protects beta cells against cell death induced by elevated glucose and/or non-esterified fatty acids. METHODS: Human islets and INS832/13 cells were cultured at glucose concentrations of 5 or 25 mmol/l in the presence or absence of palmitate. Apoptosis was evaluated by monitoring DNA fragmentation and chromatin condensation. Wild-type and protein kinase B mutants were overexpressed in INS832/13 cells using adenoviruses. Nuclear factor-kappa B DNA binding was assayed by electrophoretic mobility shift assay. RESULTS: In human pancreatic beta cells and INS832/13 cells, glucagon-like peptide-1 prevented beta cell apoptosis induced by elevated concentrations of (i) glucose (glucotoxicity), (ii) palmitate (lipotoxicity) and (iii) both glucose and palmitate (glucolipotoxicity). Overexpression of a dominant-negative protein kinase B suppressed the anti-apoptotic action of glucagon-like peptide-1 in INS832/13 cells, whereas a constitutively active protein kinase B prevented beta cell apoptosis induced by elevated glucose and palmitate. Glucagon-like peptide-1 enhanced nuclear factor-kappa B DNA binding activity and stimulated the expression of inhibitor of apoptosis protein-2 and Bcl-2, two anti-apoptotic genes under the control of nuclear factor-kappa B. Inhibition of nuclear factor-kappa B by BAY 11-7082 abolished the prevention of glucolipotoxicity by glucagon-like peptide-1. CONCLUSIONS/INTERPRETATION: The results demonstrate a potent protective effect of glucagon-like peptide-1 on beta cell gluco-, lipo- and glucolipotoxicity. This effect is mediated via protein kinase B activation and possibly its downstream target nuclear factor-kappa B.

Apoptosis↗

PubMed2003

AMPK as a metabolic switch in rat muscle, liver and adipose tissue after exercise.

UNLABELLED: An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK)-activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol-3-phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5-aminoimidazole 4-carboxamide-riboside (AICAR), further indicating that the exercise-induced alterations in these enzymes were AMPK-mediated. CONCLUSIONS: (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post-exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.

Acetyl-CoA Carboxylase↗

PubMed2002

The effect of chronic insulin delivery via the intraperitoneal versus the subcutaneous route on hepatic triglyceride secretion rate in streptozotocin diabetic rats.

Chronic intraperitoneal or subcutaneous insulin administration increases triglyceride secretion rate (TGSR) in normal rats. We wished to determine the effect of this treatment on TGSR and the hepatic lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in diabetic rats. Streptozotocin-diabetic rats, untreated (D), diabetic rats treated with insulin (3 U/day for 21 days) intraperitoneally (IP) or subcutaneously (SC) and non-diabetic rats (N) were studied. TGSR was determined using Triton WR-1339. Fasting glucose and triglyceride levels, high in D, were normalized by insulin treatment regardless of route. Peripheral insulin levels were lowest in D and highest in SC, portal insulin levels were lowest in D and highest in IP. Non-esterified fatty acid levels were not elevated in D, presumably due to adipose tissue depletion. TGSR was reduced in D (P<0.05) and was normalized following insulin administration, regardless of route. ACC activity was normal, but FAS was decreased in D (P<0.05). ACC and FAS were normal in both IP and SC. Thus, in streptozotocin-diabetic rats, chronic intraperitoneal or subcutaneous insulin treatment increases TGSR and FAS activity from their low levels in insulin-deficient rats to levels equal to but not higher than those in normal rats.

Acetyl-CoA Carboxylase↗

PubMed2002

The antilipolytic agent 3,5-dimethylpyrazole inhibits insulin release in response to both nutrient secretagogues and cyclic adenosine monophosphate agonists in isolated rat islets.

This study intended to test the hypothesis that intracellular lipolysis in the pancreatic beta cells is implicated in the regulation of insulin secretion stimulated by nutrient secretagogues or cyclic adenosine monophosphate (cAMP) agonists. Indeed, although lipid signaling molecules were repeatedly reported to influence beta-cell function, the contribution of intracellular triglycerides to the generation of these molecules has remained elusive. Thus, we have studied insulin secretion of isolated rat pancreatic islets in response to various secretagogues in the presence or absence of 3,5-dimethylpyrazole (DMP), a water-soluble and highly effective antilipolytic agent, as previously shown in vivo. In vitro exposure of islets to DMP resulted in an inhibition (by approximately 50%) of the insulin release stimulated not only by high glucose, but also by another nutrient secretagogue, 2-ketoisocaproate, as well as the cAMP agonists 3-isobutyl-1-methylxanthine and glucagon. The inhibitory effect of DMP, which was not due to alteration of islet glucose oxidation, could be reversed upon addition of sn-1,2-dioctanoylglycerol, a synthetic diglyceride, which activates protein kinase C. The results provide direct pharmacologic evidence supporting the concept that endogenous beta-cell lipolysis plays an important role in the generation of lipid signaling molecules involved in the control of insulin secretion in response to both fuel stimuli and cAMP agonists.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes.

Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.

Acyl Coenzyme A↗

PubMed2001

Protein kinase Czeta activation mediates glucagon-like peptide-1-induced pancreatic beta-cell proliferation.

Glucagon-like peptide-1 (GLP-1), an insulinotropic and glucoincretin hormone, is a potentially important therapeutic agent in the treatment of diabetes. We previously provided evidence that GLP-1 induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol-3 kinase (PI-3K)-dependent manner. In the present study, we investigated the downstream effectors of PI-3K to determine the precise signal transduction pathways that mediate the action of GLP-1 on beta-cell proliferation. GLP-1 increased extracellular signal-related kinase 1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase B activities nonadditively with glucose in pancreatic beta(INS 832/13) cells. GLP-1 also caused nuclear translocation of the atypical protein kinase C (aPKC) zeta isoform in INS as well as in dissociated normal rat beta-cells as shown by immunolocalization and Western immunoblotting analysis. Tritiated thymidine incorporation measurements showed that the p38 MAPK inhibitor SB203580 suppressed GLP-1-induced beta-cell proliferation. Further investigation was performed using isoform-specific pseudosubstrates of classical (alpha, beta, and gamma) or zeta aPKC isoforms. The PKCzeta pseudosubstrate suppressed the proliferative action of GLP-1, whereas the inhibitor of classical PKC isoforms had no effect. Overexpression of a kinase-dead PKCzeta acting as a dominant negative protein suppressed GLP-1-induced proliferation. In addition, ectopic expression of a constitutively active PKCzeta mutant stimulated tritiated thymidine incorporation to the same extent as GLP-1, and the glucoincretin had no growth-promoting action under this condition. The data indicate that GLP-1-induced activation of PKCzeta is implicated in the beta-cell proliferative signal of the insulinotropic hormone. The results are consistent with a model in which GLP-1-induced PI-3K activation results in PKCzeta translocation to the nucleus, which may play a role in the pleiotropic effects (DNA synthesis, metabolic enzymes, and insulin gene expression) of the glucoincretin.

Animals↗

PubMed2001

Uncoupling protein 2: a possible link between fatty acid excess and impaired glucose-induced insulin secretion?

The mechanism by which long-term exposure of the beta-cell to elevated concentrations of fatty acid alters glucose-induced insulin secretion has been examined. Exposure of INS-1 beta-cells to 0.4 mmol/l oleate for 72 h increased basal insulin secretion and decreased insulin release in response to high glucose, but not in response to agents acting at the level of the K(ATP) channel (tolbutamide) or beyond (elevated KCl). This also suppressed the glucose-induced increase in the cellular ATP-to-ADP ratio. The depolarization of the plasma membrane promoted by glucose was decreased after oleate exposure, whereas the response to KCl was unchanged. Cells exposed to free fatty acids displayed a lower mitochondrial membrane potential and a decreased glucose-induced hyperpolarization. The possible implication of uncoupling protein (UCP)-2 in the altered secretory response was examined by measuring UCP2 gene expression after chronic exposure of the cells to fatty acids. UCP2 mRNA and protein were increased twofold by oleate. Palmitate and the nonoxidizable fatty acid bromopalmitate had similar effects on UCP2 mRNA, suggesting that UCP2 gene induction by fatty acids does not require their metabolism. The data are compatible with a role of UCP2 and partial mitochondrial uncoupling in the decreased secretory response to glucose observed after chronic exposure of the beta-cell to elevated fatty acids, and suggest that the expression and/or activity of the protein may modulate insulin secretion in response to glucose.

Adenosine Diphosphate↗

PubMed2001

A role for hormone-sensitive lipase in glucose-stimulated insulin secretion: a study in hormone-sensitive lipase-deficient mice.

Endogenous lipid stores are thought to be involved in the mechanism whereby the beta-cell adapts its secretory capacity in obesity and diabetes. In addition, hormone-sensitive lipase (HSL) is expressed in beta-cells and may provide fatty acids necessary for the generation of coupling factors linking glucose metabolism to insulin release. We have recently created HSL-deficient mice that were used to directly assess the role of HSL in insulin secretion and action. HSL(-/-) mice were normoglycemic and normoinsulinemic under basal conditions, but showed an approximately 30% reduction of circulating free fatty acids (FFAs) with respect to control and heterozygous animals after an overnight fast. An intraperitoneal glucose tolerance test revealed that HSL-null mice were glucose-intolerant and displayed a lack of a rise in plasma insulin after a glucose challenge. Examination of plasma glucose during an insulin tolerance test suggested that HSL-null mice were insulin-resistant, because plasma glucose was barely lowered after the injection of insulin. Freshly isolated islets from HSL-deficient mice displayed elevated secretion at low (3 mmol/l) glucose, failed to release insulin in response to high (20 mmol/l) glucose, but had a normal secretion when challenged with elevated KCl. The phenotype of heterozygous mice with respect to the measured parameters in vitro was similar to that of wild type. Finally, the islet triglyceride content of HSL(-/-) mice was 2-2.5 fold that in HSL(-/+) and HSL(+/+) animals. The results demonstrate an important role of HSL and endogenous beta-cell lipolysis in the coupling mechanism of glucose-stimulated insulin secretion. The data also provide direct support for the concept that some lipid molecule(s), such as FFAs, fatty acyl-CoA or their derivatives, are implicated in beta-cell glucose signaling.

Animals↗

PubMed2000

Glucose down-regulates the expression of the peroxisome proliferator-activated receptor-alpha gene in the pancreatic beta -cell.

To better understand the action of glucose on fatty acid metabolism in the beta-cell and the link between chronically elevated glucose or fatty acids and beta-cell decompensation in adipogenic diabetes, we investigated whether glucose regulates peroxisomal proliferator-activated receptor (PPAR) gene expression in the beta-cell. Islets or INS(832/13) beta-cells exposed to high glucose show a 60-80% reduction in PPARalpha mRNA expression. Oleate, either in the absence or presence of glucose, has no effect. The action of glucose is dose-dependent in the 6-20 mm range and maximal after 6 h. Glucose also causes quantitatively similar reductions in PPARalpha protein and DNA binding activity of this transcription factor. The effect of glucose is blocked by the glucokinase inhibitor mannoheptulose, is partially mimicked by 2-deoxyglucose, and is not blocked by the 3-O-methyl or the 6-deoxy analogues of the sugar that are not phosphorylated. Chronic elevated glucose reduces the expression levels of the PPAR target genes, uncoupling protein 2 and acyl-CoA oxidase, which are involved in fat oxidation and lipid detoxification. A 3-day exposure of INS-1 cells to elevated glucose results in a permanent rise in malonyl-CoA, the inhibition of fat oxidation, and the promotion of fatty acid esterification processes and causes elevated insulin secretion at low glucose. The results suggest that a reduction in PPARalpha gene expression together with a rise in malonyl-CoA plays a role in the coordinated adaptation of beta-cell glucose and lipid metabolism to hyperglycemia and may be implicated in the mechanism of beta-cell "glucolipotoxicity."

Animals↗

PubMed2000

Oleate activates phosphatidylinositol 3-kinase and promotes proliferation and reduces apoptosis of MDA-MB-231 breast cancer cells, whereas palmitate has opposite effects.

Epidemiological studies and experiments using animal models and cultured breast cancer cells have suggested that a high intake of dietary fat could increase breast cancer risk. Little is known about the biochemical pathways by which various free fatty acids (FFAs) influence breast cancer cell proliferation and apoptosis. The present study was designed to investigate the effects of the two most abundant circulating FFAs, oleate and palmitate, on established human breast cancer cell lines after a short period of serum starvation. The unsaturated FFA oleate (C:18:1) stimulated cell proliferation, whereas the saturated FFA palmitate (C:16) dose dependently inhibited it. The half maximal effective concentrations of oleate and palmitate in the presence of albumin were 5 and 25 microM, respectively. The growth-inhibitory effect of palmitate in MDA-MB-231 cells was related to the induction of apoptosis as indicated by morphological and biochemical criteria. Moreover, oleate protected cells against the proapoptotic action of palmitate. Oleate and palmitate increased and decreased phophatidylinositol 3-kinase (PI3-K) activity, respectively, and the actions of the two FFAs on the enzyme were antagonistic. The PI3-K inhibitors wortmannin and LY294002 completely blocked the proliferative action of oleate. 2-Bromopalmitate, a nonmetabolizable analogue, did not affect MDA-MB-231 cell proliferation, suggesting that palmitate must be metabolized to exert its effect. Thus, various types of fatty acids are not equivalent with respect to their actions on breast cancer cell proliferation and apoptosis. The results support the concept that PI3-K is implicated in the control of breast cancer cell growth by FFAs and that PI3-K may provide a link between fat and cancer. The data are also consistent with the view that the type of FFA and their ratios in the diet in addition to the total amount of fat influence mammary carcinogenesis.

Apoptosis↗

PubMed2000

Characterization of rat liver malonyl-CoA decarboxylase and the study of its role in regulating fatty acid metabolism.

In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic beta-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54. 7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.7 kDa in size. To address this, we transiently transfected a mutated MCD expression plasmid (second ATG to GCG) into COS-7 cells and performed Western blot analysis using our anti-MCD antibody. Western blot analysis revealed that two isoforms of MCD were still present, demonstrating that the second ATG may not be responsible for translation of the 50.7 kDa isoform of MCD. These data also suggest that the smaller isoform of MCD may originate from intracellular processing. To ascertain the functional role of the 50. 7 kDa isoform of rat liver MCD, we measured liver MCD activity and expression in rats subjected to conditions which are known to alter fatty acid metabolism. The activity of MCD was significantly elevated under conditions in which hepatic fatty acid oxidation is known to increase, such as streptozotocin-induced diabetes or following a 48 h fast. A 2-fold increase in expression was observed in the streptozotocin-diabetic rats compared with control rats. In addition, MCD activity was shown to be enhanced by alkaline phosphatase treatment, suggesting phosphorylation-related control of the enzyme. Taken together, our data demonstrate that rat liver expresses a 50.7 kDa form of MCD which does not originate from the second methionine of the cDNA sequence. This MCD is regulated by at least two mechanisms (only one of which is phosphorylation), and its activity and expression are increased under conditions where fatty acid oxidation increases.

Alkaline Phosphatase↗

PubMed2000

Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contraction and the AMP-activated protein kinase activator 5-aminoimidazole-4-carboxamide-1-beta -D-ribofuranoside.

Alterations in the concentration of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, have been linked to the regulation of fatty acid oxidation in skeletal muscle. During contraction decreases in muscle malonyl-CoA concentration have been related to activation of AMP-activated protein kinase (AMPK), which phosphorylates and inhibits acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in malonyl-CoA formation. We report here that the activity of malonyl-CoA decarboxylase (MCD) is increased in contracting muscle. Using either immunopurified enzyme or enzyme partially purified by (NH(4))(2)SO(4) precipitation, 2-3-fold increases in the V(max) of MCD and a 40% decrease in its K(m) for malonyl-CoA (190 versus 119 micrometer) were observed in rat gastrocnemius muscle after 5 min of contraction, induced by electrical stimulation of the sciatic nerve. The increase in MCD activity was markedly diminished when immunopurified enzyme was treated with protein phosphatase 2A or when phosphatase inhibitors were omitted from the homogenizing solution and assay mixture. Incubation of extensor digitorum longus muscle for 1 h with 2 mm 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, a cell-permeable activator of AMPK, increased MCD activity 2-fold. Here, too, addition of protein phosphatase 2A to the immunopellets reversed the increase of MCD activity. The results strongly suggest that activation of AMPK during muscle contraction leads to phosphorylation of MCD and an increase in its activity. They also suggest a dual control of malonyl-CoA concentration by ACC and MCD, via AMPK, during exercise.

Adenylate Kinase↗

PubMed2000

Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells).

Non-insulin-dependent diabetes mellitus is associated with, in addition to impaired insulin release, elevated levels of free fatty acids (FFA) in the blood. Insulin release is stimulated when beta-cells are acutely exposed to FFA, whereas chronic exposure may inhibit glucose-induced insulin secretion. In the present study we investigated the direct effects of long chain acyl-CoA (LC-CoA), the active intracellular form of FFA, on insulin exocytosis. Palmitoyl-CoA stimulated both insulin release from streptolysin-O-permeabilized HIT cells and fusion of secretory granules to the plasma membrane of mouse pancreatic beta-cells, as measured by cell capacitance. The LC-CoA effect was chain length-dependent, requiring chain lengths of at least 14 carbons. LC-CoA needed to be present to stimulate insulin release, and consequently there was no effect following its removal. The stimulatory effect was observed after inhibition of protein kinase activity and in the absence of ATP, even though both kinases and ATP, themselves, modulate exocytosis. The effect of LC-CoA was inhibited by cerulenin, which has been shown to block protein acylation. The data suggest that altered LC-CoA levels, resulting from FFA or glucose metabolism, may act directly on the exocytotic machinery to stimulate insulin release by a mechanism involving LC-CoA protein binding.

Animals↗

PubMed2000

Metabolic control of beta-cell function.

Glucose-induced insulin secretion is pulsatile. Glucose metabolism generates oscillations in the ATP/ADP ratio which lead to opening and closing of ATP-sensitive K(+)-channels producing subsequent oscillations in membrane potential, cytoplasmic calcium and insulin release. Metabolic signals derived from glucose can also stimulate insulin release independent of their effects on ATP-sensitive K(+)-channels. The ATP/ADP ratio may mediate both ATP-sensitive K(+)-channel-dependent and -independent pathways of secretion. Glucose metabolism also results in an increase in long-chain acyl-CoA, which is proposed to act as an effector molecule in the beta -cell. Long-chain acyl-CoA has a variety of effects in the beta -cell that may effect insulin secretion including opening ATP-sensitive K(+)-channels, activating endoplasmic reticulum Ca(2+)-ATPases and stimulating classical protein kinase C activity. In addition to stimulating insulin release, nutrients also effect gene expression, protein synthesis and beta -cell proliferation. Gene expression is effected by nutrient induction of a variety of immediate early response genes. Glucose stimulates proinsulin biosynthesis both at the translational and transcriptional level. beta -cell proliferation, as a result of insulin-like growth factor and growth hormone mitogenic pathways, is also glucose dependent. Thus, many beta -cell functions in addition to secretion are controlled by nutrient metabolism.

Acyl Coenzyme A↗

PubMed2000

The role of long-chain fatty acyl-CoA esters in beta-cell signal transduction.

Glucose-induced insulin secretion is associated with inhibition of free fatty acid (FFA) oxidation, increased esterification and complex lipid formation by pancreatic beta-cells. Abundant evidence favors a role for cytosolic long-chain acyl-CoA (LC-CoA), including the rapid rise in malonyl CoA, the inhibitory effect of hydroxycitrate or acetyl CoA carboxylase knockout, both of which prevent malonyl CoA formation, and the stimulatory effect of exogenous FFA. On the other hand, some evidence opposes the concept, including the fall in total LC-CoA levels in response to glucose, the stimulatory effect of LC-CoA on K(ATP) channels and the lack of inhibition of glucose-stimulated secretion either by overexpression of malonyl CoA decarboxylase, which markedly lowers malonyl CoA levels, or by triacsin C, which blocks FFA conversion to LC-CoA. Alternative explanations for these data are presented. A revised model of nutrient-stimulated secretion involving two arms of signal transduction that occur simultaneously is proposed. One arm depends on modulation of the K(ATP) channel evoked by changes in the ATP/ADP ratio. The other arm depends upon anaplerotic input into the tricarboxylic acid cycle, generation of excess citrate, and increases in cytosolic malonyl-CoA. Input from this arm is increased LC-CoA. Signaling through both arms would be required for normal secretion. LC-CoA esters and products formed from them are potent regulators of enzymes and channels. It is hypothesized that their elevations directly modulate the activity of enzymes, genes and various beta-cell functions or modify the acylation state of key proteins involved in regulation of ion channels and exocytosis.

Acyl Coenzyme A↗

PubMed2000

Dehydroepiandrosterone sulfate and beta-cell function: enhanced glucose-induced insulin secretion and altered gene expression in rodent pancreatic beta-cells.

Administration of dehydroepiandrosterone (DHEA), or its sulfated form (DHEAS), controls hyperglycemia in diabetic rodents without directly altering insulin sensitivity. We show that DHEAS enhanced glucose-stimulated insulin secretion when administered in vivo to rats or in vitro to beta-cell lines, without changing cellular insulin content. Insulin secretion increased from 3 days of steroid exposure in vitro, suggesting that DHEAS did not directly activate the secretory processes. DHEAS selectively increased the beta-cell mRNA expression of acyl CoA synthetase-2 and peroxisomal acyl CoA oxidase in a time-dependent manner. Although DHEAS is a peroxisomal proliferator, it did not alter the mRNA expression of peroxisomal proliferator-activated receptor (PPAR) alpha or beta, or enhance the activity of transfected PPAR alpha, beta, or gamma in vitro. Thus, DHEAS directly affected the beta-cell to enhance glucose-stimulated insulin secretion and increased the mRNA expression of specific beta-cell mitochondrial and peroxisomal lipid metabolic enzymes. This effect of DHEAS on insulin secretion may contribute to the amelioration of hyperglycemia seen in various rodent models of diabetes.

Acyl-CoA Oxidase↗

PubMed2000

Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.

The biochemical mechanisms involved in regulation of insulin secretion are not completely understood. The rat INS-1 cell line has been used to gain insight in this area because it secretes insulin in response to glucose concentrations in the physiological range. However, the magnitude of the response is far less than that seen in freshly isolated rat islets. In the current study, we have stably transfected INS-1 cells with a plasmid containing the human proinsulin gene. After antibiotic selection and clonal expansion, 67% of the resultant clones were found to be poorly responsive to glucose in terms of insulin secretion (< or =2-fold stimulation by 15 mmol/l compared with 3 mmol/l glucose), 17% of the clones were moderately responsive (2- to 5-fold stimulation), and 16% were strongly responsive (5- to 13-fold stimulation). The differences in responsiveness could not be ascribed to differences in insulin content. Detailed analysis of one of the strongly responsive lines (832/13) revealed that its potent response to glucose (average of 10-fold) was stable over 66 population doublings (approximately 7.5 months of tissue culture) with half-maximal stimulation at 6 mmol/l glucose. Furthermore, in the presence of 15 mmol/l glucose, insulin secretion was potentiated significantly by 100 pmol/l isobutylmethylxanthine (320%), 1 mmol/l oleate/palmitate (77%), and 50 nmol/l glucagon-like peptide 1 (60%), whereas carbachol had no effect. Glucose-stimulated insulin secretion was also potentiated by the sulfonylurea tolbutamide (threefold at 3 mmol/l glucose and 50% at 15 mmol/l glucose) and was abolished by diazoxide, which demonstrates the operation of the ATP-sensitive K+ channel (K(ATP)) in 832/13 cells. Moreover, when the K(ATP) channel was bypassed by incubation of cells in depolarizing K+ (35 mmol/l), insulin secretion was more effectively stimulated by glucose in 832/13 cells than in parental INS-1 cells, which demonstrates the presence of a K(ATP) channel-independent pathway of glucose sensing. We conclude that clonal selection of INS-1 cells allows isolation of cell lines that exhibit markedly enhanced and stable responsiveness to glucose and several of its known potentiators. These lines may be attractive new vehicles for studies of beta-cell function.

Adenosine Triphosphate↗