Identification of a mammalian glycerol-3-phosphate phosphatase: Role in metabolism and signaling in pancreatic ß-cells and hepatocytes.

Obesity, and the associated disturbed glycerolipid/fatty acid (GL/FA) cycle, contribute to insulin resistance, islet ß-cell failure, and type 2 diabetes. Flux through the GL/FA cycle is regulated by the availability of glycerol-3-phosphate (Gro3P) and fatty acyl-CoA. We describe here a mammalian Gro3P phosphatase (G3PP), which was not known to exist in mammalian cells, that can directly hydrolyze Gro3P to glycerol. We identified that mammalian phosphoglycolate phosphatase, with an uncertain function, acts in fact as a G3PP. We found that G3PP, by controlling Gro3P levels, regulates glycolysis and glucose oxidation, cellular redox and ATP production, gluconeogenesis, glycerolipid synthesis, and fatty acid oxidation in pancreatic islet ß-cells and hepatocytes, and that glucose stimulated insulin secretion and the response to metabolic stress, e.g., glucolipotoxicity, in ß-cells. In vivo overexpression of G3PP in rat liver lowers body weight gain and hepatic glucose production from glycerol and elevates plasma HDL levels. G3PP is expressed at various levels in different tissues, and its expression varies according to the nutritional state in some tissues. As Gro3P lies at the crossroads of glucose, lipid, and energy metabolism, control of its availability by G3PP adds a key level of metabolic regulation in mammalian cells, and G3PP offers a potential target for type 2 diabetes and cardiometabolic disorders.

Identification of the signals for glucose-induced insulin secretion in INS1 (832/13) ß-cells using metformin-induced metabolic deceleration as a model.

Metabolic deceleration in pancreatic ß-cells is associated with inhibition of glucose-induced insulin secretion (GIIS), but only in the presence of intermediate/submaximal glucose concentrations. Here, we used acute metformin treatment as a tool to induce metabolic deceleration in INS1 (832/13) ß-cells, with the goal of identifying key pathways and metabolites involved in GIIS. Metabolites and pathways previously implicated as signals for GIIS were measured in the cells at 2-25 mm glucose, with or without 5 mm metformin. We defined three criteria to identify candidate signals: 1) glucose-responsiveness, 2) sensitivity to metformin-induced inhibition of the glucose effect at intermediate glucose concentrations, and 3) alleviation of metformin inhibition by elevated glucose concentrations. Despite the lack of recovery from metformin-induced impairment of mitochondrial energy metabolism (glucose oxidation, O2 consumption, and ATP production), insulin secretion was almost completely restored at elevated glucose concentrations. Meeting the criteria for candidates involved in promoting GIIS were the following metabolic indicators and metabolites: cytosolic NAD+/NADH ratio (inferred from the dihydroxyacetone phosphate:glycerol-3-phosphate ratio), mitochondrial membrane potential, ADP, Ca2+, 1-monoacylglycerol, diacylglycerol, malonyl-CoA, and HMG-CoA. On the contrary, most of the purine and nicotinamide nucleotides, acetoacetyl-CoA, H2O2, reduced glutathione, and 2-monoacylglycerol were not glucose-responsive. Overall these results underscore the significance of mitochondrial energy metabolism-independent signals in GIIS regulation; in particular, the candidate lipid signaling molecules 1-monoacylglycerol, diacylglycerol, and malonyl-CoA; the predominance of KATP/Ca2+ signaling control by low ADP·Mg2+ rather than by high ATP levels; and a role for a more oxidized state (NAD+/NADH) in the cytosol during GIIS that favors high glycolysis rates.

Metabolic fate of glucose and candidate signaling and excess-fuel detoxification pathways in pancreatic ß-cells.

Glucose metabolism promotes insulin secretion in ß-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes ß-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the ß-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in ß-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in ß-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters.

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/ß-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

A beta cell ATGL-lipolysis/adipose tissue axis controls energy homeostasis and body weight via insulin secretion in mice.

AIMS/HYPOTHESIS: To directly assess the role of beta cell lipolysis in insulin secretion and whole-body energy homeostasis, inducible beta cell-specific adipose triglyceride lipase (ATGL)-deficient (B-Atgl-KO) mice were studied under normal diet (ND) and high-fat diet (HFD) conditions. METHODS: Atgl flox/flox mice were cross-bred with Mip-Cre-ERT mice to generate Mip-Cre-ERT/+;Atgl flox/flox mice. At 8 weeks of age, these mice were injected with tamoxifen to induce deletion of beta cell-specific Atgl (also known as Pnpla2), and the mice were fed an ND or HFD. RESULTS: ND-fed male B-Atgl-KO mice showed decreased insulinaemia and glucose-induced insulin secretion (GSIS) in vivo. Changes in GSIS correlated with the islet content of long-chain saturated monoacylglycerol (MAG) species that have been proposed to be metabolic coupling factors for insulin secretion. Exogenous MAGs restored GSIS in B-Atgl-KO islets. B-Atgl-KO male mice fed an HFD showed reduced insulinaemia, glycaemia in the fasted and fed states and after glucose challenge, as well as enhanced insulin sensitivity. Moreover, decreased insulinaemia in B-Atgl-KO mice was associated with increased energy expenditure, and lipid metabolism in brown (BAT) and white (WAT) adipose tissues, leading to reduced fat mass and body weight. CONCLUSIONS/INTERPRETATION: ATGL in beta cells regulates insulin secretion via the production of signalling MAGs. Decreased insulinaemia due to lowered GSIS protects B-Atgl-KO mice from diet-induced obesity, improves insulin sensitivity, increases lipid mobilisation from WAT and causes BAT activation. The results support the concept that fuel excess can drive obesity and diabetes via hyperinsulinaemia, and that an islet beta cell ATGL-lipolysis/adipose tissue axis controls energy homeostasis and body weight via insulin secretion.

Adipose triglyceride lipase is implicated in fuel- and non-fuel-stimulated insulin secretion.

Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous beta-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL(-/-) mice indicated the presence of other TG lipase(s) in the beta-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K(ATP)-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL(-/-) mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL(-/-) mice. Accordingly, isolated islets from ATGL(-/-) mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL(-/-) islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.

Adipose ABHD6 regulates tolerance to cold and thermogenic programs.

Enhanced energy expenditure in brown (BAT) and white adipose tissues (WAT) can be therapeutic against metabolic diseases. We examined the thermogenic role of adipose α/ß-hydrolase domain 6 (ABHD6), which hydrolyzes monoacylglycerol (MAG), by employing adipose-specific ABHD6-KO mice. Control and KO mice showed similar phenotypes at room temperature and thermoneutral conditions. However, KO mice were resistant to hypothermia, which can be accounted for by the simultaneously increased lipolysis and lipogenesis of the thermogenic glycerolipid/free fatty acid (GL/FFA) cycle in visceral fat, despite unaltered uncoupling protein 1 expression. Upon cold stress, nuclear 2-MAG levels increased in visceral WAT of the KO mice. Evidence is provided that 2-MAG causes activation of PPARα in white adipocytes, leading to elevated expression and activity of GL/FFA cycle enzymes. In the ABHD6-ablated BAT, glucose and oxidative metabolism were elevated upon cold induction, without changes in GL/FFA cycle and lipid turnover. Moreover, response to in vivo ß3-adrenergic stimulation was comparable between KO and control mice. Our data reveal a MAG/PPARα/GL/FFA cycling metabolic signaling network in visceral adipose tissue, which contributes to cold tolerance, and that adipose ABHD6 is a negative modulator of adaptive thermogenesis.

EIF4A2 is a positional candidate gene at the 3q27 locus linked to type 2 diabetes in French families.

One of the most replicated loci influencing type 2 diabetes-related quantitative traits (quantitative trait loci [QTL]) is on chromosome 3q27 and modulates both type 2 diabetes-and metabolic syndrome-associated phenotypes. A QTL for type 2 diabetes age of onset (logarithm of odds [LOD] score = 3.01 at D3S3686, P = 0.0001) was identified in a set of French families. To assess genetic variation underlying both age-of-onset QTL and our previous type 2 diabetes linkage in a 3.87-Mb interval, we explored 36 single nucleotide polymorphisms (SNPs) in two biologically relevant candidate genes for glucose homeostasis, kininogen (KNG1), and eukaryotic translation initiation factor 4alpha2 (EIF4A2). Analysis of 148 families showed significant association of a frequent SNP, rs266714, located 2.47 kb upstream of EIF4A2, with familial type 2 diabetes (family-based association test, P = 0.0008) and early age of onset (P = 0.0008). This SNP also contributes to both age-of-onset QTL (1.13 LOD score decrease P = 0.02) and type 2 diabetes linkage (genotype identical-by-descent sharing test, P = 0.02). However, no association was observed in three independent European diabetic cohorts. EIF4A2 controls specific mRNA translation and protein synthesis rate in pancreatic beta-cells, and our data indicates that EIF4A2 is downregulated by high glucose in rat beta-INS832/13 cells. The potential role of EIF4A2 in glucose homeostasis and its putative contribution to type 2 diabetes in the presence of metabolic stress will require further investigation.

Differential Insulin Secretion of High-Fat Diet-Fed C57BL/6NN and C57BL/6NJ Mice: Implications of Mixed Genetic Background in Metabolic Studies.

Many metabolic studies employ tissue-specific gene knockout mice, which requires breeding of floxed gene mice, available mostly on C57BL/6N (NN) genetic background, with cre or Flp recombinase-expressing mice, available on C57BL/6J (JJ) background, resulting in the generation of mixed C57BL/6NJ (NJ) genetic background mice. Recent awareness of many genetic differences between NN and JJ strains including the deletion of nicotinamide nucleotide transhydrogenase (nnt), necessitates examination of the consequence of mixed NJ background on glucose tolerance, beta cell function and other metabolic parameters. Male mice with NN and NJ genetic background were fed with normal or high fat diets (HFD) for 12 weeks and glucose and insulin homeostasis were studied. Genotype had no effect on body weight and food intake in mice fed normal or high fat diets. Insulinemia in the fed and fasted states and after a glucose challenge was lower in HFD-fed NJ mice, even though their glycemia and insulin sensitivity were similar to NN mice. NJ mice showed mild glucose intolerance. Moreover, glucose- but not KCl-stimulated insulin secretion in isolated islets was decreased in HFD-fed NJ vs NN mice without changes in insulin content and beta cell mass. Under normal diet, besides reduced fed insulinemia, NN and NJ mice presented similar metabolic parameters. However, HFD-fed NJ mice displayed lower fed and fasted insulinemia and glucose-induced insulin secretion in vivo and ex vivo, as compared to NN mice. These results strongly caution against using unmatched mixed genetic background C57BL/6 mice for comparisons, particularly under HFD conditions.

Pancreatic ß-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression.

Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of ß-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany ß-cell failure in HDR islets. The ß-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression.

α/ß-Hydrolase Domain 6 Deletion Induces Adipose Browning and Prevents Obesity and Type 2 Diabetes.

Suppression of α/ß-domain hydrolase-6 (ABHD6), a monoacylglycerol (MAG) hydrolase, promotes glucose-stimulated insulin secretion by pancreatic ß cells. We report here that high-fat-diet-fed ABHD6-KO mice show modestly reduced food intake, decreased body weight gain and glycemia, improved glucose tolerance and insulin sensitivity, and enhanced locomotor activity. ABHD6-KO mice also show increased energy expenditure, cold-induced thermogenesis, brown adipose UCP1 expression, fatty acid oxidation, and white adipose browning. Adipose browning and cold-induced thermogenesis are replicated by the ABHD6 inhibitor WWL70 and by antisense oligonucleotides targeting ABHD6. Our evidence suggests that one mechanism by which the lipolysis derived 1-MAG signals intrinsic and cell-autonomous adipose browning is via PPARα and PPARγ activation, and that ABHD6 regulates adipose browning by controlling signal competent 1-MAG levels. Thus, ABHD6 regulates energy homeostasis, brown adipose function, and white adipose browning and is a potential therapeutic target for obesity and type 2 diabetes.

Adenosine 5'-monophosphate-activated protein kinase and p38 mitogen-activated protein kinase participate in the stimulation of glucose uptake by dinitrophenol in adult cardiomyocytes.

During metabolic stress, such as ischemia or hypoxia, glucose becomes the principal energy source for the heart. It has been shown that increased cardiac glucose uptake during metabolic stress has a protective effect on cell survival and heart function. Despite its physiological importance, only limited data are available on the molecular mechanisms regulating glucose uptake under these conditions. We used 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, as a model to mimic hypoxia and gain insight into the signaling pathway underlying metabolic stress-induced glucose uptake in primary cultures of rat adult cardiomyocytes. The results demonstrate that 0.1 mM DNP induces 2.2- and 9-fold increases in AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation, respectively. This is associated with a 2.3-fold increase in glucose uptake in these cells. To further delineate the role of AMPK in the regulation of glucose uptake, we used two complementary approaches: pharmacological inhibition of the enzyme with adenine 9-beta-D arabinofuranoside and adenoviral infection with a dominant-negative AMPK (DN-AMPK) mutant. Our results show that overexpression of DN-AMPK completely suppressed DNP-mediated phosphorylation of acetyl coenzyme A carboxylase, a downstream target of AMPK. Inhibition of AMPK with either 9-beta-D arabinofuranoside or DN-AMPK also abolished DNP-mediated p38 MAPK phosphorylation. Importantly, AMPK inhibition only partially decreased DNP-stimulated glucose uptake in cardiomyocytes. Inhibition of p38 MAPK with the pharmacological agent PD169316 also partially reduced (70%) glucose uptake in response to DNP. In conclusion, our results indicate that p38 MAPK acts downstream of AMPK in cardiomyocytes and that activation of the AMPK/p38 MAPK signaling cascade is essential for maximal stimulation of glucose uptake in response to DNP in adult cardiomyocytes.

Glucagon-like peptide 1 induces pancreatic beta-cell proliferation via transactivation of the epidermal growth factor receptor.

We previously provided evidence that glucagon-like peptide 1 (GLP-1) induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol (PI) 3-kinase- and protein kinase C zeta-dependent manner. However, the exact mechanism by which the GLP-1 receptor (GLP-1R), a member of the G protein-coupled receptor (GPCR) superfamily, activates the PI 3-kinase signaling pathway to promote beta-cell growth remains unknown. We hypothesized that the GLP-1R could activate PI 3-kinase and promote beta-cell proliferation through transactivation of the epidermal growth factor (EGF) receptor (EGFR), an event possibly linked to GPCRs via activation of c-Src and the production of putative endogenous EGF-like ligands. Both the c-Src inhibitor PP1 and the EGFR-specific inhibitor AG1478 blocked GLP-1-induced [(3)H]thymidine incorporation in INS(832/13) cells as well as in isolated rat islets, while only AG1478 inhibited the proliferative action of betacellulin (BTC), an EGFR agonist. Both compounds also suppressed GLP-1-induced PI 3-kinase activation. A time-dependent increase in tyrosine phosphorylation of the EGFR in response to GLP-1 was observed in INS(832/13) cells. This transactivation of the EGFR was sensitive to both the pharmacological agents PP1 and AG1478. The action of GLP-1 and BTC on INS cell proliferation was found to be not additive. Overexpression of a dominant-negative EGFR in INS cells with a retroviral expression vector curtailed GLP-1-induced beta-cell proliferation. GLP-1 treatment of INS cells caused a decrease in cell surface-associated BTC, as shown by FACS analysis. Also, the metalloproteinase inhibitor GM6001 and an anti-BTC neutralizing antibody suppressed the GLP-1 proliferative effect. Finally, coculturing the prostatic cancer cell line LNCaP that lacks GLP-1 responsiveness with INS cells increased LNCaP cell proliferation in the presence of GLP-1, thus revealing that INS cells secrete a growth factor in response to GLP-1. GM6001 and an anti-BTC neutralizing antibody suppressed increased LNCaP cell proliferation in the presence of GLP-1 in the coculture experiments. The results are consistent with a model in which GLP-1 increases PI 3-kinase activity and enhances beta-cell proliferation via transactivation of the EGFR that would require the proteolytic processing of membrane-anchored BTC or other EGF-like ligands.

Malonyl-CoA signaling, lipid partitioning, and glucolipotoxicity: role in beta-cell adaptation and failure in the etiology of diabetes.

Beta-cells possess inherent mechanisms to adapt to overnutrition and the prevailing concentrations of glucose, fatty acids, and other fuels to maintain glucose homeostasis. However, this is balanced by potentially harmful actions of the same nutrients. Both glucose and fatty acids may cause good/adaptive or evil/toxic actions on the beta-cell, depending on their concentrations and the time during which they are elevated. Chronic high glucose dramatically influences beta-cell lipid metabolism via substrate availability, changes in the activity and expression of enzymes of glucose and lipid metabolism, and modifications in the expression level of key transcription factors. We discuss here the emerging view that beta-cell "glucotoxicity" is in part indirectly caused by "lipotoxicity," and that beta-cell abnormalities will become particularly apparent when both glucose and circulating fatty acids are high. We support the concept that elevated glucose and fatty acids synergize in causing toxicity in islets and other organs, a process that may be instrumental in the pleiotropic defects associated with the metabolic syndrome and type 1 and type 2 diabetes. The mechanisms by which hyperglycemia and hyperlipidemia alter insulin secretion are discussed and a model of beta-cell "glucolipotoxicity" that implicates alterations in beta-cell malonyl-CoA concentrations; peroxisome proliferator-activated receptor-alpha and -gamma and sterol regulatory element binding protein-1c expression; and lipid partitioning is proposed.

Hormone-sensitive lipase has a role in lipid signaling for insulin secretion but is nonessential for the incretin action of glucagon-like peptide 1.

We previously reported decreased glucose-stimulated insulin secretion (GSIS) in hormone-sensitive lipase-null mice (HSL(-/-)), both in vivo and in vitro. The focus of the current study was to gain further insight into the signaling role and regulation of lipolysis in islet tissue. The effect of glucagon-like peptide 1 (GLP-1) on GSIS was also studied, as GLP-1 could augment GSIS via protein kinase A activation of HSL and lipolysis. Freshly isolated islets from fasted and fed male HSL(-/-) and wild-type (HSL(+/+)) mice were studied at ages 4 and 7 months. Neutral cholesteryl ester hydrolase activity was markedly reduced in islets from both 4- and 7-month-old male HSL(-/-) mice, whereas a marked deficiency in triglyceride lipase activity became evident only in the older mice. The deficiencies in lipase activities were associated with higher islet triglyceride content and reduced lipolysis at basal glucose levels. Lipolysis was stimulated by high glucose in islets of both wild-type and HSL-null mice. Severe deficiencies in GSIS were found, but only in islets from 7-month-old, fasted, male HSL(-/-) mice. GSIS was less affected in 4-month-old fasted male HSL(-/-) mice and not reduced in female mice. Exogenous delivery of free fatty acids (FFAs) rescued GSIS, supporting the view that the lack of endogenous FFA supply for lipid-signaling processes in HSL(-/-) mice was responsible for the loss of GSIS. GLP-1 also rescued GSIS in HSL(-/-) mice, indicating that signaling via HSL is not a major pathway for its incretin effect. Thus, the secretory phenotype of HSL-null mice is gender dependent, increases with age, and is influenced by the nutritional state. Under most circumstances, the major determinant of lipolytic flux in the beta-cell involves an enzyme(s) other than HSL that is acutely activated by glucose. Our results support the view that the availability of endogenous FFA through HSL and an additional enzyme(s) is involved in providing lipid moieties for beta-cell signaling for secretion in response to glucose.

Malonyl-CoA decarboxylase is present in the cytosolic, mitochondrial and peroxisomal compartments of rat hepatocytes.

A role for cytosolic malonyl-CoA decarboxylase (MCD) as a regulator of fatty acid oxidation has been postulated. However, there is no direct evidence that MCD is present in the cytosol. To address this issue, we performed cell fractionation and electron microscopic colloidal gold studies of rat liver to determine the location and activity of MCD. By both methods, substantial amounts of MCD protein and activity were found in the cytosol, mitochondria and peroxisomes, the latter with the highest specific activity. MCD species with different electrophoretic mobility were observed in the three fractions. The data demonstrate that active MCD is present in the cytosol, mitochondria and peroxisomes of rat liver, consistent with the view that MCD participates in the regulation of cytosolic malonyl-CoA levels and of hepatic fatty acid oxidation.

α/ß-Hydrolase domain-6 and saturated long chain monoacylglycerol regulate insulin secretion promoted by both fuel and non-fuel stimuli.

OBJECTIVE: α/ß-Hydrolase domain-6 (ABHD6) is a newly identified monoacylglycerol (MAG) lipase. We recently reported that it negatively regulates glucose stimulated insulin secretion (GSIS) in the ß cells by hydrolyzing lipolysis-derived MAG that acts as a metabolic coupling factor and signaling molecule via exocytotic regulator Munc13-1. Whether ABHD6 and MAG play a role in response to all classes of insulin secretagogues, in particular various fuel and non-fuel stimuli, is unknown. METHODS: Insulin secretion in response to various classes of secretagogues, exogenous MAG and pharmacological agents was measured in islets of mice deficient in ABHD6 specifically in the ß cell (BKO). Islet perifusion experiments and determinations of glucose and fatty acid metabolism, cytosolic Ca(2+) and MAG species levels were carried out. RESULTS: Deletion of ABHD6 potentiated insulin secretion in response to the fuels glutamine plus leucine and α-ketoisocaproate and to the non-fuel stimuli glucagon-like peptide 1, carbamylcholine and elevated KCl. Fatty acids amplified GSIS in control and BKO mice to the same extent. Exogenous 1-MAG amplified insulin secretion in response to fuel and non-fuel stimuli. MAG hydrolysis activity was greatly reduced in BKO islets without changes in total diacylglycerol and triacylglycerol lipase activity. ABHD6 deletion induced insulin secretion independently from KATP channels and did not alter the glucose induced rise in intracellular Ca(2+). Perifusion studies showed elevated insulin secretion during second phase of GSIS in BKO islets that was not due to altered cytosolic Ca(2+) signaling or because of changes in glucose and fatty acid metabolism. Glucose increased islet saturated long chain 1-MAG species and ABHD6 deletion caused accumulation of these 1-MAG species at both low and elevated glucose. CONCLUSION: ABHD6 regulates insulin secretion in response to fuel stimuli at large and some non-fuel stimuli by controlling long chain saturated 1-MAG levels that synergize with other signaling pathways for secretion.

Saturated fatty acids synergize with elevated glucose to cause pancreatic beta-cell death.

We have proposed the "glucolipotoxicity" hypothesis in which elevated free fatty acids (FFAs) together with hyperglycemia are synergistic in causing islet beta-cell damage because high glucose inhibits fat oxidation and consequently lipid detoxification. The effects of 1-2 d culture of both rat INS 832/13 cells and human islet beta-cells were investigated in medium containing glucose (5, 11, 20 mM) in the presence or absence of various FFAs. A marked synergistic effect of elevated concentrations of glucose and saturated FFA (palmitate and stearate) on inducing beta-cell death by apoptosis was found in both INS 832/13 and human islet beta-cells. In comparison, linoleate (polyunsaturated) synergized only modestly with high glucose, whereas oleate (monounsaturated) was not toxic. Treating cells with the acyl-coenzyme A synthase inhibitor triacsin C, or the AMP kinase activators metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside that redirect lipid partitioning to oxidation, curtailed glucolipotoxicity. In contrast, the fat oxidation inhibitor etomoxir, like glucose, markedly enhanced palmitate-induced cell death. The data indicate that FFAs must be metabolized to long chain fatty acyl-CoA to exert toxicity, the effect of which can be reduced by activating fatty acid oxidation. The results support the glucolipotoxicity hypothesis of beta-cell failure proposing that elevated FFAs are particularly toxic in the context of hyperglycemia.

Defective insulin secretory response to intravenous glucose in C57Bl/6J compared to C57Bl/6N mice.

OBJECTIVE: The C57Bl/6J (Bl/6J) mouse is the most widely used strain in metabolic research. This strain carries a mutation in nicotinamide nucleotide transhydrogenase (Nnt), a mitochondrial enzyme involved in NADPH production, which has been suggested to lead to glucose intolerance and beta-cell dysfunction. However, recent reports comparing Bl/6J to Bl/6N (carrying the wild-type Nnt allele) under normal diet have led to conflicting results using glucose tolerance tests. Thus, we assessed glucose-stimulated insulin secretion (GSIS), insulin sensitivity, clearance and central glucose-induced insulin secretion in Bl/6J and N mice using gold-standard methodologies. METHODS: GSIS was measured using complementary tests (oral and intravenous glucose tolerance tests) and hyperglycemic clamps. Whole-body insulin sensitivity was assessed using euglycemic-hyperinsulinemic clamps. Neurally-mediated insulin secretion was measured during central hyperglycemia. RESULTS: Bl/6J mice have impaired GSIS compared to Bl/6N when glucose is administered intravenously during both a tolerance test and hyperglycemic clamp, but not in response to oral glucose. First and second phases of GSIS are altered without changes in whole body insulin sensitivity, insulin clearance, beta-cell mass or central response to glucose, thereby demonstrating defective beta-cell function in Bl/6J mice. CONCLUSIONS: The Bl/6J mouse strain displays impaired insulin secretion. These results have important implications for choosing the appropriate test to assess beta-cell function and background strain in genetically modified mouse models.

α/ß-Hydrolase domain-6-accessible monoacylglycerol controls glucose-stimulated insulin secretion.

Glucose metabolism in pancreatic ß cells stimulates insulin granule exocytosis, and this process requires generation of a lipid signal. However, the signals involved in lipid amplification of glucose-stimulated insulin secretion (GSIS) are unknown. Here we show that in ß cells, glucose stimulates production of lipolysis-derived long-chain saturated monoacylglycerols, which further increase upon inhibition of the membrane-bound monoacylglycerol lipase α/ß-Hydrolase Domain-6 (ABHD6). ABHD6 expression in ß cells is inversely proportional to GSIS. Exogenous monoacylglycerols stimulate ß cell insulin secretion and restore GSIS suppressed by the pan-lipase inhibitor orlistat. Whole-body and ß-cell-specific ABHD6-KO mice exhibit enhanced GSIS, and their islets show elevated monoacylglycerol production and insulin secretion in response to glucose. Inhibition of ABHD6 in diabetic mice restores GSIS and improves glucose tolerance. Monoacylglycerol binds and activates the vesicle priming protein Munc13-1, thereby inducing insulin exocytosis. We propose saturated monoacylglycerol as a signal for GSIS and ABHD6 as a negative modulator of insulin secretion.