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.

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.

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.

Silencing ANGPTL8 reduces mouse preadipocyte differentiation and insulin signaling.

ANGPTL8, expressed mainly in the liver and adipose tissue, regulates the activity of lipoprotein lipase (LPL) present in the extracellular space and triglyceride (TG) metabolism through its interaction with ANGPTL3 and ANGPTL4. Whether intracellular ANGPTL8 can also exert effects in tissues where it is expressed is uncertain. ANGPTL8 expression was low in preadipocytes and much increased during differentiation. To better understand the role of intracellular ANGPTL8 in adipocytes and assess whether it may play a role in adipocyte differentiation, we knocked down its expression in normal mouse subcutaneous preadipocytes. ANGPTL8 knockdown reduced adipocyte differentiation, cellular TG accumulation and also isoproterenol-stimulated lipolysis at day 7 of differentiation. RNA-Seq analysis of ANGPTL8 siRNA or control siRNA transfected SC preadipocytes on days 0, 2, 4 and 7 of differentiation showed that ANGPTL8 knockdown impeded the early (day 2) expression of adipogenic and insulin signaling genes, PPARγ, as well as genes related to extracellular matrix and NF-κB signaling. Insulin mediated Akt phosphorylation was reduced at an early stage during adipocyte differentiation. This study based on normal primary cells shows that ANGPTL8 has intracellular actions in addition to effects in the extracellular space, like modulating LPL activity. Preadipocyte ANGPTL8 expression modulates their differentiation possibly via changes in insulin signaling gene expression.

Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs are key regulators of gene expression involved in health and disease. The goal of our study was to investigate the global changes in beta cell microRNA expression occurring in two models of obesity-associated type 2 diabetes and to assess their potential contribution to the development of the disease. METHODS: MicroRNA profiling of pancreatic islets isolated from prediabetic and diabetic db/db mice and from mice fed a high-fat diet was performed by microarray. The functional impact of the changes in microRNA expression was assessed by reproducing them in vitro in primary rat and human beta cells. RESULTS: MicroRNAs differentially expressed in both models of obesity-associated type 2 diabetes fall into two distinct categories. A group including miR-132, miR-184 and miR-338-3p displays expression changes occurring long before the onset of diabetes. Functional studies indicate that these expression changes have positive effects on beta cell activities and mass. In contrast, modifications in the levels of miR-34a, miR-146a, miR-199a-3p, miR-203, miR-210 and miR-383 primarily occur in diabetic mice and result in increased beta cell apoptosis. These results indicate that obesity and insulin resistance trigger adaptations in the levels of particular microRNAs to allow sustained beta cell function, and that additional microRNA deregulation negatively impacting on insulin-secreting cells may cause beta cell demise and diabetes manifestation. CONCLUSIONS/INTERPRETATION: We propose that maintenance of blood glucose homeostasis or progression toward glucose intolerance and type 2 diabetes may be determined by the balance between expression changes of particular microRNAs.

Degradation of oxytetracycline and doxycycline by ozonation: Degradation pathways and toxicity assessment.

Tetracyclines are one of the antibiotics widely employed worldwide and frequently detected in surface waters because of incomplete removal from wastewater treatment. Various advanced oxidation processes have been investigated for tetracyclines degradation and their transformation products (TPs) have recently gained more attention. Studies on ozonation are however seldom for the degradation of oxytetracycline (OTC) and doxycycline (DTC). In the present study, a lower O3 inlet gas concentration (4.67 ± 0.13 mg/L), supplied at a flow rate of 0.27 L/min, was shown to be more effective at removing OTC than the same dose of ozone applied at higher inlet gas concentration (up to 6.29 mg/L) over a shorter time at the same flow rate. The use of pCBA and t-BuOH indicated that ozone plays a more important role in the degradation of OTC than HO•. The DTC degradation was less efficient than for OTC, with 99 % removal requiring twice the amount of ozone. OTC had almost no inhibition of Vibrio fischeri, however, the inhibition ratio was increased to 37 % (5-min) and 46 % (15-min) within 1 min of ozonation. Contrastly, DTC had toxic effects on V. fischeri (inhibition rate5min of 84 %) and sustained toxicity in samples treated for up to 40-min. The observed toxicities after treatment could be explained by the identified TPs (26 TPs for OTC and 23 for DTC, some identified for the first time) and their quantitative structure-activity relationship analysis data. Several TPs showed toxic or extremely toxic predicted effects on fish, daphnid, and green algae, corresponding with the V. fischeri inhibition results. Among the possible degradation pathways, aromatic ring hydroxylation and ring-opening pathways could lead to the formation of TPs less harmful to the environment.

A peripherally restricted cannabinoid-1 receptor inverse agonist promotes insulin secretion and protects from cytokine toxicity in human pancreatic islets.

The cannabinoid receptor CB1R is expressed in pancreatic ß-cells; CB1R increased activity is associated with diabetes, obesity, cardiovascular disorders as well as decreased insulin secretion and insulin resistance. CB1R was shown to signal through G-protein coupling as well as ß-arrestins in ß-cells. Peripherally restricted CB1R inverse agonists purportedly have beneficial effects on insulin secretion in ß-cells, without the unwanted effects in the central nervous system. Here we show that a peripherally restricted CB1R inverse agonist, MRI-1891, augments glucose stimulated insulin secretion in isolated human pancreatic islets and mouse islets. The insulin secretion enhancing effect of MRI-1891 is comparable to exendin-4, an analogue of the glucagon like peptide-1 (GLP1). Moreover, MRI-1891 treatment protects isolated human islet cells against cytokine-induced apoptosis, similar to exendin-4. Thus, MRI-1891, a new class of CB1R inverse agonist, may be considered a potential therapeutic for both type 1 and type 2 diabetes because of its ability to protect pancreatic ß-cells from cytokine toxicity and to promote insulin secretion.

Voluntary running exercise prevents ß-cell failure in susceptible islets of the Zucker diabetic fatty rat.

Physical activity improves glycemic control in type 2 diabetes (T2D), but its contribution to preserving ß-cell function is uncertain. We evaluated the role of physical activity on ß-cell secretory function and glycerolipid/fatty acid (GL/FA) cycling in male Zucker diabetic fatty (ZDF) rats. Six-week-old ZDF rats engaged in voluntary running for 6 wk (ZDF-A). Inactive Zucker lean and ZDF (ZDF-I) rats served as controls. ZDF-I rats displayed progressive hyperglycemia with ß-cell failure evidenced by falling insulinemia and reduced insulin secretion to oral glucose. Isolated ZDF-I rat islets showed reduced glucose-stimulated insulin secretion expressed per islet and per islet protein. They were also characterized by loss of the glucose regulation of fatty acid oxidation and GL/FA cycling, reduced mRNA expression of key ß-cell genes, and severe reduction of insulin stores. Physical activity prevented diabetes in ZDF rats through sustaining ß-cell compensation to insulin resistance shown in vivo and in vitro. Surprisingly, ZDF-A islets had persistent defects in fatty acid oxidation, GL/FA cycling, and ß-cell gene expression. ZDF-A islets, however, had preserved islet insulin mRNA and insulin stores compared with ZDF-I rats. Physical activity did not prevent hyperphagia, dyslipidemia, or obesity in ZDF rats. In conclusion, islets of ZDF rats have a susceptibility to failure that is possibly due to altered ß-cell fatty acid metabolism. Depletion of pancreatic islet insulin stores is a major contributor to islet failure in this T2D model, preventable by physical activity.

Glycerol-3-phosphate phosphatase operates a glycerol shunt in pancreatic ß-cells that controls insulin secretion and metabolic stress.

OBJECTIVE: The recently identified glycerol-3-phosphate (Gro3P) phosphatase (G3PP) in mammalian cells, encoded by the PGP gene, was shown to regulate glucose, lipid and energy metabolism by hydrolyzing Gro3P and to control glucose-stimulated insulin secretion (GSIS) in ß-cells, in vitro. However, whether G3PP regulates ß-cell function and insulin secretion in vivo is not known. METHODS: We now examined the role of G3PP in the control of insulin secretion in vivo, ß-cell function and glucotoxicity in inducible ß-cell specific G3PP-KO (BKO) mice. Inducible BKO mice were generated by crossing floxed-G3PP mice with Mip-Cre-ERT (MCre) mice. All the in vivo studies were done using BKO and control mice fed normal diet and the ex vivo studies were done using pancreatic islets from these mice. RESULTS: BKO mice, compared to MCre controls, showed increased body weight, adiposity, fed insulinemia, enhanced in vivo GSIS, reduced plasma triglycerides and mild glucose intolerance. Isolated BKO mouse islets incubated at high (16.7 mM), but not at low or intermediate glucose (3 and 8 mM), showed elevated GSIS, Gro3P content as well as increased levels of metabolites and signaling coupling factors known to reflect ß-cell activation for insulin secretion. BKO islets also showed reduced glycerol release and increased O2 consumption and ATP production at high glucose only. BKO islets chronically exposed to elevated glucose levels showed increased apoptosis, reduced insulin content and decreased mRNA expression of ß-cell differentiation markers, Pdx-1, MafA and Ins-2. CONCLUSIONS: The results demonstrate that ß-cells are endowed with a "glycerol shunt", operated by G3PP that regulates ß-cell metabolism, signaling and insulin secretion in vivo, primarily at elevated glucose concentrations. We propose that the glycerol shunt plays a role in preventing insulin hypersecretion and excess body weight gain and contributes to ß-cell mass preservation in the face of hyperglycemia.

Hepatic glycerol shunt and glycerol-3-phosphate phosphatase control liver metabolism and glucodetoxification under hyperglycemia.

OBJECTIVE: Glycerol-3-phosphate (Gro3P) phosphatase (G3PP) hydrolyzes Gro3P to glycerol that exits the cell, thereby operating a "glycerol shunt", a metabolic pathway that we identified recently in mammalian cells. We have investigated the role of G3PP and the glycerol shunt in the regulation of glucose metabolism and lipogenesis in mouse liver. METHODS: We generated hepatocyte-specific G3PP-KO mice (LKO), by injecting AAV8-TBG-iCre to male G3PPfl/fl mice. Controls received AAV8-TBG-eGFP. Both groups were fed chow diet for 10 weeks. Hyperglycemia (16-20 mM) was induced by glucose infusion for 55 h. Hepatocytes were isolated from normoglycemic mice for ex vivo studies and targeted metabolomics were measured in mice liver after glucose infusion. RESULTS: LKO mice showed no change in body weight, food intake, fed and fasted glycemia but had increased fed plasma triglycerides. Hepatic glucose production from glycerol was increased in fasted LKO mice. LKO mouse hepatocytes displayed reduced glycerol production, elevated triglyceride and lactate production at high glucose concentration. Hyperglycemia in LKO mice led to increased liver weight and accumulation of triglycerides, glycogen and cholesterol together with elevated levels of Gro3P, dihydroxyacetone phosphate, acetyl-CoA and some Krebs cycle intermediates in liver. Hyperglycemic LKO mouse liver showed elevated expression of proinflammatory cytokines and M1-macrophage markers accompanied by increased plasma triglycerides, LDL/VLDL, urea and uric acid and myocardial triglycerides. CONCLUSIONS: The glycerol shunt orchestrated by G3PP acts as a glucose excess detoxification pathway in hepatocytes by preventing metabolic disturbances that contribute to enhanced liver fat, glycogen storage, inflammation and lipid build-up in the heart. We propose G3PP as a novel therapeutic target for hepatic disorders linked to nutrient excess.

Substantial PCSK9 inactivation in ß-cells does not modify glucose homeostasis or insulin secretion in mice.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by promoting the degradation of the LDL receptor (LDLR). PCSK9 loss-of-function mutations are associated with increased fasting plasma glucose levels and slightly elevated risk of type 2-diabetes. Considering the known detrimental effects of cholesterol accumulation in ß-cell, and the widespread use of PCSK9 inhibitors to treat hypercholesterolemia, it is important to gain insight into the role of pancreatic PCSK9 in glucose homeostasis and ß-cell function. We generated the first ß-cell-specific KO of PCSK9 (ßKO). PCSK9 mRNA and protein expression were reduced by 48% and 78% in ßKO islets, respectively, indicating that ß-cells constitute a major site of PCSK9 expression. In islets, loss of ß-cell PCSK9 resulted in unchanged LDLR protein levels, but reduced LDLR mRNA, indicating that cholesterol internalization is enhanced and that ß-cell PCSK9 promotes LDLR degradation. In contrast, whole body PCSK9 KO mice exhibited 2-fold higher LDLR protein levels in islets and a stable expression of cholesterogenic genes. Whole body KO and ßKO mice presented normal glucose tolerance, insulin release in response to glucose load and insulin sensitivity. Ex vivo glucose-stimulated insulin secretion in presence or absence of fatty acids was similar in WT and KO islets. Like KO mice, individuals carrying loss-of-function PCSK9 variants may be protected from cholesterol-induced toxicity due to reduced circulating cholesterol levels. Using both whole body KO or ßKO models, our data demonstrate that PCSK9 deletion in mouse does not have any toxic effect on ß-cell function and glucose homeostasis.

New Mammalian Glycerol-3-Phosphate Phosphatase: Role in ß-Cell, Liver and Adipocyte Metabolism.

Cardiometabolic diseases, including type 2 diabetes, obesity and non-alcoholic fatty liver disease, have enormous impact on modern societies worldwide. Excess nutritional burden and nutri-stress together with sedentary lifestyles lead to these diseases. Deranged glucose, fat, and energy metabolism is at the center of nutri-stress, and glycolysis-derived glycerol-3-phosphate (Gro3P) is at the crossroads of these metabolic pathways. Cellular levels of Gro3P can be controlled by its synthesis, utilization or hydrolysis. The belief that mammalian cells do not possess an enzyme that hydrolyzes Gro3P, as in lower organisms and plants, is challenged by our recent work showing the presence of a Gro3P phosphatase (G3PP) in mammalian cells. A previously described phosphoglycolate phosphatase (PGP) in mammalian cells, with no established physiological function, has been shown to actually function as G3PP, under physiological conditions, particularly at elevated glucose levels. In the present review, we summarize evidence that supports the view that G3PP plays an important role in the regulation of gluconeogenesis and fat storage in hepatocytes, glucose stimulated insulin secretion and nutri-stress in ß-cells, and lipogenesis in adipocytes. We provide a balanced perspective on the pathophysiological significance of G3PP in mammals with specific reference to cardiometabolic diseases.

Dietary citrate acutely induces insulin resistance and markers of liver inflammation in mice.

Citrate is widely used as a food additive being part of virtually all processed foods. Although considered inert by most of the regulatory agencies in the world, plasma citrate has been proposed to play immunometabolic functions in multiple tissues through altering a plethora of cellular pathways. Here, we used a short-term alimentary intervention (24 hours) with standard chow supplemented with citrate in amount corresponding to that found in processed foods to evaluate its effects on glucose homeostasis and liver physiology in C57BL/6J mice. Animals supplemented with dietary citrate showed glucose intolerance and insulin resistance as revealed by glucose and insulin tolerance tests. Moreover, animals supplemented with citrate in their food displayed fed and fasted hyperinsulinemia and enhanced insulin secretion during an oral glucose tolerance test. Citrate treatment also amplified glucose-induced insulin secretion in vitro in INS1-E cells. Citrate supplemented animals had increased liver PKCα activity and altered phosphorylation at serine or threonine residues of components of insulin signaling including IRS-1, Akt, GSK-3 and FoxO1. Furthermore, citrate supplementation enhanced the hepatic expression of lipogenic genes suggesting increased de novo lipogenesis, a finding that was reproduced after citrate treatment of hepatic FAO cells. Finally, liver inflammation markers were higher in citrate supplemented animals. Overall, the results demonstrate that dietary citrate supplementation in mice causes hyperinsulinemia and insulin resistance both in vivo and in vitro, and therefore call for a note of caution on the use of citrate as a food additive given its potential role in metabolic dysregulation.

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.

Nutrient-Induced Metabolic Stress, Adaptation, Detoxification, and Toxicity in the Pancreatic ß-Cell.

Paraphrasing the Swiss physician and father of toxicology Paracelsus (1493-1541) on chemical agents used as therapeutics, "the dose makes the poison," it is now realized that this aptly applies to the calorigenic nutrients. The case here is the pancreatic islet ß-cell presented with excessive levels of nutrients such as glucose, lipids, and amino acids. The short-term effects these nutrients exert on the ß-cell are enhanced insulin biosynthesis and secretion and changes in glucose sensitivity. However, chronic fuel surfeit triggers additional compensatory and adaptive mechanisms by ß-cells to cope with the increased insulin demand or to protect itself. When these mechanisms fail, toxicity due to the nutrient surplus ensues, leading to ß-cell dysfunction, dedifferentiation, and apoptosis. The terms glucotoxicity, lipotoxicity, and glucolipotoxicity have been widely used, but there is some confusion as to what they mean precisely and which is most appropriate for a given situation. Here we address the gluco-, lipo-, and glucolipo-toxicities in ß-cells by assessing the evidence both for and against each of them. We also discuss potential mechanisms and defend the view that many of the identified "toxic" effects of nutrient excess, which may also include amino acids, are in fact beneficial adaptive processes. In addition, candidate fuel-excess detoxification pathways are evaluated. Finally, we propose that a more general term should be used for the in vivo situation of overweight-associated type 2 diabetes reflecting both the adaptive and toxic processes to mixed calorigenic nutrients excess: "nutrient-induced metabolic stress" or, in brief, "nutri-stress."

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.

Novel materials for catalytic ozonation of wastewater for disinfection and removal of micropollutants.

The purpose of this work was to identify novel materials that could be used for the catalytic ozonation of wastewater for improved removal of micropollutants and disinfection. The materials chosen for investigation were selected based on their commercial availability and composition characteristics that suggested that they could act as catalysts. Synthetic wastewater (SWW) was used to mimic municipal secondary effluent wastewater while obtaining constant and reproducible matrix characteristics. Polonite®, wollastonite, zeolite, TiO2-Al2O3 (8%/92%), and AL-1010S (AlO2-based) were tested for their potential impact on efficiency of disinfection, based on the removal of E. coli bacteria and removal of contaminants of emerging concern (CECs), relative to conventional ozonation. Atrazine (ATZ), ibuprofen (IBP), naproxen (NPX), and gemfibrozil (GBZ) were used as indicator compounds. Zeolite and wollastonite did not promote disinfection and CECs removal; TiO2-Al2O3 and AL-1010S provided improvement for both criteria, but to a lesser extent in SWW than in Milli-Q water; and Polonite® did not enhance the removal of CECs but led to the higher E. coli inactivation. These results suggest that Polonite®, AL-1010S, and TiO2-Al2O3 can act as catalysts and provide mechanisms to lower the ozone dose required to reach disinfection. The apparent kinetic constant of the reaction for the catalytic ozonation of ATZ, in the ultrapure water, was determined for Polonite®, TiO2-Al2O3 and AL-1010S. The reusability of the catalysts was demonstrated over four consecutive cycles of 6 h of treatment in a continuous flow ozonation system.

Contribution of the Long Noncoding RNA H19 to ß-Cell Mass Expansion in Neonatal and Adult Rodents.

Pancreatic ß-cell expansion throughout the neonatal period is essential to generate the appropriate mass of insulin-secreting cells required to maintain blood glucose homeostasis later in life. Hence, defects in this process can predispose to diabetes development during adulthood. Global profiling of transcripts in pancreatic islets of newborn and adult rats revealed that the transcription factor E2F1 controls expression of the long noncoding RNA H19, which is profoundly downregulated during the postnatal period. H19 silencing decreased ß-cell expansion in newborns, whereas its re-expression promoted proliferation of ß-cells in adults via a mechanism involving the microRNA let-7 and the activation of Akt. The offspring of rats fed a low-protein diet during gestation and lactation display a small ß-cell mass and an increased risk of developing diabetes during adulthood. We found that the islets of newborn rats born to dams fed a low-protein diet express lower levels of H19 than those born to dams that did not eat a low-protein diet. Moreover, we observed that H19 expression increases in islets of obese mice under conditions of increased insulin demand. Our data suggest that the long noncoding RNA H19 plays an important role in postnatal ß-cell mass expansion in rats and contributes to the mechanisms compensating for insulin resistance in obesity.

Fatty acid signaling in the beta-cell and insulin secretion.

Fatty acids (FAs) and other lipid molecules are important for many cellular functions, including vesicle exocytosis. For the pancreatic beta-cell, while the presence of some FAs is essential for glucose-stimulated insulin secretion, FAs have enormous capacity to amplify glucose-stimulated insulin secretion, which is particularly operative in situations of beta-cell compensation for insulin resistance. In this review, we propose that FAs do this via three interdependent processes, which we have assigned to a "trident model" of beta-cell lipid signaling. The first two arms of the model implicate intracellular metabolism of FAs, whereas the third is related to membrane free fatty acid receptor (FFAR) activation. The first arm involves the AMP-activated protein kinase/malonyl-CoA/long-chain acyl-CoA (LC-CoA) signaling network in which glucose, together with other anaplerotic fuels, increases cytosolic malonyl-CoA, which inhibits FA partitioning into oxidation, thus increasing the availability of LC-CoA for signaling purposes. The second involves glucose-responsive triglyceride (TG)/free fatty acid (FFA) cycling. In this pathway, glucose promotes LC-CoA esterification to complex lipids such as TG and diacylglycerol, concomitant with glucose stimulation of lipolysis of the esterification products, with renewal of the intracellular FFA pool for reactivation to LC-CoA. The third arm involves FFA stimulation of the G-protein-coupled receptor GPR40/FFAR1, which results in enhancement of glucose-stimulated accumulation of cytosolic Ca2+ and consequently insulin secretion. It is possible that FFA released by the lipolysis arm of TG/FFA cycling is partly "secreted" and, via an autocrine/paracrine mechanism, is additive to exogenous FFAs in activating the FFAR1 pathway. Glucose-stimulated release of arachidonic acid from phospholipids by calcium-independent phospholipase A2 and/or from TG/FFA cycling may also be involved. Improved knowledge of lipid signaling in the beta-cell will allow a better understanding of the mechanisms of beta-cell compensation and failure in diabetes.

Identification of islet-enriched long non-coding RNAs contributing to ß-cell failure in type 2 diabetes.

OBJECTIVE: Non-coding RNAs constitute a major fraction of the ß-cell transcriptome. While the involvement of microRNAs is well established, the contribution of long non-coding RNAs (lncRNAs) in the regulation of ß-cell functions and in diabetes development remains poorly understood. The aim of this study was to identify novel islet lncRNAs differently expressed in type 2 diabetes models and to investigate their role in ß-cell failure and in the development of the disease. METHODS: Novel transcripts dysregulated in the islets of diet-induced obese mice were identified by high throughput RNA-sequencing coupled with de novo annotation. Changes in the level of the lncRNAs were assessed by real-time PCR. The functional role of the selected lncRNAs was determined by modifying their expression in MIN6 cells and primary islet cells. RESULTS: We identified about 1500 novel lncRNAs, a number of which were differentially expressed in obese mice. The expression of two lncRNAs highly enriched in ß-cells, ßlinc2, and ßlinc3, correlated to body weight gain and glycemia levels in obese mice and was also modified in diabetic db/db mice. The expression of both lncRNAs was also modulated in vitro in isolated islet cells by glucolipotoxic conditions. Moreover, the expression of the human orthologue of ßlinc3 was altered in the islets of type 2 diabetic patients and was associated to the BMI of the donors. Modulation of the level of ßlinc2 and ßlinc3 by overexpression or downregulation in MIN6 and mouse islet cells did not affect insulin secretion but increased ß-cell apoptosis. CONCLUSIONS: Taken together, the data show that lncRNAs are modulated in a model of obesity-associated type 2 diabetes and that variations in the expression of some of them may contribute to ß-cell failure during the development of the disease.