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.

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.

14-3-3ζ Constrains insulin secretion by regulating mitochondrial function in pancreatic ß cells.

While critical for neurotransmitter synthesis, 14-3-3 proteins are often assumed to have redundant functions due to their ubiquitous expression, but despite this assumption, various 14-3-3 isoforms have been implicated in regulating metabolism. We previously reported contributions of 14-3-3ζ in ß cell function, but these studies were performed in tumor-derived MIN6 cells and systemic KO mice. To further characterize the regulatory roles of 14-3-3ζ in ß cell function, we generated ß cell-specific 14-3-3ζ-KO mice. Although no effects on ß cell mass were detected, potentiated glucose-stimulated insulin secretion (GSIS), mitochondrial function, and ATP synthesis were observed. Deletion of 14-3-3ζ also altered the ß cell transcriptome, as genes associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute 14-3-3 protein inhibition in mouse and human islets recapitulated the enhancements in GSIS and mitochondrial function, suggesting that 14-3-3ζ is the critical isoform in ß cells. In dysfunctional db/db islets and human islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3ζ, was inversely associated with insulin secretion, and pan-14-3-3 protein inhibition led to enhanced GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3ζ in the regulation of ß cell function and provides a deeper understanding of how insulin secretion is controlled in ß cells.

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.

Scaffold Proteins: From Coordinating Signaling Pathways to Metabolic Regulation.

Among their pleiotropic functions, scaffold proteins are required for the accurate coordination of signaling pathways. It has only been within the past 10 years that their roles in glucose homeostasis and metabolism have emerged. It is well appreciated that changes in the expression or function of signaling effectors, such as receptors or kinases, can influence the development of chronic diseases such as diabetes and obesity. However, little is known regarding whether scaffolds have similar roles in the pathogenesis of metabolic diseases. In general, scaffolds are often underappreciated in the context of metabolism or metabolic diseases. In the present review, we discuss various scaffold proteins and their involvement in signaling pathways related to metabolism and metabolic diseases. The aims of the present review were to highlight the importance of scaffold proteins and to raise awareness of their physiological contributions. A thorough understanding of how scaffolds influence metabolism could aid in the discovery of novel therapeutic approaches to treat chronic conditions, such as diabetes, obesity, and cardiovascular disease, for which the incidence of all continue to increase at alarming rates.

Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA-splicing factors during adipogenesis.

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope-tagged 14-3-3ζ molecule. After inducing adipogenesis, TAP-14-3-3ζ complexes were purified, followed by MS analysis to determine the 14-3-3ζ interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis (i.e. Ptrf/Cavin1) within the 14-3-3ζ interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3ζ interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of Pparg and the adipogenesis-associated gene Lpin1 In summary, we have identified novel adipogenic factors within the 14-3-3ζ interactome. Further characterization of additional proteins within the 14-3-3ζ interactome may help identify novel targets to block obesity-associated expansion of adipose tissues.

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.

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.

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.

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.

Metabolic inflexibility impairs insulin secretion and results in MODY-like diabetes in triple FoxO-deficient mice.

Pancreatic ß cell failure in type 2 diabetes is associated with functional abnormalities of insulin secretion and deficits of ß cell mass. It's unclear how one begets the other. We have shown that loss of ß cell mass can be ascribed to impaired FoxO1 function in different models of diabetes. Here we show that ablation of the three FoxO genes (1, 3a, and 4) in mature ß cells results in early-onset, maturity-onset diabetes of the young (MODY)-like diabetes, with abnormalities of the MODY networks Hnf4α, Hnf1α, and Pdx1. FoxO-deficient ß cells are metabolically inflexible, i.e., they preferentially utilize lipids rather than carbohydrates as an energy source. This results in impaired ATP generation and reduced Ca(2+)-dependent insulin secretion. The present findings demonstrate a secretory defect caused by impaired FoxO activity that antedates dedifferentiation. We propose that defects in both pancreatic ß cell function and mass arise through FoxO-dependent mechanisms during diabetes progression.

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.

Palmitate induces C-reactive protein expression in human aortic endothelial cells. Relevance to fatty acid-induced endothelial dysfunction.

Circulating levels of free fatty acids are commonly elevated in patients with the metabolic syndrome and exert, through activating proinflammatory pathways, harmful effects of the vascular endothelium. In this study, we examined the effect of palmitate (PA) on endothelial C-reactive protein (CRP) expression and the role of CRP in PA-induced nitric oxide (NO) inhibition. Palmitate increased, in a dose-dependent manner, CRP protein expression and production in human aortic endothelial cells (HAECs). Induction of CRP protein was mimicked by ceramide, whereas bromopalmitate and other common free fatty acids such as oleate or linoleate were ineffective. Palmitate also elicited reactive oxygen species production in HAECs, an effect prevented by protein kinase C (PKC) inhibition and adenosine monophosphate-activated kinase (AMPK) activation. Palmitate-treated HAECs showed increased CRP messenger RNA expression and nuclear factor (NF)-κB activation. Induction of CRP expression by PA was prevented by antioxidants and normalized by PKC and mitogen-activated protein kinase inhibitors. Disrupting NF-κB and Janus kinase/signal transducers and activators of transcription pathways or inducing AMPK activation also suppressed the stimulatory effect of PA on CRP messenger RNA expression. Finally, in HAECs, PA reduced NO release, an effect reversed by anti-CRP antibody. These data demonstrate that PA-induced endothelial CRP expression involves PKC-driven oxidative stress, possibly through AMPK inhibition, and activation of downstream redox-sensitive signaling pathways, including NF-κB. They further support a role for endothelial cell-derived CRP as mediator of the suppressive effect of PA on NO production.

The connection between C-reactive protein (CRP) and diabetic vasculopathy. Focus on preclinical findings.

Current evidence supports a central role of inflammation in the pathogenesis of atherosclerosis and diabetes. Type 2 diabetes is an inflammatory atherothrombotic condition associated with a high prevalence of cardiovascular disease. In patients with type 2 diabetes, low grade inflammation is reflected by increased plasma levels of several biomarkers of inflammation such as C-reactive protein (CRP). Small increases in CRP predict the likelihood of developing cardiovascular events both in diabetic and nondiabetic populations. In addition, in apparently healthy subjects, increased levels of CRP predict the risk of developing type 2 diabetes. There is some evidence that CRP, besides its predictive role in determining cardiovascular risk, may represent an active participant in atherogenesis. CRP is expressed in human atherosclerotic plaques and both vascular cells and monocytes/macrophages appear to represent a significant source of CRP in the inflammatory vessel wall. By activating the main cell types present in the atherosclerotic lesions, CRP generated within the coronary plaques may contribute to the development and progression of atherosclerosis. Data on vascular CRP regulation are scarce. Current evidence suggests that inflammatory and metabolic factors associated with diabetes, such as high glucose, adipokines, modified lipoproteins and free fatty acids may trigger CRP production by endothelial cells, smooth muscle cells and monocytes/macrophages. These data suggest that local CRP concentration in diabetic atherosclerotic plaques could be higher than in nondiabetic ones. Given the possible correlation between local CRP production and the degree of severity of coronary artery disease or the nature of the lesion, such alteration may contribute to the accelerated development of vascular disease in patients with type 2 diabetes.