Fads2 knockout mice reveal that ALA prevention of hepatic steatosis is dependent on delta-6 desaturase activity.

The production of the omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from alpha-linolenic acid (ALA) relies on the delta-6 desaturase (D6D) enzyme encoded by the Fads2 gene. While EPA and DHA reduce hepatic triacylglycerol (TAG) storage and regulate lipogenesis, the independent impact of ALA is less understood. To address this gap in knowledge, hepatic fatty acid metabolism was investigated in male wildtype (WT) and Fads2 knockout (KO) mice fed diets (16% kcal from fat) containing either lard (no n-3 LCPUFA), flaxseed oil (ALA rich), or menhaden oil (EPA/DHA rich) for 21 weeks. Fat content and composition, as well as markers of lipogenesis, glyceroneogenesis, and TAG synthesis, were analyzed using histology, gas chromatography, and reverse transcription quantitative PCR (RT-qPCR). Mice fed the menhaden diet had significantly lower hepatic TAG compared to both lard- and flax-fed mice, concomitant with changes in n-3 and n-6 LCPUFA in both TAG and phospholipid (PL) fractions (all p < 0.05). Flax-fed WT mice had lower liver TAG content compared to their KO counterparts. Menhaden-fed mice had significantly lower expression of key lipogenic (Scd1, Srebp-1c, Fasn, Fads1, Fads2), glyceroneogenic (Pck1), and TAG synthesis (Agpat3) genes compared to lard, with flax-fed mice showing some intermediate effects. Gene expression effects were independent of D6D activity, since no differences were detected between WT and KO mice fed the same diet. This study demonstrates that EPA/DHA and not ALA itself is critical for the prevention of hepatic steatosis.

An obesogenic diet impairs uncoupled substrate oxidation and promotes whitening of the brown adipose tissue in rats.

Brown adipose tissue (BAT) is rich in mitochondria containing uncoupling protein 1 (UCP1), and dissipates energy through thermogenesis. However, even though BAT mass and its UCP1 content increase in rodents chronically fed a high-fat sucrose-enriched (HFS) diet, marked expansion of adiposity still occurs in these animals, suggesting insufficient BAT-mediated HFS diet-induced thermogenesis. Thus, the objective of this study was to investigate the metabolic and molecular mechanisms that regulate BAT thermogenesis in HFS-induced obesity. To accomplish this, rats were fed either a standard chow or HFS diet for 8 weeks. Subsequently, glucose and fatty acid metabolism and the molecular mechanisms underlying these processes were assessed in freshly isolated primary BAT adipocytes. Despite increasing BAT mass and its UCP1 content, the HFS diet reduced uncoupled glucose and palmitate oxidation in BAT adipocytes. It also markedly diminished tyrosine hydroxylase content and lipolysis in these cells. Conversely, glucose uptake, lactate production, glycerol incorporation into lipids, palmitate incorporation into triacylglycerol (TAG), phosphoenolpyruvate carboxykinase and glycerol kinase levels, and lipoprotein lipase and cluster of differentiation 36 gene expression were increased. In summary, a HFS diet enhanced glyceroneogenesis and shifted BAT metabolism toward TAG synthesis by impairing UCP1-mediated substrate oxidation and by enhancing fatty acid esterification in intact brown adipocytes. These adaptive metabolic responses to chronic HFS feeding attenuated BAT thermogenic capacity and favoured the development of obesity. KEY POINTS: Despite increasing brown adipose tissue (BAT) mass and levels of thermogenic proteins such as peroxisome proliferator-activated receptor γ coactivator 1α, carnitine palmitoyltransferase 1B and uncoupling protein 1 (UCP1), an obesogenic high-fat sucrose-enriched (HFS) diet attenuated uncoupled glucose and fatty acid oxidation in brown adipocytes. Brown adipocytes diverted glycerol and fatty acids toward triacylglycerol (TAG) synthesis by elevating the cellular machinery that promotes fatty acid uptake along with phosphoenolpyruvate carboxykinase and glycerol kinase levels. The HFS diet increased glucose uptake that supported lactate production and provided substrate for glyceroneogenesis and TAG synthesis in brown adipocytes. Impaired UCP-1-mediated thermogenic capacity and enhanced TAG storage in BAT adipocytes were consistent with reduced adipose triglyceride lipase and tyrosine hydroxylase levels in HFS diet-fed animals.

The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells.

Cancer cells are reprogrammed to consume large amounts of glucose to support anabolic biosynthetic pathways. However, blood perfusion and consequently the supply with glucose are frequently inadequate in solid cancers. PEPCK-M (PCK2), the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK), has been shown by us and others to be functionally expressed and to mediate gluconeogenesis, the reverse pathway of glycolysis, in different cancer cells. Serine and ribose synthesis have been identified as downstream pathways fed by PEPCK in cancer cells. Here, we report that PEPCK-M-dependent glycerol phosphate formation from noncarbohydrate precursors (glyceroneogenesis) occurs in starved lung cancer cells and supports de novo glycerophospholipid synthesis. Using stable isotope-labeled glutamine and lactate, we show that PEPCK-M generates phosphoenolpyruvate and 3-phosphoglycerate, which are at least partially converted to glycerol phosphate and incorporated into glycerophospholipids (GPL) under glucose and serum starvation. This pathway is required to maintain levels of GPL, especially phosphatidylethanolamine (PE), as shown by stable shRNA-mediated silencing of PEPCK-M in H23 lung cancer cells. PEPCK-M shRNA led to reduced colony formation after starvation, and the effect was partially reversed by the addition of dioleyl-PE. Furthermore, PEPCK-M silencing abrogated cancer growth in a lung cancer cell xenograft model. In conclusion, glycerol phosphate formation for de novo GPL synthesis via glyceroneogenesis is a newly characterized anabolic pathway in cancer cells mediated by PEPCK-M under conditions of severe nutrient deprivation.

Reactive oxygen species-dependent regulation of pyruvate dehydrogenase kinase-4 in white adipose tissue.

Reactive oxygen species (ROS) are important signaling molecules mediating the exercise-induced adaptations in skeletal muscle. Acute exercise also drives the expression of genes involved in reesterification and glyceroneogenesis in white adipose tissue (WAT), but whether ROS play any role in this effect has not been explored. We speculated that exercise-induced ROS would regulate acute exercise-induced responses in WAT. To address this question, we utilized various models to alter redox signaling in WAT. We examined basal and exercise-induced gene expression in a genetically modified mouse model of reduced mitochondrial ROS emission [mitochondrial catalase overexpression (MCAT)]. Additionally, H2O2, various antioxidants, and the ß3-adrenergic receptor agonist CL316243 were used to assess gene expression in white adipose tissue culture. MCAT mice have reduced ROS emission from WAT, enlarged WAT depots and adipocytes, and greater pyruvate dehydrogenase kinase-4 (Pdk4) gene expression. In WAT culture, H2O2 reduced glyceroneogenic gene expression. In wild-type mice, acute exercise induced dramatic but transient increases in Pdk4 and phosphoenolpyruvate carboxykinase (Pck1) mRNA in both subcutaneous inguinal WAT and epididymal WAT depots, which was almost completely absent in MCAT mice. Furthermore, the induction of Pdk4 and Pck1 in WAT culture by CL316243 was markedly reduced in the presence of antioxidants N-acetyl-cysteine or vitamin E. Genetic and nutritional approaches that attenuate redox signaling prevent exercise- and ß-agonist-induced gene expression within WAT. Combined, these data suggest that ROS represent important mediators of gene expression within WAT.

Impact of skeletal muscle IL-6 on subcutaneous and visceral adipose tissue metabolism immediately after high- and moderate-intensity exercises.

White adipose tissue is a major energy reserve for the body and is essential for providing fatty acids for other tissues when needed. Skeletal muscle interleukin-6 (IL-6) has been shown to be secreted from the working muscle and has been suggested to signal to adipose tissue and enhance lipolysis. The aim of the present study was to investigate the role of skeletal muscle IL-6 in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) lipolysis and glyceroneogenesis with prolonged moderate-intensity exercise and high-intensity exercise in mice. Female inducible muscle-specific IL-6 knockout (IL-6 iMKO) mice and littermate control (Floxed) mice performed a single exercise bout for either 120 min at 16 m/min and 10° slope (moderate intensity) or 30 min at 20 m/min and 10° slope (high intensity), or they remained rested (rest). Visceral and subcutaneous adipose tissues, quadriceps muscles, and blood were quickly obtained. Plasma IL-6 increased in Floxed mice but not in IL-6 iMKO mice with high-intensity exercise. VAT signal transducer and activator of transcription (STAT)3Tyr705 phosphorylation was lower, and VAT hormone-sensitive lipase (HSL)Ser563 phosphorylation was higher in IL-6 iMKO mice than in Floxed mice at rest. Furthermore, HSLSer563 and HSLSer660 phosphorylation increased in VAT and phosphoenolpyruvate carboxykinase protein decreased in SAT with moderate-intensity exercise in both genotypes. On the other hand, both exercise protocols increased pyruvate dehydrogenaseSer232 phosphorylation in VAT only in IL-6 iMKO mice and decreased tumor necrosis factor-α messenger RNA in SAT and VAT only in Floxed mice. In conclusion, the present findings suggest that skeletal muscle IL-6 regulates markers of lipolysis in VAT in the basal state and pyruvate availability for glyceroneogenesis in VAT with exercise. Moreover, skeletal muscle IL-6 may contribute to exercise-induced anti-inflammatory effects in SAT and VAT.

Cytosolic phosphoenolpyruvate carboxykinase as a cataplerotic pathway in the small intestine.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme that is highly expressed in the liver and kidney but is also expressed at lower levels in a variety of other tissues where it may play adjunct roles in fatty acid esterification, amino acid metabolism, and/or TCA cycle function. PEPCK is expressed in the enterocytes of the small intestine, but it is unclear whether it supports a gluconeogenic rate sufficient to affect glucose homeostasis. To examine potential roles of intestinal PEPCK, we generated an intestinal PEPCK knockout mouse. Deletion of intestinal PEPCK ablated ex vivo gluconeogenesis but did not significantly affect glycemia in chow, high-fat diet, or streptozotocin-treated mice. In contrast, postprandial triglyceride secretion from the intestine was attenuated in vivo, consistent with a role in fatty acid esterification. Intestinal amino acid profiles and 13C tracer appearance into these pools were significantly altered, indicating abnormal amino acid trafficking through the enterocyte. The data suggest that the predominant role of PEPCK in the small intestine of mice is not gluconeogenesis but rather to support nutrient processing, particularly with regard to lipids and amino acids. NEW & NOTEWORTHY The small intestine expresses gluconeogenic enzymes for unknown reasons. In addition to glucose synthesis, the nascent steps of this pathway can be used to support amino acid and lipid metabolisms. When phosphoenolpyruvate carboxykinase, an essential gluconeogenic enzyme, is knocked out of the small intestine of mice, glycemia is unaffected, but mice inefficiently absorb dietary lipid, have abnormal amino acid profiles, and inefficiently catabolize glutamine. Therefore, the initial steps of intestinal gluconeogenesis are used for processing dietary triglycerides and metabolizing amino acids but are not essential for maintaining blood glucose levels.

Dicarbonyl stress in clinical obesity.

The glyoxalase system in the cytoplasm of cells provides the primary defence against glycation by methylglyoxal catalysing its metabolism to D-lactate. Methylglyoxal is the precursor of the major quantitative advanced glycation endproducts in physiological systems - arginine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. Glyoxalase 1 of the glyoxalase system was linked to anthropometric measurements of obesity in human subjects and to body weight in strains of mice. Recent conference reports described increased weight gain on high fat diet-fed mouse with lifelong deficiency of glyoxalase 1 deficiency, compared to wild-type controls, and decreased weight gain in glyoxalase 1-overexpressing transgenic mice, suggesting a functional role of glyoxalase 1 and dicarbonyl stress in obesity. Increased methylglyoxal, dicarbonyl stress, in white adipose tissue and liver may be a mediator of obesity and insulin resistance and thereby a risk factor for development of type 2 diabetes and non-alcoholic fatty liver disease. Increased methylglyoxal formation from glyceroneogenesis on adipose tissue and liver and decreased glyoxalase 1 activity in obesity likely drives dicarbonyl stress in white adipose tissue increasing the dicarbonyl proteome and related dysfunction. The clinical significance will likely emerge from on-going clinical evaluation of inducers of glyoxalase 1 expression in overweight and obese subjects. Increased transcapillary escape rate of albumin and increased total body interstitial fluid volume in obesity likely makes levels of glycation of plasma protein unreliable indicators of glycation status in obesity as there is a shift of albumin dwell time from plasma to interstitial fluid, which decreases overall glycation for a given glycemic exposure.

A role for mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) in the regulation of hepatic gluconeogenesis.

Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism.

The nuclear retinoid-related orphan receptor-α regulates adipose tissue glyceroneogenesis in addition to hepatic gluconeogenesis.

Circadian rhythms have an essential role in feeding behavior and metabolism. RORα is a nuclear receptor involved in the interface of the circadian system and metabolism. The adipocyte glyceroneogenesis pathway derives free fatty acids (FFA) liberated by lipolysis to reesterification into triglycerides, thus regulating FFA homeostasis and fat mass. Glyceroneogenesis shares with hepatic gluconeogenesis the key enzyme phosphoenolpyruvate carboxykinase c (PEPCKc), whose gene is a RORα target in the liver. RORα-deficient mice (staggerer, ROR(sg/sg)) have been shown to exhibit a lean phenotype and fasting hypoglycemia for unsolved reasons. In the present study, we investigated whether adipocyte glyceroneogenesis might also be a target pathway of RORα, and we further evaluated the role of RORα in hepatocyte gluconeogenesis. In vivo investigations comparing ROR(sg/sg) mice with their wild-type (WT) littermates under fasting conditions demonstrated that, in the absence of RORα, the release of FFA into the bloodstream was altered and the rise in glycemia in response to pyruvate reduced. The functional analysis of each pathway, performed in adipose tissue or liver explants, confirmed the impairment of adipocyte glyceroneogenesis and liver gluconeogenesis in the ROR(sg/sg) mice; these reductions of FFA reesterification or glucose production were associated with decreases in PEPCKc mRNA and protein levels. Treatment of explants with RORα agonist or antagonist enhanced or inhibited these pathways, respectively, in tissues isolated from WT but not ROR(sg/sg) mice. Our results indicated that both adipocyte glyceroneogenesis and hepatocyte gluconeogenesis were regulated by RORα. This study demonstrates the physiological function of RORα in regulating both glucose and FFA homeostasis.

Metabolism of glycerol, glucose, and lactate in the citric acid cycle prior to incorporation into hepatic acylglycerols.

During hepatic lipogenesis, the glycerol backbone of acylglycerols originates from one of three sources: glucose, glycerol, or substrates passing through the citric acid cycle via glyceroneogenesis. The relative contribution of each substrate source to glycerol in rat liver acylglycerols was determined using (13)C-enriched substrates and NMR. Animals received a fixed mixture of glucose, glycerol, and lactate; one group received [U-(13)C6]glucose, another received [U-(13)C3]glycerol, and the third received [U-(13)C3]lactate. After 3 h, the livers were harvested to extract fats, and the glycerol moiety from hydrolyzed acylglycerols was analyzed by (13)C NMR. In either fed or fasted animals, glucose and glycerol provided the majority of the glycerol backbone carbons, whereas the contribution of lactate was small. In fed animals, glucose contributed >50% of the total newly synthesized glycerol backbone, and 35% of this contribution occurred after glucose had passed through the citric acid cycle. By comparison, the glycerol contribution was ~40%, and of this, 17% of the exogenous glycerol passed first through the cycle. In fasted animals, exogenous glycerol became the major contributor to acylglycerols. The contribution from exogenous lactate did increase in fasted animals, but its overall contribution remained small. The contributions of glucose and glycerol that had passed through the citric acid cycle first increased in fasted animals from 35 to 71% for glucose and from 17 to 24% for glycerol. Thus, a substantial fraction from both substrate sources passed through the cycle prior to incorporation into the glycerol moiety of acylglycerols in the liver.

The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice.

Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2h and was 1.7-fold greater than that observed in the control group after 6h. The up-regulation of GPD1 began 2h after administering ethanol, and significantly increased 6h later with the concomitant escalation in the glycolytic gene expression. The incorporation of (14)C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.

The mitochondrial pyruvate carrier regulates adipose glucose partitioning in female mice.

OBJECTIVE: The mitochondrial pyruvate carrier (MPC) occupies a critical node in intermediary metabolism, prompting interest in its utility as a therapeutic target for the treatment of obesity and cardiometabolic disease. Dysregulated nutrient metabolism in adipose tissue is a prominent feature of obesity pathophysiology, yet the functional role of adipose MPC has not been explored. We investigated whether the MPC shapes the adaptation of adipose tissue to dietary stress in female and male mice. METHODS: The impact of pharmacological and genetic disruption of the MPC on mitochondrial pathways of triglyceride assembly (lipogenesis and glyceroneogenesis) was assessed in 3T3L1 adipocytes and murine adipose explants, combined with analyses of adipose MPC expression in metabolically compromised humans. Whole-body and adipose-specific glucose metabolism were subsequently investigated in male and female mice lacking adipocyte MPC1 (Mpc1AD-/-) and fed either standard chow, high-fat western style, or high-sucrose lipid restricted diets for 24 weeks, using a combination of radiolabeled tracers and GC/MS metabolomics. RESULTS: Treatment with UK5099 or siMPC1 impaired the synthesis of lipids and glycerol-3-phosphate from pyruvate and blunted triglyceride accumulation in 3T3L1 adipocytes, whilst MPC expression in human adipose tissue was negatively correlated with indices of whole-body and adipose tissue metabolic dysfunction. Mature adipose explants from Mpc1AD-/- mice were intrinsically incapable of incorporating pyruvate into triglycerides. In vivo, MPC deletion restricted the incorporation of circulating glucose into adipose triglycerides, but only in female mice fed a zero fat diet, and this associated with sex-specific reductions in tricarboxylic acid cycle pool sizes and compensatory transcriptional changes in lipogenic and glycerol metabolism pathways. However, whole-body adiposity and metabolic health were preserved in Mpc1AD-/- mice regardless of sex, even under conditions of zero dietary fat. CONCLUSIONS: These findings highlight the greater capacity for mitochondrially driven triglyceride assembly in adipose from female versus male mice and expose a reliance upon MPC-gated metabolism for glucose partitioning in female adipose under conditions of dietary lipid restriction.

Sex-dependent adipose glucose partitioning by the mitochondrial pyruvate carrier.

Objective: The mitochondrial pyruvate carrier (MPC) occupies a critical node in intermediary metabolism, prompting interest in its utility as a therapeutic target for the treatment of obesity and cardiometabolic disease. Dysregulated nutrient metabolism in adipose tissue is a prominent feature of obesity pathophysiology, yet the functional role of adipose MPC has not been explored. We investigated whether the MPC shapes the adaptation of adipose tissue to dietary stress in female and male mice. Methods: The impact of pharmacological and genetic disruption of the MPC on mitochondrial pathways of triglyceride assembly (lipogenesis and glyceroneogenesis) was assessed in 3T3L1 adipocytes and murine adipose explants, combined with analyses of adipose MPC expression in metabolically compromised humans. Whole-body and adipose-specific glucose metabolism were subsequently investigated in male and female mice lacking adipocyte MPC1 (Mpc1AD-/-) and fed either standard chow, high-fat western style, or high-sucrose lipid restricted diets for 24 weeks, using a combination of radiolabeled tracers and GC/MS metabolomics. Results: Treatment with UK5099 or siMPC1 impaired the synthesis of lipids and glycerol-3-phosphate from pyruvate and blunted triglyceride accumulation in 3T3L1 adipocytes, whilst MPC expression in human adipose tissue was negatively correlated with indices of whole-body and adipose tissue metabolic dysfunction. Mature adipose explants from Mpc1AD-/- mice were intrinsically incapable of incorporating pyruvate into triglycerides. In vivo, MPC deletion restricted the incorporation of circulating glucose into adipose triglycerides, but only in female mice fed a zero fat diet, and this associated with sex-specific reductions in tricarboxylic acid cycle pool sizes and compensatory transcriptional changes in lipogenic and glycerol metabolism pathways. However, whole-body adiposity and metabolic health were preserved in Mpc1AD-/- mice regardless of sex, even under conditions of zero dietary fat. Conclusion: These findings highlight the greater capacity for mitochondrially driven triglyceride assembly in adipose from female versus male mice and expose a reliance upon MPC-gated metabolism for glucose partitioning in female adipose under conditions of dietary lipid restriction.

Resveratrol supplementation improves white adipose tissue function in a depot-specific manner in Zucker diabetic fatty rats.

Resveratrol (RSV) is a polyphenolic compound suggested to have anti-diabetic properties. Surprisingly, little is known regarding the effects of RSV supplementation on adipose tissue (AT) metabolism in vivo. The purpose of this study was to assess the effects of RSV on mitochondrial content and respiration, glyceroneogenesis (GNG), and adiponectin secretion in adipose tissue from Zucker diabetic fatty (ZDF) rats. Five-week-old ZDF rats were fed a chow diet with (ZDF RSV) or without (ZDF chow) RSV (200 mg/kg body wt) for 6 wk. Changes in adipose tissue metabolism were assessed in subcutaneous (scAT) and intra-abdominal [retroperitoneal (rpWAT), epididymal (eWAT)] adipose tissue depots. ZDF RSV rats showed lower fasting glucose and higher circulating adiponectin, as well as lower glucose area under the curve during intraperitoneal glucose and insulin tolerance tests than ZDF chow. [¹4C]pyruvate incorporation into triglycerides and adiponectin secretion were higher in scAT from ZDF RSV rats, concurrent with increases in adipose tissue triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and the phosphorylation of pyruvate dehydrogenase-E1α (PDH) (Ser293) protein content in this depot. Moreover, uncoupled mitochondrial respiration and complex I and II-supported respiration were increased in both scAT and rpWAT, which correlated with increases in cytochrome c oxidase subunit IV (COX4) protein content. In vitro treatment of scAT with RSV (50 µmol/l; 24 h) induced pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α) mRNA expression. Collectively, these data demonstrate that RSV can induce adipose tissue mitochondrial biogenesis in parallel with increases in GNG and adiponectin secretion.

Ketosis Alters Transcriptional Adaptations of Subcutaneous White Adipose Tissue in Holstein Cows during the Transition Period.

Ketosis is a common nutritional, metabolic disease during the perinatal period in dairy cows characterized by elevated blood ß-hydroxybutyrate (BHBA). In this study, RNA sequencing (RNA-seq) was performed to investigate adaptive changes in adipose tissue during the perinatal period of dairy cows. Blood and tailhead subcutaneous white adipose tissue (sWAT) were obtained from ketotic cows (Ket = 8, BHBA ≥ 1.4 mmol/L) and non-ketotic cows (Nket = 6, BHBA < 1.4 mmol/L) 21 d pre-partum and 10 d post-partum. Compared with pre-partum, decreased lipid synthesis due to down-regulation of PCK1 may be in a strong association with clinical ketosis. Simultaneously, PCK2 was downregulated in the Ket postnatally compared to its expression prenatally, and the expression of PCK2 was 2.7~4.2 times higher than that of PCK1, implying a more severe lipid storage impairment in the Ket. Moreover, compared to pre-partum, the upregulated differentially expressed genes post-partum in the Ket were enriched in the inflammatory response biological process. The higher expression of TNC (tenascin C) in the post-partum Ket relative to the Nket suggested that the adipose tissue of ketotic cows might also be accompanied by tissue fibrosis. Notably, pre-partum CD209 was higher in the Ket than in the Nket, which might be used as a candidate marker for the pre-partum prediction of ketosis. Combined with published gene expression traits, these results suggested that inflammation leads to a more widespread downregulation of the lipid synthesis gene network in adipose tissue in ketotic cows. Additionally, sWAT in post-partum cows with ketosis might also be accompanied by tissue fibrosis which could make the treatment of ketosis more difficult.

A randomized clinical trial evaluating the effect of empagliflozin on triglycerides in obese adults: Role of visceral fat.

BACKGROUND: Empagliflozin, a sodium glucose cotransporter 2 inhibitor, is a medication to treat type 2 diabetes. The effect of empagliflozin in persons without diabetes has received less attention. Here we conducted a randomized, double-blind placebo-controlled clinical trial to examine the effect of empagliflozin on plasma triglycerides in obese non-diabetic adults. METHODS: Participants (n = 35; BMI ≥ 30 kg/m2) underwent body composition assessments using MRI, and were randomly assigned to either placebo or empagliflozin (10 mg/d) for three months. At the baseline and post-treatment visit, after an overnight fast, blood was drawn for biochemical analysis. Participants received [U-13C3]glycerol orally followed by multiple blood draws over 3 h to examine glycerol incorporation into triglycerides using NMR spectroscopy. RESULTS: The changes in blood triglyceride concentration with empagliflozin therapy related to the mass of baseline visceral adipose tissue (VAT; r = 0.53, p = 0.04). Empagliflozin slightly lowered triglycerides in obese subjects with low VAT, but increased triglycerides in the subjects with high VAT. Consistently, empagliflozin effectively suppressed triglyceride synthesis following [U-13C3]glycerol administration in the subjects with low VAT (p < 0.05), but not in the subjects with high VAT. The subjects with high VAT lost body weight after three months of empagliflozin treatment. In all subjects, about 20% of the triglyceride backbone originated from mitochondrial metabolism of glycerol. CONCLUSIONS: The effect of empagliflozin on triglycerides in obese adults differed depending on VAT. Empagliflozin suppressed triglyceride synthesis in the subjects with low VAT, but tended to increase triglycerides in those with high VAT.

Phosphoenolpyruvate carboxykinase in cell metabolism: Roles and mechanisms beyond gluconeogenesis.

BACKGROUND: Phosphoenolpyruvate carboxykinase (PCK) has been almost exclusively recognized as a critical enzyme in gluconeogenesis, especially in the liver and kidney. Accumulating evidence has shown that the enhanced activity of PCK leads to increased glucose output and exacerbation of diabetes, whereas the defects of PCK result in lethal hypoglycemia. Genetic mutations or polymorphisms are reported to be related to the onset and progression of diabetes in humans. SCOPE OF REVIEW: Recent studies revealed that the PCK pathway is more complex than just gluconeogenesis, depending on the health or disease condition. Dysregulation of PCK may contribute to the development of obesity, cardiac hypertrophy, stroke, and cancer. Moreover, a regulatory network with multiple layers, from epigenetic regulation, transcription regulation, to posttranscription regulation, precisely tunes the expression of PCK. Deciphering the molecular basis that regulates PCK may pave the way for developing practical strategies to treat metabolic dysfunction. MAJOR CONCLUSIONS: In this review, we summarize the metabolic and non-metabolic roles of the PCK enzyme in cells, especially beyond gluconeogenesis. We highlight the distinct functions of PCK isoforms (PCK1 and PCK2), depict a detailed network regulating PCK's expression, and discuss its clinical relevance. We also discuss the therapeutic potential targeting PCK and the future direction that is highly in need to better understand PCK-mediated signaling under diverse conditions.

PPARγ-induced upregulation of subcutaneous fat adiponectin secretion, glyceroneogenesis and BCAA oxidation requires mTORC1 activity.

The nutrient sensors peroxisome proliferator-activated receptor γ (PPARγ) and mechanistic target of rapamycin complex 1 (mTORC1) closely interact in the regulation of adipocyte lipid storage. The precise mechanisms underlying this interaction and whether this extends to other metabolic processes and the endocrine function of adipocytes are still unknown. We investigated herein the involvement of mTORC1 as a mediator of the actions of the PPARγ ligand rosiglitazone in subcutaneous inguinal white adipose tissue (iWAT) mass, endocrine function, lipidome, transcriptome and branched-chain amino acid (BCAA) metabolism. Mice bearing regulatory associated protein of mTOR (Raptor) deletion and therefore mTORC1 deficiency exclusively in adipocytes and littermate controls were fed a high-fat diet supplemented or not with the PPARγ agonist rosiglitazone (30 mg/kg/day) for 8 weeks and evaluated for iWAT mass, lipidome, transcriptome (Rnaseq), respiration and BCAA metabolism. Adipocyte mTORC1 deficiency not only impaired iWAT adiponectin transcription, synthesis and secretion, PEPCK mRNA levels, triacylglycerol synthesis and BCAA oxidation and mRNA levels of related proteins but also completely blocked the upregulation in these processes induced by pharmacological PPARγ activation with rosiglitazone. Mechanistically, adipocyte mTORC1 deficiency impairs PPARγ transcriptional activity by reducing PPARγ protein content, as well as by downregulating C/EBPα, a co-partner and facilitator of PPARγ. In conclusion, mTORC1 and PPARγ are essential partners involved in the regulation of subcutaneous adipose tissue adiponectin production and secretion and BCAA oxidative metabolism.

Impact of training state on fasting-induced regulation of adipose tissue metabolism in humans.

Recruitment of fatty acids from adipose tissue is increased during fasting. However, the molecular mechanisms behind fasting-induced metabolic regulation in human adipose tissue and the potential impact of training state in this are unknown. Therefore the aim of the present study was to investigate 1) fasting-induced regulation of lipolysis and glyceroneogenesis in human adipose tissue as well as 2) the impact of training state on basal oxidative capacity and fasting-induced metabolic regulation in human adipose tissue. Untrained [maximal oxygen uptake (V̇o2max) < 45 ml·min-1·kg-1] and trained subjects (V̇o2max > 55 ml·min-1·kg-1) fasted for 36 h, and abdominal subcutaneous adipose tissue biopsies were obtained 2, 12, 24, and 36 h after a standardized meal. Adipose tissue oxidative phosphorylation complexes, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase (PDH)-E1α protein as well as PDH kinase (PDK) 2, PDK4, and PDH phosphatase 2 mRNA content were higher in trained subjects than in untrained subjects. In addition, trained subjects had higher adipose tissue hormone-sensitive lipase Ser660 phosphorylation and adipose triglyceride lipase protein content as well as higher plasma free fatty acid concentration than untrained subjects during fasting. Moreover, adipose tissue PDH phosphorylation increased with fasting only in trained subjects. Taken together, trained subjects seem to possess higher basal adipose tissue oxidative capacity as well as higher capacity for regulation of lipolysis and for providing substrate for glyceroneogenesis in adipose tissue during fasting than untrained subjects. NEW & NOTEWORTHY This study shows for the first time higher protein content of lipolytic enzymes and higher oxidative phosphorylation protein in adipose tissue from trained subjects than from untrained subjects during fasting. Furthermore, trained subjects had higher capacity for adipose tissue glyceroneogenesis than untrained subjects.

Differential regulation of glyceroneogenesis by glucocorticoids in epididymal and retroperitoneal white adipose tissue from rats.

PURPOSE: Investigate the glycerol-3-phosphate generation pathways in epididymal (EPI) and retroperitoneal (RETRO) adipose tissues from dexamethasone-treated rats. METHODS: Rats were treated with dexamethasone for 7 days. Glycerol-3-phosphate generation pathways via glycolysis, glyceroneogenesis and direct phosphorylation of glycerol were evaluated, respectively, by 2-deoxyglucose uptake, phosphoenolpyruvate carboxykinase (PEPCK-C) activity and pyruvate incorporation into triacylglycerol (TAG)-glycerol, and glycerokinase activity and glycerol incorporation into TAG-glycerol. RESULTS: Dexamethasone treatment markedly decreased the body weight, but increased the weight and lipid content of EPI and RETRO and plasma insulin, glucose, non-esterified fatty acid and TAG levels. EPI and RETRO from dexamethasone-treated rats showed increased rates of de novo fatty acid synthesis (80 and 100%) and basal lipolysis (20%). In EPI, dexamethasone decreased the 2-deoxyglucose uptake (50%), as well as glyceroneogenesis, evidenced by a decrease of PEPCK-C activity (39%) and TAG-glycerol synthesis from pyruvate (66%), but increased the glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (72%) in this tissue. In spite of a similar reduction in 2-deoxyglucose uptake in RETRO, dexamethasone treatment increased glyceroneogenesis, evidenced by PEPCK activity (96%), and TAG-glycerol synthesis from pyruvate (110%), accompanied by a decrease in glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (50%). Dexamethasone effects on RETRO were accompanied by a decrease in p-Akt content and by lower insulin effects on the rates of glycerol release in the presence of isoproterenol and on the rates of glucose uptake in isolated adipocytes. CONCLUSION: Our data demonstrated differential regulation of glyceroneogenesis and direct phosphorylation of glycerol by glucocorticoids in EPI and RETRO from rats.