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Abstract Adiponectin is a secretory protein, primarily produced in adipocytes. However, low but detectable expression of adiponectin can be observed in cell types beyond adipocytes, particularly in kidney tubular cells, but its local renal role is unknown. We assessed the impact of renal adiponectin by utilizing male inducible kidney tubular cell-specific adiponectin overexpression or knockout mice. Kidney-specific adiponectin overexpression induces a doubling of phosphoenolpyruvate carboxylase expression and enhanced pyruvate-mediated glucose production, tricarboxylic acid cycle intermediates and an upregulation of fatty acid oxidation (FAO). Inhibition of FAO reduces the adiponectin-induced enhancement of glucose production, highlighting the role of FAO in the induction of renal gluconeogenesis. In contrast, mice lacking adiponectin in the kidney exhibit enhanced glucose tolerance, lower utilization and greater accumulation of lipid species. Hence, renal adiponectin is an inducer of gluconeogenesis by driving enhanced local FAO and further underlines the important systemic contribution of renal gluconeogenesis.

Summary: The liver coordinates the systemic response to nutrient deprivation and availability by producing glucose from gluconeogenesis during fasting and synthesizing lipids via de novo lipogenesis (DNL) when carbohydrates are abundant. Mitochondrial pyruvate metabolism is thought to play important roles in both gluconeogenesis and DNL. We examined the effects of hepatocyte-specific mitochondrial pyruvate carrier (MPC) deletion on the fasting-refeeding response. Rates of DNL during refeeding were impaired by hepatocyte MPC deletion, but this did not reduce intrahepatic lipid content. During fasting, glycerol is converted to glucose by two pathways; a direct cytosolic pathway and an indirect mitochondrial pathway requiring the MPC. Hepatocyte MPC deletion reduced the incorporation of 13C-glycerol into TCA cycle metabolites, but not into new glucose. Furthermore, suppression of glycerol and alanine metabolism did not affect glucose concentrations in fasted hepatocyte-specific MPC-deficient mice, suggesting multiple layers of redundancy in glycemic control in mice.

BACKGROUND Hepatic de novo lipogenesis (DNL) and β-oxidation are tightly coordinated, and their dysregulation is thought to contribute to the pathogenesis of nonalcoholic fatty liver (NAFL). Fasting normally relaxes DNL-mediated inhibition of hepatic β-oxidation, dramatically increasing ketogenesis and decreasing reliance on the TCA cycle. Thus, we tested whether aberrant oxidative metabolism in fasting NAFL subjects is related to the inability to halt fasting DNL.METHODS Forty consecutive nondiabetic individuals with and without a history of NAFL were recruited for this observational study. After phenotyping, subjects fasted for 24 hours, and hepatic metabolism was interrogated using a combination of 2H2O and 13C tracers, magnetic resonance spectroscopy, and high-resolution mass spectrometry.RESULTS Within a subset of subjects, DNL was detectable after a 24-hour fast and was more prominent in those with NAFL, though it was poorly correlated with steatosis. However, fasting DNL negatively correlated with hepatic β-oxidation and ketogenesis and positively correlated with citrate synthesis. Subjects with NAFL but undetectable fasting DNL (25th percentile) were comparatively normal. However, those with the highest fasting DNL (75th percentile) were intransigent to the effects of fasting on the concentration of insulin, non-esterified fatty acid, and ketones. Additionally, they sustained glycogenolysis and were spared the loss of oxaloacetate to gluconeogenesis in favor of citrate synthesis, which correlated with DNL and diminished ketogenesis.CONCLUSION Metabolic flux analysis in fasted subjects indicates that shared metabolic mechanisms link the dysregulations of hepatic DNL, ketogenesis, and the TCA cycle in NAFL.TRIAL REGISTRATION Data were obtained during the enrollment/non-intervention phase of Effect of Vitamin E on Non-Alcoholic Fatty Liver Disease, ClinicalTrials.gov NCT02690792.FUNDING This work was supported by the University of Texas Southwestern NORC Quantitative Metabolism Core (NIH P30DK127984), the NIH/National Institute of Diabetes and Digestive and Kidney Diseases (R01DK078184, R01DK128168, R01DK087977, R01DK132254, and K01DK133630), the NIH/National Institute on Alcohol Abuse and Alcoholism (K01AA030327), and the Robert A. Welch Foundation (I-1804).

Background & Aims: Substitution of lysine for glutamic acid at residu 167 in Transmembrane 6 superfamily member 2 (TM6SF2) is associated with fatty liver disease and reduced plasma lipid levels. Tm6sf2-/- mice replicate the human phenotype but were not suitable for detailed mechanistic studies. As an alternative model, we generated Tm6sf2-/- rats to determine the subcellular location and function of TM6SF2. Methods: Two lines of Tm6sf2-/- rats were established using gene editing. Lipids from tissues and from newly secreted very low density lipoproteins (VLDLs) were quantified using enzymatic assays and mass spectrometry. Neutral lipids were visualized in tissue sections using Oil Red O staining. The rate of dietary triglyceride (TG) absorption and hepatic VLDL-TG secretion were compared in Tm6sf2-/- mice and in their wild-type littermates. The intracellular location of TM6SF2 was determined by cell fractionation. Finally, TM6SF2 was immunoprecipitated from liver and enterocytes to identify interacting proteins. Results: Tm6sf2-/- rats had a 6-fold higher mean hepatic TG content (56.1 ± 28.9 9 vs 9.8 ± 3.9 mg/g; P < .0001) and lower plasma cholesterol levels (99.0 ± 10.5 vs 110.6 ± 14.0 mg/dL; P = .0294) than their wild-type littermates. Rates of appearance of dietary and hepatic TG into blood were reduced significantly in Tm6sf2-/- rats (P < .001 and P < .01, respectively). Lipid content of newly secreted VLDLs isolated from perfused livers was reduced by 53% (TG) and 62% (cholesterol) (P = .005 and P = .01, respectively) in Tm6sf2-/- mice. TM6SF2 was present predominantly in the smooth endoplasmic reticulum and endoplasmic reticulum–Golgi intermediate compartments, but not in Golgi. Both apolipoprotein B-48 and acyl-CoA synthetase long chain family member 5 physically interacted with TM6SF2. Conclusions: TM6SF2 acts in the smooth endoplasmic reticulum to promote bulk lipidation of apolipoprotein B–containing lipoproteins, thus preventing fatty liver disease.

Fat synthesis is necessary for normal physiology, but its dysregulation contributes to the pathology of many diseases. Here, the authors report a high-resolution mass spectrometry approach that quantifies fat synthesis flux in humans and mice following a brief and low dose of deuterated water.

Objective: Excessive hepatic gluconeogenesis is a defining feature of type 2 diabetes (T2D). Most gluconeogenic flux is routed through mitochondria. The mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytosol into the mitochondrial matrix, thereby gating pyruvate-driven gluconeogenesis. Disruption of the hepatocyte MPC attenuates hyperglycemia in mice during high fat diet (HFD)-induced obesity but exerts minimal effects on glycemia in normal chow diet (NCD)-fed conditions. The goal of this investigation was to test whether hepatocyte MPC disruption provides sustained protection from hyperglycemia during long-term HFD and the differential effects of hepatocyte MPC disruption on TCA cycle metabolism in NCD versus HFD conditions. Method: We utilized long-term high fat feeding, serial measurements of postabsorptive blood glucose and metabolomic profiling and 13C-lactate/13C-pyruvate tracing to investigate the contribution of the MPC to hyperglycemia and altered hepatic TCA cycle metabolism during HFD-induced obesity. Results: Hepatocyte MPC disruption resulted in long-term attenuation of hyperglycemia induced by HFD. HFD increased hepatic mitochondrial pyruvate utilization and TCA cycle capacity in an MPC-dependent manner. Furthermore, MPC disruption decreased progression of fibrosis and levels of transcript markers of inflammation. Conclusions: By contributing to chronic hyperglycemia, fibrosis, and TCA cycle expansion, the hepatocyte MPC is a key mediator of the pathophysiology induced in the HFD model of T2D.

Dysregulated mitochondrial metabolism during hepatic insulin resistance may contribute to pathophysiologies ranging from elevated glucose production to hepatocellular oxidative stress and inflammation. Given that obesity impairs insulin action but paradoxically activates mTORC1, we tested whether insulin action and mammalian target of rapamycin complex 1 (mTORC1) contribute to altered in vivo hepatic mitochondrial metabolism. Loss of hepatic insulin action for 2 weeks caused increased gluconeogenesis, mitochondrial anaplerosis, tricarboxylic acid (TCA) cycle oxidation, and ketogenesis. However, activation of mTORC1, induced by the loss of hepatic Tsc1, suppressed these fluxes. Only glycogen synthesis was impaired by both loss of insulin receptor and mTORC1 activation. Mice with a double knockout of the insulin receptor and Tsc1 had larger livers, hyperglycemia, severely impaired glycogen storage, and suppressed ketogenesis, as compared to those with loss of the liver insulin receptor alone. Thus, activation of hepatic mTORC1 opposes the catabolic effects of impaired insulin action under some nutritional states.

Intracellular lipids and their synthesis contribute to the mechanisms and complications of obesity-associated diseases. We describe an NMR approach that provides an abbreviated lipidomic analysis with concurrent lipid biosynthetic fluxes. Following deuterated water administration, positional isotopomer analysis by deuterium NMR of specific lipid species was used to examine flux through de novo lipogenesis (DNL), FA elongation, desaturation, and TG-glycerol synthesis. The NMR method obviated certain assumptions regarding sites of enrichment and exchangeable hydrogens required by mass isotope methods. The approach was responsive to genetic and pharmacological gain or loss of function of DNL, elongation, desaturation, and glyceride synthesis. BDF1 mice consuming a high-fat diet (HFD) or matched low-fat diet for 35 weeks were examined across feeding periods to determine how flux through these pathways contributes to diet induced fatty liver and obesity. HFD mice had increased rates of FA elongation and glyceride synthesis. However DNL was markedly suppressed despite insulin resistance and obesity. We conclude that most hepatic TGs in the liver of HFD mice were formed from the reesterification of existing or ingested lipids, not DNL.

Carrier-facilitated pyruvate transport across the inner mitochondrial membrane plays an essential role in anabolic and catabolic intermediary metabolism. Mitochondrial pyruvate carrier 2 (Mpc2) is believed to be a component of the complex that facilitates mitochondrial pyruvate import. Complete MPC2 deficiency resulted in embryonic lethality in mice. However, a second mouse line expressing an N-terminal truncated MPC2 protein (Mpc2Δ16) was viable but exhibited a reduced capacity for mitochondrial pyruvate oxidation. Metabolic studies demonstrated exaggerated blood lactate concentrations after pyruvate, glucose, or insulin challenge in Mpc2Δ16 mice. Additionally, compared with wild-type controls, Mpc2Δ16 mice exhibited normal insulin sensitivity but elevated blood glucose after bolus pyruvate or glucose injection. This was attributable to reduced glucose-stimulated insulin secretion and was corrected by sulfonylurea KATP channel inhibitor administration. Collectively, these data are consistent with a role for MPC2 in mitochondrial pyruvate import and suggest that Mpc2 deficiency results in defective pancreatic β cell glucose sensing.

The manner in which insulin resistance impinges on hepatic mitochondrial function is complex. Although liver insulin resistance is associated with respiratory dysfunction, the effect on fat oxidation remains controversial, and biosynthetic pathways that traverse mitochondria are actually increased. The tricarboxylic acid (TCA) cycle is the site of terminal fat oxidation, chief source of electrons for respiration, and a metabolic progenitor of gluconeogenesis. Therefore, we tested whether insulin resistance promotes hepatic TCA cycle flux in mice progressing to insulin resistance and fatty liver on a high-fat diet (HFD) for 32 weeks using standard biomolecular and in vivo 2H/13C tracer methods. Relative mitochondrial content increased, but respiratory efficiency declined by 32 weeks of HFD. Fasting ketogenesis became unresponsive to feeding or insulin clamp, indicating blunted but constitutively active mitochondrial β-oxidation. Impaired insulin signaling was marked by elevated in vivo gluconeogenesis and anaplerotic and oxidative TCA cycle flux. The induction of TCA cycle function corresponded to the development of mitochondrial respiratory dysfunction, hepatic oxidative stress, and inflammation. Thus, the hepatic TCA cycle appears to enable mitochondrial dysfunction during insulin resistance by increasing electron deposition into an inefficient respiratory chain prone to reactive oxygen species production and by providing mitochondria-derived substrate for elevated gluconeogenesis.

Fasting promotes triglyceride (TG) accumulation in lean tissues of some animals, but the effect in humans is unknown. Additionally, fasting lipolysis is sexually dimorphic in humans, suggesting that lean tissue TG accumulation and metabolism may differ between women and men. This study investigated lean tissue TG content and metabolism in women and men during extended fasting. Liver and muscle TG content were measured by magnetic resonance spectroscopy during a 48-h fast in healthy men and women. Whole-body and hepatic carbohydrate, lipid, and energy metabolism were also evaluated using biochemical, calorimetric, and stable isotope tracer techniques. As expected, postabsorptive plasma fatty acids (FAs) were higher in women than in men but increased more rapidly in men with the onset of early starvation. Concurrently, sexual dimorphism was apparent in lean tissue TG accumulation during the fast, occurring in livers of men but in muscles of women. Despite differences in lean tissue TG distribution, men and women had identical fasting responses in whole-body and hepatic glucose and oxidative metabolism. In conclusion, TG accumulated in livers of men but in muscles of women during extended fasting. This sexual dimorphism was related to differential fasting plasma FA concentrations but not to whole body or hepatic utilization of this substrate.

Sources of blood glucose can be determined after oral ingestion of 2H2O followed by isolation of plasma glucose and measurement of the relative 2H enrichments in select positions within the glucose molecule. Typically, 2H enrichments are obtained by mass spectrometry but 2H NMR offers an alternative. Here it is demonstrated that the entire analysis may be automated by Bayesian analysis of a 2H free induction decay signal of monoacetone glucose to obtain a direct readout of the relative contributions of glycogenolysis, glycerol, and phosphoenol pyruvate to plasma glucose production. Furthermore, Markov Chain Monte Carlo (MCMC) simulations of the posterior probability density provide uncertainties in all metabolic parameters from a single patient, thereby allowing comparisons in glucose metabolism from one individual to another. The combined MCMC Bayesian methodology is operationally simple and requires little intervention from the operator. Magn Reson Med 50:659–663, 2003. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract The heart requires a continuous supply of energy but has little capacity for energy storage and thus relies on exogenous metabolic sources. We previously showed that cardiac MED13 modulates systemic energy homeostasis in mice. Here, we sought to define the extra‐cardiac tissue(s) that respond to cardiac MED13 signaling. We show that cardiac overexpression of MED13 in transgenic (MED13cTg) mice confers a lean phenotype that is associated with increased lipid uptake, beta‐oxidation and mitochondrial content in white adipose tissue (WAT) and liver. Cardiac expression of MED13 decreases metabolic gene expression in the heart but enhances them in WAT. Although exhibiting increased energy expenditure in the fed state, MED13cTg mice metabolically adapt to fasting. Furthermore, MED13cTg hearts oxidize fuel that is readily available, rendering them more efficient in the fed state. Parabiosis experiments in which circulations of wild‐type and MED13cTg mice are joined, reveal that circulating factor(s) in MED13cTg mice promote enhanced metabolism and leanness. These findings demonstrate that MED13 acts within the heart to promote systemic energy expenditure in extra‐cardiac energy depots and point to an unexplored metabolic communication system between the heart and other tissues.

PGC-1β plays pleiotropic roles in regulating intermediary metabolism and has been shown to regulate both catabolic and anabolic processes in liver. We sought to evaluate the effects of PGC-1β on liver energy metabolism by generating mice with postnatal, liver-specific deletion of PGC-1β (LS-PGC-1β(-/-) mice). LS-PGC-1β(-/-) mice were outwardly normal, but exhibited a significant increase in hepatic triglyceride content at 6 weeks of age. Hepatic steatosis was due, at least in part, to impaired capacity for fatty acid oxidation and marked mitochondrial dysfunction. Mitochondrial DNA content and the expression of genes encoding multiple steps in mitochondrial fatty acid oxidation and oxidative phosphorylation pathways were significantly diminished in LS-PGC-1β(-/-) mice. Liquid chromatography mass spectrometry-based analyses also revealed that acetylcarnitine and butyrylcarnitine levels were depleted whereas palmitoylcarnitine content was increased in LS-PGC-1β(-/-) liver, which is consistent with attenuated rates of fatty acid oxidation. Interestingly, loss of PGC-1β also significantly impaired inducible expression of glycolytic and lipogenic enzymes that occurs with high carbohydrate diet refeeding after a prolonged fast. These results suggest that PGC-1β plays dual roles in regulating hepatic fatty acid metabolism by controlling the expression of programs of genes involved in both fatty acid oxidation and de novo fatty acid synthesis.