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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.

Hepatic gluconeogenesis from amino acids contributes significantly to diabetic hyperglycemia, but the molecular mechanisms involved are incompletely understood. Alanine transaminases (ALT1 and ALT2) catalyze the interconversion of alanine and pyruvate, which is required for gluconeogenesis from alanine. We find that ALT2 is overexpressed in the liver of diet-induced obese and db/db mice and that the expression of the gene encoding ALT2 (GPT2) is downregulated following bariatric surgery in people with obesity. The increased hepatic expression of Gpt2 in db/db liver is mediated by activating transcription factor 4, an endoplasmic reticulum stress-activated transcription factor. Hepatocyte-specific knockout of Gpt2 attenuates incorporation of

[Image: see text] Intracellular nucleotides and acyl-CoAs are metabolites that are central to the regulation of energy metabolism. They set the cellular energy charge and redox state, act as allosteric regulators, modulate signaling and transcription factors, and thermodynamically activate substrates for oxidation or biosynthesis. Unfortunately, no method exists to simultaneously quantify these biomolecules in tissue extracts. A simple method was developed using ion-pairing reversed-phase high-performance liquid chromatography–electrospray-ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to simultaneously quantify adenine nucleotides (AMP, ADP, and ATP), pyridine dinucleotides (NAD(+) and NADH), and short-chain acyl-CoAs (acetyl, malonyl, succinyl, and propionyl). Quantitative analysis of these molecules in mouse liver was achieved using stable-isotope-labeled internal standards. The method was extensively validated by determining the linearity, accuracy, repeatability, and assay stability. Biological responsiveness was confirmed in assays of liver tissue with variable durations of ischemia, which had substantial effects on tissue energy charge and redox state. We conclude that the method provides a simple, fast, and reliable approach to the simultaneous analysis of nucleotides and short-chain acyl-CoAs.

De novo lipogenesis (DNL) is disrupted in a wide range of human disease. Thus, quantification of DNL may provide insight into mechanisms and guide interventions if it can be performed rapidly and noninvasively. DNL flux is commonly measured by 2H incorporation into fatty acids following deuterated water (2H2O) administration. However, the sensitivity of this approach is limited by the natural abundance of 13C, which masks detection of 2H by mass spectrometry. Here we report that high-resolution Orbitrap gas-chromatography mass-spectrometry resolves 2H and 13C fatty acid mass isotopomers, allowing DNL to be quantified using lower 2H2O doses and shorter experimental periods than previously possible. Serial measurements over 24-hrs in mice detects the nocturnal activation of DNL and matches a 3H-water method in mice with genetic activation of DNL. Most importantly, DNL is detected in overnight-fasted humans in less than an hour and is responsive to feeding during a 4-h study. Thus, 2H specific MS provides the ability to study DNL in settings that are currently impractical., 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.

The pyruvate dehydrogenase complex (PDC) is a key control point of energy metabolism and is subject to regulation by multiple mechanisms, including posttranslational phosphorylation by pyruvate dehydrogenase kinase (PDK). Pharmacological modulation of PDC activity could provide a new treatment for diabetic cardiomyopathy, as dysregulated substrate selection is concomitant with decreased heart function. Dichloroacetate (DCA), a classic PDK inhibitor, has been used to treat diabetic cardiomyopathy, but the lack of specificity and side effects of DCA indicate a more specific inhibitor of PDK is needed. This study was designed to determine the effects of a novel and highly selective PDK inhibitor, 2((2,4-dihydroxyphenyl)sulfonyl) isoindoline-4,6-diol (designated PS10), on pyruvate oxidation in diet-induced obese (DIO) mouse hearts compared with DCA-treated hearts. Four groups of mice were studied: lean control, DIO, DIO + DCA, and DIO + PS10. Both DCA and PS10 improved glucose tolerance in the intact animal. Pyruvate metabolism was studied in perfused hearts supplied with physiological mixtures of long chain fatty acids, lactate, and pyruvate. Analysis was performed using conventional (1)H and (13)C isotopomer methods in combination with hyperpolarized [1-(13)C]pyruvate in the same hearts. PS10 and DCA both stimulated flux through PDC as measured by the appearance of hyperpolarized [(13)C]bicarbonate. DCA but not PS10 increased hyperpolarized [1-(13)C]lactate production. Total carbohydrate oxidation was reduced in DIO mouse hearts but increased by DCA and PS10, the latter doing so without increasing lactate production. The present results suggest that PS10 is a more suitable PDK inhibitor for treatment of diabetic cardiomyopathy.

delta-5 desaturase and delta-6 desaturase are enzymes known to be involved in the synthesis of highly unsaturated fatty acids. In this issue, Kim et al. (2019) show that production of NAD(+) by this desaturase reaction is an adaptive response to NAD(+) depletion that may regulate cellular REDOX status.

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., Graphical abstract Image 1, Highlights • Hepatic MPC disruption protects from hyperglycemia during long-term HFD. • HFD increases TCA cycle metabolite pool capacity and flux. • Hepatic MPC disruption abrogates HFD-induced TCA cycle expansion.

The heart oxidizes fatty acids, carbohydrates, and ketone bodies inside the tricarboxylic acid (TCA) cycle to generate the reducing equivalents needed for ATP production. Competition between these substrates makes it difficult to estimate the extent of pyruvate oxidation. Previously, hyperpolarized pyruvate detected propionate-mediated activation of carbohydrate oxidation, even in the presence of acetate. In this report, the optimal concentration of propionate for the activation of glucose oxidation was measured in mouse hearts perfused in Langendorff mode. This study was performed with a more physiologically relevant perfusate than the previous work. Increasing concentrations of propionate did not cause adverse effects on myocardial metabolism, as evidenced by unchanged O2consumption, TCA cycle flux, and developed pressures. Propionate at 1 mM was sufficient to achieve significant increases in pyruvate dehydrogenase flux (3×), and anaplerosis (6×), as measured by isotopomer analysis. These results further demonstrate the potential of propionate as an aid for the correct estimation of total carbohydrate oxidative capacity in the heart. However, liquid chromotography/mass spectroscopy-based metabolomics detected large changes (~30-fold) in malate and fumarate pool sizes. This observation leads to a key observation regarding mass balance in the TCA cycle; flux through a portion of the cycle can be drastically elevated without changing the O2consumption.

Computational models based on the metabolism of stable isotope tracers can yield valuable insight into the metabolic basis of disease. The complexity of these models is limited by the number of tracers and the ability to characterize tracer labeling in downstream metabolites. NMR spectroscopy is ideal for multiple tracer experiments since it precisely detects the position of tracer nuclei in molecules, but it lacks sensitivity for detecting low-concentration metabolites. GC-MS detects stable isotope mass enrichment in low-concentration metabolites, but lacks nuclei and positional specificity. We performed liver perfusions and in vivo infusions of (2)H and (13)C tracers, yielding complex glucose isotopomers that were assigned by NMR and fit to a newly developed metabolic model. Fluxes regressed from (2)H and (13)C NMR positional isotopomer enrichments served to validate GC-MS-based flux estimates obtained from the same experimental samples. NMR-derived fluxes were largely recapitulated by modeling the mass isotopomer distributions of six glucose fragment ions measured by GC-MS. Modest differences related to limited fragmentation coverage of glucose C1–C3 were identified, but fluxes such as gluconeogenesis, glycogenolysis, cataplerosis and TCA cycle flux were tightly correlated between the methods. Most importantly, modeling of GC-MS data could assign fluxes in primary mouse hepatocytes, an experiment that is impractical with (2)H or (13)C NMR.

Rats selectively bred for high capacity running (HCR) or low capacity running (LCR) display divergence for intrinsic aerobic capacity and hepatic mitochondrial oxidative capacity, both factors associated with susceptibility for nonalcoholic fatty liver disease. Here, we tested if HCR and LCR rats display differences in susceptibility for hepatic steatosis after 16 wk of high-fat diets (HFD) with either 45% or 60% of kcals from fat. HCR rats were protected against HFD-induced hepatic steatosis, whereas only the 60% HFD induced steatosis in LCR rats, as marked by a doubling of liver triglycerides. Hepatic complete fatty acid oxidation (FAO) and mitochondrial respiratory capacity were all lower in LCR compared with HCR rats. LCR rats also displayed lower hepatic complete and incomplete FAO in the presence of etomoxir, suggesting a reduced role for noncarnitine palmitoyltransferase-1-mediated lipid catabolism in LCR versus HCR rats. Hepatic complete FAO and mitochondrial respiration were largely unaffected by either chronic HFD; however, 60% HFD feeding markedly reduced 2-pyruvate oxidation, a marker of tricarboxylic acid (TCA) cycle flux, and mitochondrial complete FAO only in LCR rats. LCR rats displayed lower levels of hepatic long-chain acylcarnitines than HCR rats but maintained similar levels of hepatic acetyl-carnitine levels, further supporting lower rates of β-oxidation, and TCA cycle flux in LCR than HCR rats. Finally, only LCR rats displayed early reductions in TCA cycle genes after the acute initiation of a HFD. In conclusion, intrinsically high aerobic capacity confers protection against HFD-induced hepatic steatosis through elevated hepatic mitochondrial oxidative capacity.

SummaryDysregulated 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.

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 and13C 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.

Inhibiting lipogenesis prevents hepatic steatosis in rodents with insulin resistance. To determine if reducing lipogenesis functions similarly in humans, we developed MK-4074, a liver-specific inhibitor of acetyl-CoA carboxylase (ACC1) and (ACC2), enzymes that produce malonyl-CoA for fatty acid synthesis. MK-4074 administered to subjects with hepatic steatosis for 1 month lowered lipogenesis, increased ketones, and reduced liver triglycerides by 36%. Unexpectedly, MK-4074 increased plasma triglycerides by 200%. To further investigate, mice that lack ACC1 and ACC2 in hepatocytes (ACC dLKO) were generated. Deletion of ACCs decreased polyunsaturated fatty acid (PUFA) concentrations in liver due to reduced malonyl-CoA, which is required for elongation of essential fatty acids. PUFA deficiency induced SREBP-1c, which increased GPAT1 expression and VLDL secretion. PUFA supplementation or siRNA-mediated knockdown of GPAT1 normalized plasma triglycerides. Thus, inhibiting lipogenesis in humans reduced hepatic steatosis, but inhibiting ACC resulted in hypertriglyceridemia due to activation of SREBP-1c and increased VLDL secretion.

Glycogenolysis and gluconeogenesis are sensitive to nutritional state, and the net direction of flux is controlled by multiple enzymatic steps. This delicate balance in the liver is disrupted by a variety of pathological states including cancer and diabetes mellitus. Hyperpolarized carbon-13 magnetic resonance is a new metabolic imaging technique that can probe intermediary metabolism nondestructively. There are currently no methods to rapidly distinguish livers in a gluconeogenic from glycogenolytic state. Here we use the gluconeogenic precursor dihydroxyacetone (DHA) to deliver hyperpolarized carbon-13 to the perfused mouse liver. DHA enters gluconeogenesis at the level of the trioses. Perfusion conditions were designed to establish either a gluconeogenic or a glycogenolytic state. Unexpectedly, we found that [2-(13)C]DHA was metabolized within a few seconds to the common intermediates and end products of both glycolysis and gluconeogenesis under both conditions, including [2,5-(13)C]glucose, [2-(13)C]glycerol 3-phosphate, [2-(13)C]phosphoenolpyruvate (PEP), [2-(13)C]pyruvate, [2-(13)C]alanine, and [2-(13)C]lactate. [2-(13)C]Phosphoenolpyruvate, a key branch point in gluconeogenesis and glycolysis, was monitored in functioning tissue for the first time. Observation of [2-(13)C]PEP was not anticipated as the free energy difference between PEP and pyruvate is large. Pyruvate kinase is the only regulatory step of the common glycolytic-gluconeogenic pathway that appears to exert significant control over the kinetics of any metabolites of DHA. A ratio of glycolytic to gluconeogenic products distinguished the gluconeogenic from glycogenolytic state in these functioning livers.

SummaryCarrier-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.

Bile acid sequestrants are nonabsorbable resins designed to treat hypercholesterolemia by preventing ileal uptake of bile acids, thus increasing catabolism of cholesterol into bile acids. However, sequestrants also improve hyperglycemia and hyperinsulinemia through less characterized metabolic and molecular mechanisms. Here, we demonstrate that the bile acid sequestrant, colesevelam, significantly reduced hepatic glucose production by suppressing hepatic glycogenolysis in diet-induced obese mice and that this was partially mediated by activation of the G protein-coupled bile acid receptor TGR5 and glucagon-like peptide-1 (GLP-1) release. A GLP-1 receptor antagonist blocked suppression of hepatic glycogenolysis and blunted but did not eliminate the effect of colesevelam on glycemia. The ability of colesevelam to induce GLP-1, lower glycemia, and spare hepatic glycogen content was compromised in mice lacking TGR5. In vitro assays revealed that bile acid activation of TGR5 initiates a prolonged cAMP signaling cascade and that this signaling was maintained even when the bile acid was complexed to colesevelam. Intestinal TGR5 was most abundantly expressed in the colon, and rectal administration of a colesevelam/bile acid complex was sufficient to induce portal GLP-1 concentration but did not activate the nuclear bile acid receptor farnesoid X receptor (FXR). The beneficial effects of colesevelam on cholesterol metabolism were mediated by FXR and were independent of TGR5/GLP-1. We conclude that colesevelam administration functions through a dual mechanism, which includes TGR5/GLP-1-dependent suppression of hepatic glycogenolysis and FXR-dependent cholesterol reduction.

The Mediator complex governs gene expression by linking upstream signaling pathways with the basal transcriptional machinery. However, how individual Mediator subunits may function in different tissues remains to be investigated. Through skeletal muscle-specific deletion of the Mediator subunit MED13 in mice, we discovered a gene regulatory mechanism by which skeletal muscle modulates the response of the liver to a high-fat diet. Skeletal muscle-specific deletion of MED13 in mice conferred resistance to hepatic steatosis by activating a metabolic gene program that enhances muscle glucose uptake and storage as glycogen. The consequent insulin-sensitizing effect within skeletal muscle lowered systemic glucose and insulin levels independently of weight gain and adiposity and prevented hepatic lipid accumulation. MED13 suppressed the expression of genes involved in glucose uptake and metabolism in skeletal muscle by inhibiting the nuclear receptor NURR1 and the MEF2 transcription factor. These findings reveal a fundamental molecular mechanism for the governance of glucose metabolism and the control of hepatic lipid accumulation by skeletal muscle. Intriguingly, MED13 exerts opposing metabolic actions in skeletal muscle and the heart, highlighting the customized, tissue-specific functions of the Mediator complex.

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 (2)H/(13)C 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.

In the heart, detection of hyperpolarized [ 13 C]bicarbonate and 13 CO 2 by magnetic resonance (MR) after administration of hyperpolarized [1- 13 C]pyruvate is caused exclusively by oxidative decarboxylation of pyruvate via the pyruvate dehydrogenase complex (PDH). However, liver mitochondria possess alternative anabolic pathways accessible by [1- 13 C]pyruvate, which may allow a wider diagnostic range for hyperpolarized MR compared with other tissue. Metabolism of hyperpolarized [1- 13 C]pyruvate in the tricarboxylic acid (TCA) cycle was monitored in the isolated perfused liver from fed and fasted mice. Hyperpolarized [1- 13 C]pyruvate was rapidly converted to [1- 13 C]lactate, [1- 13 C]alanine, [1- 13 C]malate, [4- 13 C]malate, [1- 13 C]aspartate, [4- 13 C]aspartate, and [ 13 C]bicarbonate. Livers from fasted animals had increased lactate:alanine, consistent with elevated NADH:NAD + . The appearance of asymmetrically enriched malate and aspartate indicated high rates of anaplerotic pyruvate carboxylase activity and incomplete equilibration with fumarate. Hyperpolarized [ 13 C]bicarbonate was also detected, consistent with multiple mechanisms, including cataplerotic decarboxylation of [4- 13 C]oxaloacetate via phosphoenolpyruvate carboxykinase (PEPCK), forward TCA cycle flux of [4- 13 C]oxaloacetate to generate 13 CO 2 at isocitrate dehydrogenase, or decarboxylation of [1- 13 C]pyruvate by PDH. Isotopomer analysis of liver glutamate confirmed that anaplerosis was sevenfold greater than flux through PDH. In addition, signal from [4- 13 C]malate and [4- 13 C]aspartate was markedly blunted and signal from [ 13 C]bicarbonate was completely abolished in livers from PEPCK KO mice, indicating that the major pathway for entry of hyperpolarized [1- 13 C]pyruvate into the hepatic TCA cycle is via pyruvate carboxylase, and that cataplerotic flux through PEPCK is the primary source of [ 13 C]bicarbonate. We conclude that MR detection of hyperpolarized TCA intermediates and bicarbonate is diagnostic of pyruvate carboxylase and PEPCK flux in the liver.

Elevation of postprandial glucose stimulates release of insulin from granules stored in pancreatic islet β-cells. We demonstrate here that divalent zinc ions coreleased with insulin from β-cells in response to high glucose are readily detected by MRI using the Zn 2+ -responsive T 1 agent, GdDOTA-diBPEN. Image contrast was significantly enhanced in the mouse pancreas after injection of a bolus of glucose followed by a low dose of the Zn 2+ sensor. Images of the pancreas were not enhanced by the agent in mice without addition of glucose to stimulate insulin release, nor were images enhanced in streptozotocin-treated mice with or without added glucose. These observations are consistent with MRI detection of Zn 2+ released from β-cells only during glucose-stimulated insulin secretion. Images of mice fed a high-fat (60%) diet over a 12-wk period and subjected to this same imaging protocol showed a larger volume of contrast-enhanced pancreatic tissue, consistent with the expansion of pancreatic β-cell mass during fat accumulation and progression to type 2 diabetes. This MRI sensor offers the exciting potential for deep-tissue monitoring of β-cell function in vivo during development of type 2 diabetes or after implantation of islets in type I diabetic patients.

Hepatic ketogenesis provides a vital systemic fuel during fasting because ketone bodies are oxidized by most peripheral tissues and, unlike glucose, can be synthesized from fatty acids via mitochondrial β-oxidation. Since dysfunctional mitochondrial fat oxidation may be a cofactor in insulin-resistant tissue, the objective of this study was to determine whether diet-induced insulin resistance in mice results in impaired in vivo hepatic fat oxidation secondary to defects in ketogenesis. Ketone turnover (μmol/min) in the conscious and unrestrained mouse was responsive to induction and diminution of hepatic fat oxidation, as indicated by an eightfold rise during the fed (0.50+/−0.1)-to-fasted (3.8+/−0.2) transition and a dramatic blunting of fasting ketone turnover in PPARα−/−mice (1.0+/−0.1). C57BL/6 mice made obese and insulin resistant by high-fat feeding for 8 wk had normal expression of genes that regulate hepatic fat oxidation, whereas 16 wk on the diet induced expression of these genes and stimulated the function of hepatic mitochondrial fat oxidation, as indicated by a 40% induction of fasting ketogenesis and a twofold rise in short-chain acylcarnitines. Together, these findings indicate a progressive adaptation of hepatic ketogenesis during high-fat feeding, resulting in increased hepatic fat oxidation after 16 wk of a high-fat diet. We conclude that mitochondrial fat oxidation is stimulated rather than impaired during the initiation of hepatic insulin resistance in mice.

The liver plays a crucial role in mobilizing energy during nutritional deprivation. During the early stages of fasting, hepatic glycogenolysis is a primary energy source. As fasting progresses and glycogen stores are depleted, hepatic gluconeogenesis and ketogenesis become major energy sources. Here, we show that fibroblast growth factor 21 (FGF21), a hormone that is induced in liver by fasting, induces hepatic expression of peroxisome proliferator-activated receptor gamma coactivator protein-1alpha (PGC-1alpha), a key transcriptional regulator of energy homeostasis, and causes corresponding increases in fatty acid oxidation, tricarboxylic acid cycle flux, and gluconeogenesis without increasing glycogenolysis. Mice lacking FGF21 fail to fully induce PGC-1alpha expression in response to a prolonged fast and have impaired gluconeogenesis and ketogenesis. These results reveal an unexpected relationship between FGF21 and PGC-1alpha and demonstrate an important role for FGF21 in coordinately regulating carbohydrate and fatty acid metabolism during the progression from fasting to starvation.

Glucose-stimulated insulin secretion (GSIS) is central to normal control of metabolic fuel homeostasis, and its impairment is a key element of β-cell failure in type 2 diabetes. Glucose exerts its effects on insulin secretion via its metabolism in β-cells to generate stimulus/secretion coupling factors, including a rise in the ATP/ADP ratio, which serves to suppress ATP-sensitive K+(KATP) channels and activate voltage-gated Ca2+channels, leading to stimulation of insulin granule exocytosis. Whereas this KATPchannel-dependent mechanism of GSIS has been broadly accepted for more than 30 years, it has become increasingly apparent that it does not fully describe the effects of glucose on insulin secretion. More recent studies have demonstrated an important role for cyclic pathways of pyruvate metabolism in control of insulin secretion. Three cycles occur in islet β-cells: the pyruvate/malate, pyruvate/citrate, and pyruvate/isocitrate cycles. This review discusses recent work on the role of each of these pathways in control of insulin secretion and builds a case for the particular relevance of byproducts of the pyruvate/isocitrate cycle, NADPH and α-ketoglutarate, in control of GSIS.

The heart adapts to changes in nutritional status and energy demands by adjusting its relative metabolism of carbohydrates and fatty acids. Loss of this metabolic flexibility such as occurs in diabetes mellitus is associated with cardiovascular disease and heart failure. To study the long-term consequences of impaired metabolic flexibility, we have generated mice that overexpress pyruvate dehydrogenase kinase (PDK)4 selectively in the heart. Hearts from PDK4 transgenic mice have a marked decrease in glucose oxidation and a corresponding increase in fatty acid catabolism. Although no overt cardiomyopathy was observed in the PDK4 transgenic mice, introduction of the PDK4 transgene into mice expressing a constitutively active form of the phosphatase calcineurin, which causes cardiac hypertrophy, caused cardiomyocyte fibrosis and a striking increase in mortality. These results demonstrate that cardiac-specific overexpression of PDK4 is sufficient to cause a loss of metabolic flexibility that exacerbates cardiomyopathy caused by the calcineurin stress-activated pathway.

OBJECTIVE—Fluxes through mitochondrial pathways are defective in insulin-resistant skeletal muscle, but it is unclear whether similar mitochondrial defects play a role in the liver during insulin resistance and/or diabetes. The purpose of this study is to determine whether abnormal mitochondrial metabolism plays a role in the dysregulation of both hepatic fat and glucose metabolism during diabetes. RESEARCH DESIGN AND METHODS—Mitochondrial fluxes were measured using 2H/13C tracers and nuclear magnetic resonance spectroscopy in ZDF rats during early and advanced diabetes. To determine whether defects in hepatic fat oxidation can be corrected by peroxisome proliferator–activated receptor (PPAR-)-α activation, rats were treated with WY14,643 for 3 weeks before tracer administration. RESULTS—Hepatic mitochondrial fat oxidation in the diabetic liver was impaired twofold secondary to decreased ketogenesis, but tricarboxylic acid (TCA) cycle activity and pyruvate carboxylase flux were normal in newly diabetic rats and elevated in older rats. Treatment of diabetic rats with a PPAR–α agonist induced hepatic fat oxidation via ketogenesis and hepatic TCA cycle activity but failed to lower fasting glycemia or endogenous glucose production. In fact, PPAR-α agonism overstimulated mitochondrial TCA cycle flux and induced pyruvate carboxylase flux and gluconeogenesis in lean rats. CONCLUSIONS—The impairment of certain mitochondrial fluxes, but preservation or induction of others, suggests a complex defect in mitochondrial metabolism in the diabetic liver. These data indicate an important codependence between hepatic fat oxidation and gluconeogenesis in the normal and diabetic state and potentially explain the sometimes equivocal effect of PPAR-α agonists on glycemia.

When dietary carbohydrate is unavailable, glucose required to support metabolism in vital tissues is generated via gluconeogenesis in the liver. Expression of phosphoenolpyruvate carboxykinase (PEPCK), commonly considered the control point for liver gluconeogenesis, is normally regulated by circulating hormones to match systemic glucose demand. However, this regulation fails in diabetes. Because other molecular and metabolic factors can also influence gluconeogenesis, the explicit role of PEPCK protein content in the control of gluconeogenesis was unclear. In this study, metabolic control of liver gluconeogenesis was quantified in groups of mice with varying PEPCK protein content. Surprisingly, livers with a 90% reduction in PEPCK content showed only a approximately 40% reduction in gluconeogenic flux, indicating a lower than expected capacity for PEPCK protein content to control gluconeogenesis. However, PEPCK flux correlated tightly with TCA cycle activity, suggesting that under some conditions in mice, PEPCK expression must coordinate with hepatic energy metabolism to control gluconeogenesis.

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments. ispartof: Current Opinion in Biotechnology vol:34 pages:189-201 ispartof: location:England status: published

Pyruvate transport across the inner mitochondrial membrane is believed to be a prerequisite step for gluconeogenesis in hepatocytes, which is important for maintenance of normoglycemia during prolonged food deprivation, but also contributes to hyperglycemia in diabetes. To determine the requirement for mitochondrial pyruvate import in gluconeogenesis, mice with liver-specific deletion of mitochondrial pyruvate carrier 2 (LS-Mpc2−/−) were generated. Loss of MPC2 impaired, but did not completely abolish, hepatocyte pyruvate metabolism, labelled pyruvate conversion to TCA cycle intermediates and glucose, and glucose production from pyruvate. Unbiased metabolomic analyses of livers from fasted LS-Mpc2−/− mice suggested that alterations in amino acid metabolism, including pyruvate-alanine cycling, might compensate for loss of MPC2. Indeed, inhibition of pyruvate-alanine transamination further reduced mitochondrial pyruvate metabolism and glucose production by LS-Mpc2−/− hepatocytes. These data demonstrate an important role for MPC2 in controlling hepatic gluconeogenesis and illuminate a compensatory mechanism for circumventing a block in mitochondrial pyruvate import.

Mitochondria are critical for respiration in all tissues; however, in liver, these organelles also accommodate high-capacity anaplerotic/cataplerotic pathways that are essential to gluconeogenesis and other biosynthetic activities. During nonalcoholic fatty liver disease (NAFLD), mitochondria also produce ROS that damage hepatocytes, trigger inflammation, and contribute to insulin resistance. Here, we provide several lines of evidence indicating that induction of biosynthesis through hepatic anaplerotic/cataplerotic pathways is energetically backed by elevated oxidative metabolism and hence contributes to oxidative stress and inflammation during NAFLD. First, in murine livers, elevation of fatty acid delivery not only induced oxidative metabolism, but also amplified anaplerosis/cataplerosis and caused a proportional rise in oxidative stress and inflammation. Second, loss of anaplerosis/cataplerosis via genetic knockdown of phosphoenolpyruvate carboxykinase 1 (Pck1) prevented fatty acid–induced rise in oxidative flux, oxidative stress, and inflammation. Flux appeared to be regulated by redox state, energy charge, and metabolite concentration, which may also amplify antioxidant pathways. Third, preventing elevated oxidative metabolism with metformin also normalized hepatic anaplerosis/cataplerosis and reduced markers of inflammation. Finally, independent histological grades in human NAFLD biopsies were proportional to oxidative flux. Thus, hepatic oxidative stress and inflammation are associated with elevated oxidative metabolism during an obesogenic diet, and this link may be provoked by increased work through anabolic pathways.

Triglyceride turnover in the isolated C57/BL6 mouse heart was measured by dynamic 13C edit-(1)H observe NMR and the rate of fatty acid oxidation was determined by 13C NMR isotopomer analysis. In the presence of a physiological mixture of substrates, energy was produced in the citric acid cycle by oxidation of long-chain fatty acids (18%), ketones (34%), lactate (24%), pyruvate (7%), and other sources (17%). Exogenous fatty acids appeared in the triglyceride pool at 0.24 micromol/g dry wt/min, similar to the rate of oxidation of long-chain fatty acids, 0.16 micromol/g dry wt/min. Isoproterenol decreased the rate of de novo triglyceride synthesis and increased the rate of fatty acid oxidation.

Lungs harvested for transplantation utilize oxygen after procurement. We investigated the effects of storage solution substrate composition on pulmonary oxidative metabolism and energetics during the preservation interval. Rat lungs were harvested and stored at 10 degrees C in low-potassium dextran (LPD) solution. Groups of lungs were preserved with preservation solution containing 5mM carbon-13 ((13)C) labeled glucose or increasing concentrations of (13)C labeled pyruvate. Additional groups of rat lungs were studied with dichloroacetate (DCA) added to the pyruvate-modified preservation solutions. Oxidative metabolism (measured by (13)C-enrichment of glutamate) and adenine nucleotide levels were quantified. Increasing preservation solution pyruvate concentration augmented glutamate (13)C-enrichment up to a concentration of 32mM pyruvate. DCA further stimulated oxidative metabolism only at lower concentrations of pyruvate (4 and 8mM). ATP and ADP were not different among groups, but AMP levels were higher in the glucose group. These data suggest that altering the substrate composition of the preservation solution influences lung metabolism during allograft preservation for transplantation.

Background strain is known to influence the way a genetic manipulation affects mouse phenotypes. Despite data that demonstrate variations in the primary phenotype of basic inbred strains of mice, there is limited data available about specific metabolic fluxes in vivo that may be responsible for the differences in strain phenotypes. In this study, a simple stable isotope tracer/NMR spectroscopic protocol has been used to compare metabolic fluxes in ICR, FVB/N (FVB), C57BL/6J (B6), and 129S1/SvImJ (129) mouse strains. After a short-term fast in these mice, there were no detectable differences in the pathway fluxes that contribute to glucose synthesis. However, after a 24-h fast, B6 mice retain some residual glycogenolysis compared with other strains. FVB mice also had a 30% higher in vivo phospho enolpyruvate carboxykinase flux and total glucose production from the level of the TCA cycle compared with B6 and 129 strains, while total body glucose production in the 129 strain was ∼30% lower than in either FVB or B6 mice. These data indicate that there are inherent differences in several pathways involving glucose metabolism of inbred strains of mice that may contribute to a phenotype after genetic manipulation in these animals. The techniques used here are amenable to use as a secondary or tertiary tool for studying mouse models with disruptions of intermediary metabolism.

The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3′-5-triiodo-l-thyronine (T3)-treated rats and Zucker diabetic fatty (ZDF) rats ( fa/fa) after a 24-h fast. 2H2O and [U-13C3]propionate were administered intraperitoneally, and [3,4-13C2]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. 13C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both T3-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by 2H NMR analysis. In T3-treated rats, the hepatic glycogen content and hence the contribution of glycogenolysis to glucose production was essentially zero; in this case, excess glucose production was due to a dramatic increase in gluconeogenesis from TCA cycle intermediates. 13C NMR analysis also revealed increased phospho enolpyruvate carboxykinase flux (4×), increased pyruvate cycling flux (4×), and increased TCA flux (5×) in T3-treated animals. ZDF rats had substantial glycogen stores after a 24-h fast, and consequently nearly 50% of plasma glucose originated from glycogenolysis; other fluxes related to the TCA cycle were not different from controls. The differing mechanisms of excess glucose production in these models were easily distinguished by integrated 2H and 13C NMR analysis of plasma glucose.

To investigate the role of Angptl4, an inhibitor of lipoprotein lipase that is induced by >3-fold in the heart after rosiglitazone treatment, we generated transgenic mice that overexpress Angptl4 in the heart (MHC-Angptl4). We show that MHC-Angptl4 mice exhibit cardiac-restricted expression of the transgene and inhibition of cardiac lipoprotein lipase (LPL) activity. However, LPL activities in other tissues or that released into plasma by heparin are not affected. In addition, MHC-Angptl4 mice also exhibit hypertriglyceridemia after 6 h of fasting. We use echocardiography to show that MHC-Angptl4 mice develop left-ventricular dysfunction. Comparison of the metabolic profiles of isolated working hearts demonstrates that cardiac impairment in MHC-Angptl4 mice is positively associated with decreased triglyceride (TG) utilization. When bred to transgenic mice that overexpress acyl-CoA synthetase in the heart, a strain that exhibits elevated cardiac TG accumulation, cardiac TG content in double transgenic mice is reversed to that of wild-type mice. Taken together, our data support the hypothesis that induction of Angptl4 in the heart inhibits lipoprotein-derived fatty acid delivery. This mouse model will be useful to elucidate the role of reduced fatty acid supply in the pathogenesis of heart failure and related disorders.

A recently introduced tracer, [3,4-(13)C(2)]glucose, was compared to the widely used tracer, [6,6-(2)H(2)]glucose, for measurement of whole-body glucose turnover. The rate of glucose production (GP) was measured in rats after primed infusions of [3,4-(13)C(2)]glucose, [6,6-(2)H(2)]glucose, or both tracers simultaneously followed by a constant infusion of tracer(s) over 90 min. Blood glucose was purified and converted into monoacetone glucose for analysis by (13)C NMR (for [3,4-(13)C(2)]glucose) or (1)H and (2)H NMR (for [6,6-(2)H(2)]glucose). The values of GP measured during infusion of each single tracer were not significantly different. In rats infused with both tracers simultaneously, GP was identical as reported by each tracer, 42 +/- 4 micromol/kg/min. Since (2)H and (13)C enrichment in glucose is typically much less than 2% for in vivo studies, [3,4-(13)C(2)]glucose does not interfere with measurements of (13)C or (2)H enrichment patterns and therefore is valuable when multiple metabolic pathways are being evaluated simultaneously.

Type 1 and type 2 diabetes are both diseases of insulin insufficiency, although they develop by distinct pathways. The recent surge in the incidence of type 2 diabetes and the chronic ailments confronted by patients with either form of the disease highlight the need for better understanding of beta-cell biology. In this review, we present recent work focused on this goal. Our hope is that basic research being conducted in this and other laboratories will ultimately contribute to the development of methods for enhancing beta-cell function and survival in the context of both major forms of diabetes. Our strategy for understanding the beta-cell involves a multidisciplinary approach in which tools from the traditional fields of biochemistry, enzymology, and physiology are teamed with newer technologies from the fields of molecular biology, gene discovery, cell and developmental biology, and biophysical chemistry. We have focused on two important aspects of beta-cell biology in our studies: beta-cell function, specifically the metabolic regulatory mechanisms involved in glucose-stimulated insulin secretion, and beta-cell resistance to immune attack, with emphasis on resistance to inflammatory cytokines and reactive oxygen species.

Liver-specific PEPCK knockout mice, which are viable despite markedly abnormal lipid metabolism, exhibit mild hyperglycemia in response to fasting. We used isotopic tracer methods, biochemical measurements, and nuclear magnetic resonance spectroscopy to show that in mice lacking hepatic PEPCK, 1) whole-body glucose turnover is only slightly decreased; 2) whole-body gluconeogenesis from phosphoenolpyruvate, but not from glycerol, is moderately decreased; 3) tricarboxylic acid cycle activity is globally increased, even though pyruvate cycling and anaplerosis are decreased; 4) the liver is unable to synthesize glucose from lactate/pyruvate and produces only a minimal amount of glucose; and 5) glycogen synthesis in both the liver and muscle is impaired. Thus, although mice without hepatic PEPCK have markedly impaired hepatic gluconeogenesis, they are able to maintain a near-normal blood glucose concentration while fasting by increasing extrahepatic gluconeogenesis coupled with diminishing whole-body glucose utilization.

Summary Despite the different physiologic functions of FGF19 and FGF21 as hormonal regulators of fed and fasted metabolism, their pharmacologic administration causes similar increases in energy expenditure, weight loss, and enhanced insulin sensitivity in obese animals. Here, in genetic loss-of-function studies of the shared co-receptor β-Klotho, we show that these pharmacologic effects are mediated through a common, tissue-specific pathway. Surprisingly, FGF19 and FGF21 actions in liver and adipose tissue are not required for their longer-term weight loss and glycemic effects. In contrast, β-Klotho in neurons is essential for both FGF19 and FGF21 to cause weight loss and lower glucose and insulin levels. We further show an FGF21 mimetic antibody that activates the FGF receptor 1/β-Klotho complex also requires neuronal β-Klotho for its metabolic effects. These studies highlight the importance of the nervous system in mediating the beneficial weight loss and glycemic effects of endocrine FGF drugs.

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.

Flux through pyruvate dehydrogenase (PDH) in the heart may be reduced by various forms of injury to the myocardium, or by oxidation of alternative substrates in normal heart tissue. It is important to distinguish these two mechanisms because imaging of flux through PDH based on the appearance of hyperpolarized (HP) [13C]bicarbonate derived from HP [1-13C]pyruvate has been proposed as a method for identifying viable myocardium. The efficacy of propionate for increasing PDH flux in the setting of PDH inhibition by an alternative substrate was studied using isotopomer analysis paired with exams using HP [1-13C]pyruvate. Hearts from C57/bl6 mice were supplied with acetate (2 mM) and glucose (8.25 mM).13C NMR spectra were acquired in a cryogenically cooled probe at 14.1 Tesla. After addition of hyperpolarized [1-13C]pyruvate,13C NMR signals from lactate, alanine, malate, and aspartate were easily detected, in addition to small signals from bicarbonate and CO2. The addition of propionate (2 mM) increased appearance of HP [13C]bicarbonate >30-fold without change in O2consumption. Isotopomer analysis of extracts from the freeze-clamped hearts indicated that acetate was the preferred substrate for energy production, glucose contribution to energy production was minimal, and anaplerosis was stimulated in the presence of propionate. Under conditions where production of acetyl-CoA is dominated by the availability of an alternative substrate, acetate, propionate markedly stimulated PDH flux as detected by the appearance of hyperpolarized [13C]bicarbonate from metabolism of hyperpolarized [1-13C]pyruvate.

Background & Aims Hepatic gluconeogenesis helps maintain systemic energy homeostasis by compensating for discontinuities in nutrient supply. Liver-specific deletion of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) abolishes gluconeogenesis from mitochondrial substrates, deregulates lipid metabolism and affects TCA cycle. While the mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). Despite clear relevance to human physiology, the role of PEPCK-M and its gluconeogenic potential remain unknown. Here, we test the significance of PEPCK-M in gluconeogenesis and TCA cycle function in liver-specific PEPCK-C knockout and WT mice. Methods The effects of the overexpression of PEPCK-M were examined by a combination of tracer studies and molecular biology techniques. Partial PEPCK-C re-expression was used as a positive control. Metabolic fluxes were evaluated in isolated livers by NMR using 2 H and 13 C tracers. Gluconeogenic potential, together with metabolic profiling, was investigated in vivo and in primary hepatocytes. Results PEPCK-M expression partially rescued defects in lipid metabolism, gluconeogenesis and TCA cycle function impaired by PEPCK-C deletion, while ∼10% re-expression of PEPCK-C normalized most parameters. When PEPCK-M was expressed in the presence of PEPCK-C, the mitochondrial isozyme amplified total gluconeogenic capacity, suggesting autonomous regulation of oxaloacetate to phosphoenolpyruvate fluxes by the individual isoforms. Conclusions We conclude that PEPCK-M has gluconeogenic potential per se , and cooperates with PEPCK-C to adjust gluconeogenic/TCA flux to changes in substrate or energy availability, hinting at a role in the regulation of glucose and lipid metabolism in the human liver.

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.

To determine unambiguously if suppression of glucagon action will eliminate manifestations of diabetes, we expressed glucagon receptors in livers of glucagon receptor-null (GcgR −/− ) mice before and after β-cell destruction by high-dose streptozotocin. Wild type (WT) mice developed fatal diabetic ketoacidosis after streptozotocin, whereas GcgR −/− mice with similar β-cell destruction remained clinically normal without hyperglycemia, impaired glucose tolerance, or hepatic glycogen depletion. Restoration of receptor expression using adenovirus containing the GcgR cDNA restored hepatic GcgR, phospho-cAMP response element binding protein (P-CREB), and phosphoenol pyruvate carboxykinase, markers of glucagon action, rose dramatically and severe hyperglycemia appeared. When GcgR mRNA spontaneously disappeared 7 d later, P-CREB declined and hyperglycemia disappeared. In conclusion, the metabolic manifestations of diabetes cannot occur without glucagon action and, once present, disappear promptly when glucagon action is abolished. Glucagon suppression should be a major therapeutic goal in diabetes.

Background & Aims Roux-en-Y gastric bypass (RYGB) improves glucose homeostasis independently of changes in body weight by unknown mechanisms. Melanocortin-4 receptors (MC4R) have weight-independent effects on glucose homeostasis, via autonomic neurons, and also might contribute to weight loss after RYGB. We investigated whether MC4Rs mediate effects of RYGB, such as its weight-independent effects on glucose homeostasis, in mice and humans. Methods We studied C57BL/6 mice with diet-induced obesity, MC4R-deficient mice, and mice that re-express MC4R specifically in autonomic neurons after RYGB or sham surgeries. We also sequenced the MC4R locus in patients undergoing RYGB to investigate diabetes resolution in carriers of rare MC4R variants. Results MC4Rs in autonomic brainstem neurons (including the parasympathetic dorsal motor vagus) mediated improved glucose homeostasis independent of changes in body weight. In contrast, MC4Rs in cholinergic preganglionic motor neurons (sympathetic and parasympathetic) mediated RYGB-induced increased energy expenditure and weight loss. Increased energy expenditure after RYGB is the predominant mechanism of weight loss and confers resistance to weight gain from a high-fat diet, the effects of which are MC4R-dependent. MC4R-dependent effects of RYGB still occurred in mice with Mc4r haplosufficiency, and early stage diabetes resolved at a similar rate in patients with rare variants of MC4R and noncarriers. However, carriers of MC4R (I251L), a rare variant associated with increased weight loss after RYGB and increased basal activity in vitro, were more likely to have early and weight-independent resolution of diabetes than noncarriers, indicating a role for MC4Rs in the effects of RYGB. Conclusions MC4Rs in autonomic neurons mediate beneficial effects of RYGB, including weight-independent improved glucose homeostasis, in mice and humans.

Obesity is a risk factor for type 2 diabetes in cats. The risk of developing diabetes is severalfold greater for male cats than for females, even after having been neutered early in life. The purpose of this study was to investigate the role of different metabolic pathways in the regulation of endogenous glucose production (EGP) during the fasted state considering these risk factors. A triple tracer protocol using 2H2O, [U-13C3]propionate, and [3,4-13C2]glucose was applied in overnight-fasted cats (12 lean and 12 obese; equal sex distribution) fed three different diets. Compared with lean cats, obese cats had higher insulin ( P < 0.001) but similar blood glucose concentrations. EGP was lower in obese cats ( P < 0.001) due to lower glycogenolysis and gluconeogenesis (GNG; P < 0.03). Insulin, body mass index, and girth correlated negatively with EGP ( P < 0.003). Female obese cats had ∼1.5 times higher fluxes through phosphoenolpyruvate carboxykinase ( P < 0.02) and citrate synthase ( P < 0.05) than male obese cats. However, GNG was not higher because pyruvate cycling was increased 1.5-fold ( P < 0.03). These results support the notion that fasted obese cats have lower hepatic EGP compared with lean cats and are still capable of maintaining fasting euglycemia, despite the well-documented existence of peripheral insulin resistance in obese cats. Our data further suggest that sex-related differences exist in the regulation of hepatic glucose metabolism in obese cats, suggesting that pyruvate cycling acts as a controlling mechanism to modulate EGP. Increased pyruvate cycling could therefore be an important factor in modulating the diabetes risk in female cats.

We have previously demonstrated a role for pyruvate cycling in glucose-stimulated insulin secretion (GSIS). Some of the possible pyruvate cycling pathways are completed by conversion of malate to pyruvate by malic enzyme. Using INS-1-derived 832/13 cells, it has recently been shown by other laboratories that NADP-dependent cytosolic malic enzyme (MEc), but not NAD-dependent mitochondrial malic enzyme (MEm), regulates GSIS. In the current study, we show that small interfering RNA-mediated suppression of either MEm or MEc results in decreased GSIS in both 832/13 cells and a new and more glucose- and incretin-responsive INS-1-derived cell line, 832/3. The effect of MEm to suppress GSIS in these cell lines was linked to a substantial decrease in cell growth, whereas MEc suppression resulted in decreased NADPH, shown previously to be correlated with GSIS. However, adenovirus-mediated delivery of small interfering RNAs specific to MEc and MEm to isolated rat islets, while leading to effective suppression of the targets transcripts, had no effect on GSIS. Furthermore, islets isolated from MEc-null MOD1–/– mice exhibit normal glucose- and potassium-stimulated insulin secretion. These results indicate that pyruvate-malate cycling does not control GSIS in primary rodent islets.

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.

Livers from mice lacking the carbohydrate-responsive element-binding protein (ChREBP) were compared with wild type (WT) mice to determine the effect of this transcription factor on hepatic energy metabolism. The pyruvate dehydrogenase complex was considerably more active in ChREBP(-/-) mice because of diminished pyruvate dehydrogenase kinase activity. Greater pyruvate dehydrogenase complex activity caused a stimulation of lactate and pyruvate oxidation, and it significantly impaired fatty acid oxidation in perfused livers from ChREBP(-/-) mice. This shift in mitochondrial substrate utilization led to a 3-fold reduction of the free cytosolic [NAD(+)]/[NADH] ratio, a 1.7-fold increase in the free mitochondrial [NAD(+)]/[NADH] ratio, and a 2-fold decrease in the free cytosolic [ATP]/[ADP][P(i)] ratio in the ChREBP(-/-) liver compared with control. Hepatic pyruvate carboxylase flux was impaired with ChREBP deletion secondary to decreased fatty acid oxidation, increased pyruvate oxidation, and limited pyruvate availability because of reduced activity of liver pyruvate kinase and malic enzyme, which replenish pyruvate via glycolysis and pyruvate cycling. Overall, the shift from fat utilization to pyruvate and lactate utilization resulted in a decrease in the energy of ATP hydrolysis and a hypo-energetic state in the livers of ChREBP(-/-) mice.

It has been proposed that de novo synthesis of long-chain acyl-CoA (LC-CoA) is a signal for glucose-stimulated insulin secretion (GSIS). Key enzymes involved in synthesis of fatty acids from glucose include ATP-citrate lyase (CL) and fatty acid synthase (FAS). An inhibitor of CL, hydroxycitrate (HC), has been reported to inhibit insulin secretion in some laboratories but not in others. Here we show that high concentrations of NaCl created during preparation of HC by standard methods explain the inhibition of GSIS, and that removal of the excess NaCl prevents the effect. To further investigate the role of CL, two small interfering RNA adenoviruses (Ad-siCL2 and Ad-siCL3) were generated. Ad-siCL3 reduced CL mRNA levels by 92 +/- 6% and CL protein levels by 75 +/- 4% but did not affect GSIS in 832/13 cells compared with cells treated with a control adenovirus (Ad-siControl). Similar results were obtained with Ad-siCL2. Ad-siCL3-treated cells also exhibited a 52 +/- 7% reduction in cytosolic oxaloacetate, an 83 +/- 4% reduction in malonyl-CoA levels, and inhibition of [U-(14)C]glucose incorporation into lipid by 43 +/- 4%, all expected metabolic out-comes of CL suppression. Similarly, treatment of 832/13 cells with a recombinant adenovirus specific to FAS (Ad-siFAS) reduced FAS mRNA levels by 81 +/- 2% in 832/13 cells, resulting in a 59 +/- 4% decrease in [U-(14)C]glucose incorporation into lipid, without affecting GSIS. Finally, treatment of primary rat islets with Ad-siCL3 or Ad-siFAS reduced CL and FAS mRNA levels by 65 +/- 4% and 52 +/- 3%, respectively, but had no effect on GSIS relative to Ad-siControl-treated islets. These findings demonstrate that a normal rate of flux of glucose carbons through CL and FAS is not required for GSIS in insulinoma cell lines or rat islets.

Hexose-6-phosphate dehydrogenase (EC 1.1.1.47) catalyzes the conversion of glucose 6-phosphate to 6-phosphogluconolactone within the lumen of the endoplasmic reticulum, thereby generating reduced nicotinamide adenine dinucleotide phosphate. Reduced nicotinamide adenine dinucleotide phosphate is a necessary cofactor for the reductase activity of 11β-hydroxysteroid dehydrogenase type 1 (EC 1.1.1.146), which converts hormonally inactive cortisone to active cortisol (in rodents, 11-dehydrocorticosterone to corticosterone). Mice with targeted inactivation of hexose-6-phosphate dehydrogenase lack 11β-hydroxysteroid dehydrogenase type 1 reductase activity, whereas dehydrogenase activity (corticosterone to 11-dehydrocorticosterone) is increased. We now report that both glucose output and glucose use are abnormal in these mice. Mutant mice have fasting hypoglycemia. In mutant primary hepatocytes, glucose output does not increase normally in response to glucagon. Mutant animals have lower hepatic glycogen content when fed and cannot mobilize it normally when fasting. As assessed by RT-PCR, responses of hepatic enzymes to fasting are blunted; enzymes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase) are not appropriately up-regulated, and expression of glucokinase, an enzyme required for glycolysis, is not suppressed. Corticosterone has attenuated effects on expression of these enzymes in cultured mutant primary hepatocytes. Mutant mice have increased sensitivity to insulin, as assessed by homeostatic model assessment values and by increased glucose uptake by the muscle. The hypothalamic-pituitary-adrenal axis is also abnormal. Circulating ACTH, deoxycorticosterone, and corticosterone levels are increased in mutant animals, suggesting decreased negative feedback on the hypothalamic-pituitary-adrenal axis. Comparison with other animal models of adrenal insufficiency suggests that many of the observed abnormalities can be explained by blunted intracellular corticosterone actions, despite elevated circulating levels of this hormone.