Medium-Chain Fatty Acid Feeding Reduces Oxidation and Causes Panacinar Steatosis in Livers of Neonatal Pigs.

BACKGROUND: Medium-chain fatty acids (MCFAs) are commonly used to enhance the caloric content of infant formulas. We previously reported that pigs fed MCFA developed hepatic steatosis when compared to those fed isocaloric long-chain fatty acid (LCFA) rich formula. OBJECTIVES: The objectives of this study were to investigate: 1) whether MCFA and LCFA feeding affect hepatic fatty acid oxidation, and 2) how fat type alters the expression of hepatic fatty acid metabolic genes. METHODS: Twenty-six, 7-d-old pigs were fed a low-energy control (CONT) formula, or 2 isocaloric high-energy formulas rich in LCFA or MCFA for 22 days. Livers were collected for examining ex vivo fatty acid oxidation, fatty acid content, and mRNA expression of fatty acid metabolic genes. RESULTS: Liver fat was 20% for pigs in the MCFA compared with 2.9% and 4.6% for those in the CONT and LCFA groups (P < 0.05). MCFA-fed pigs had greater amounts of hepatic laurate, myristate, palmitate, and palmitoleate (14, 34, 49, and 9.3 mg · g-1) than those fed LCFA and CONT (1.8, 1.9, 19, 1.5 mg · g-1) formulas (P ≤ 0.05). Hepatic laurate and palmitate oxidation was reduced for pigs fed MCFA (29 mmol · mg-1 · h-1) compared with those fed CONT (54 mmol · mg-1 · h-1) and LCFA (51 mmol · mg-1 · h-1) formulas (P < 0.05). Expression of fatty acid synthase 3 (FASN-3), fatty acid binding protein 1 (FABP-1), and acetyl-CoA carboxylase 1 (ACACA-1) were 8-, 6-, and 2-fold greater for pigs in the MCFA than those in the LCFA and CONT groups (P < 0.05). CONCLUSIONS: Feeding MCFA resulted in hepatic steatosis compared with an isocaloric formula rich in LCFA. Steatosis occurred concomitantly with reduced fatty acid oxidation but greater mRNA expression of fatty acid synthetic and catabolic genes.

Long Chain Fatty Acids Mediate Hepatic Metabolic Flux in Preruminating Dairy Calves Fed Flaxseed Oil, High Oleic Soybean Oil, or Milk Fat.

Nutrition and physiological state affect hepatic metabolism. Our objective was to determine if feeding flaxseed oil (∼50% C18:3n-3 cis), high oleic soybean oil (∼70% C18:1 cis-9), or milk fat (∼50% C16:0) alters hepatic expression of PC, PCK1, and PCK2 and the flow of carbons from propionate and pyruvate into the TCA cycle in preruminating calves. Male Holstein calves (n = 40) were assigned to a diet of skim milk with either: 3% milk fat (MF; n = 8), 3% flaxseed oil (Flax; n = 8), 3% high oleic soybean oil (HOSO; n = 8), 1.5% MF + 1.5% high oleic soybean oil (MF-HOSO; n = 8), or 1.5% MF + 1.5% flaxseed oil (MF-Flax; n = 8) from d 14 to d 21 postnatal. At d 21 postnatal, a liver biopsy was taken for gene expression and metabolic flux analysis. Liver explants were incubated in [U-13C] propionate and [U-13C] pyruvate to trace carbon flux through TCA cycle intermediates or with [U-14C] lactate, [1-14C] palmitic acid, or [2-14C] propionate to quantify substrate oxidation to CO2 and acid soluble products. Compared with other treatments, plasma C18:3n-3 cis was 10 times higher and C18:1 cis-9 was 3 times lower in both flax (Flax and MF-Flax) treatments. PC, PCK1, and PCK2 expression and flux of [U-13C] pyruvate as well as [U-13C] propionate were not different between treatments. PC expression was negatively correlated with the enrichment of citrate M+5 and malate M+3, and PCK2 was negatively correlated with citrate M+5, suggesting that when expression of these enzymes is increased, carbon from pyruvate enters the TCA cycle via PC mediated carboxylation, and then OAA is converted to phosphoenolpyruvate via PCK2. Acid soluble product formation and PC expression were reduced in HOSO (MF-HOSO and HOSO) treatments compared with flax (MF-Flax and Flax), indicating that fatty acids regulate PC expression and carbon flux, but that fatty acid flux control points are not connected to PC, PCK1, or PCK2. In conclusion, fatty acids regulate hepatic expression of PC, PCK1, and PCK2, and carbon flux, but the point of control is distinct.

Branched-chain amino acids alter cellular redox to induce lipid oxidation and reduce de novo lipogenesis in the liver.

Metabolic and molecular interactions between branched-chain amino acid (BCAA) and lipid metabolism are evident in insulin-resistant tissues. However, it remains unclear whether insulin resistance is a prerequisite for these relationships and whether BCAAs or their metabolic intermediates can modulate hepatic lipid oxidation and synthesis. We hypothesized that BCAAs can alter hepatic oxidative function and de novo lipogenesis, independent of them being anaplerotic substrates for the mitochondria. Mice (C57BL/6NJ) were reared on a low-fat (LF), LF diet plus 1.5X BCAAs (LB), high-fat (HF) or HF diet plus 1.5X BCAAs (HB) for 12 wk. Hepatic metabolism was profiled utilizing stable isotopes coupled to mass spectrometry and nuclear magnetic resonance, together with fed-to-fasted changes in gene and protein expression. A greater induction of lipid oxidation and ketogenesis on fasting was evident in the BCAA-supplemented, insulin-sensitive livers from LB mice, whereas their rates of hepatic de novo lipogenesis remained lower than their LF counterparts. Onset of insulin resistance in HF and HB mice livers blunted these responses. Whole body turnover of BCAAs and their ketoacids, their serum concentrations, and the ketogenic flux from BCAA catabolism, all remained similar between fasted LF and LB mice. This suggested that the impact of BCAAs on lipid metabolism can occur independent of them or their degradation products fueling anaplerosis through the liver mitochondria. Furthermore, the greater induction of lipid oxidation in the LB livers accompanied higher mitochondrial NADH/NAD+ ratio and higher fed-to-fasting phosphorylation of AMPKα and ACC. Taken together, our results provide evidence that BCAA supplementation, under conditions of insulin sensitivity, improved the feeding-to-fasting induction of hepatic lipid oxidation through changes in cellular redox, thus providing a favorable biochemical environment for flux through ß-oxidation and lower de novo lipogenesis.NEW & NOTEWORTHY Branched-chain amino acids (BCAAs) have been shown to modulate lipid metabolic networks in various tissues, especially during insulin resistance. In this study we show that the dietary supplementation of BCAAs to normal, insulin-sensitive mice resulted in higher mitochondrial NADH:NAD+ ratios and AMPK activation in the liver. This change in the cellular redox status provided an optimal metabolic milieu to increase fatty acid oxidation while keeping the rates of de novo lipogenesis lower in the BCAA-supplemented mice livers.

Feeding medium-chain fatty acid-rich formula causes liver steatosis and alters hepatic metabolism in neonatal pigs.

Medium-chain fatty acids (MCFA) and long-chain fatty acids (LCFAs) are often added to enhance the caloric value of infant formulas. Evidence suggests that MCFAs promote growth and are preferred over LCFAs due to greater digestibility and ease of absorption. Our hypothesis was that MCFA supplementation would enhance neonatal pig growth to a greater extent than LCFAs. Neonatal pigs (n = 4) were fed a low-energy control (CONT) or two isocaloric high-energy formulas containing fat either from LCFAs, or MCFAs for 20 days. Pigs fed the LCFAs had greater body weight compared with CONT- and MCFA-fed pigs (P < 0.05). In addition, pigs fed the LCFAs and MCFAs had more body fat than those in the CONT group. Liver and kidney weights as a percentage of body weight were greater (P ≤ 0.05) for pigs fed the MCFAs than those fed the CONT formula, and in those fed LCFAs, liver and kidney weights as a percentage of body weight were intermediate (P ≤ 0.05). Pigs in the CONT and LCFA groups had less liver fat (12%) compared with those in the MCFA (26%) group (P ≤ 0.05). Isolated hepatocytes from these pigs were incubated in media containing [13C]tracers of alanine, glucose, glutamate, and propionate. Our data suggest alanine contribution to pyruvate is less in hepatocytes from LCFA and MCFA pigs than those in the CONT group (P < 0.05). These data suggest that a formula rich in MCFAs caused steatosis compared with an isocaloric LCFA formula. In addition, MCFA feeding can alter hepatocyte metabolism and increase total body fat without increasing lean deposition.NEW & NOTEWORTHY Our data suggest that feeding high-energy MCFA formula resulted in hepatic steatosis compared with isoenergetic LCFA or low-energy formulas. Steatosis coincided with greater laurate, myristate, and palmitate accumulation, suggesting elongation of dietary laurate. Data also suggest that hepatocytes metabolized alanine and glucose to pyruvate, but neither entered the tricarboxylic acid (TCA) cycle. In addition, the contribution of alanine and glucose was greater for the low-energy formulas compared with the high-energy formulas.

Branched chain amino acids and carbohydrate restriction exacerbate ketogenesis and hepatic mitochondrial oxidative dysfunction during NAFLD.

Mitochondrial adaptation during non-alcoholic fatty liver disease (NAFLD) include remodeling of ketogenic flux and sustained tricarboxylic acid (TCA) cycle activity, which are concurrent to onset of oxidative stress. Over 70% of obese humans have NAFLD and ketogenic diets are common weight loss strategies. However, the effectiveness of ketogenic diets toward alleviating NAFLD remains unclear. We hypothesized that chronic ketogenesis will worsen metabolic dysfunction and oxidative stress during NAFLD. Mice (C57BL/6) were kept (for 16-wks) on either a low-fat, high-fat, or high-fat diet supplemented with 1.5X branched chain amino acids (BCAAs) by replacing carbohydrate calories (ketogenic). The ketogenic diet induced hepatic lipid oxidation and ketogenesis, and produced multifaceted changes in flux through the individual steps of the TCA cycle. Higher rates of hepatic oxidative fluxes fueled by the ketogenic diet paralleled lower rates of de novo lipogenesis. Interestingly, this metabolic remodeling did not improve insulin resistance, but induced fibrogenic genes and inflammation in the liver. Under a chronic "ketogenic environment," the hepatocyte diverted more acetyl-CoA away from lipogenesis toward ketogenesis and TCA cycle, a milieu which can hasten oxidative stress and inflammation. In summary, chronic exposure to ketogenic environment during obesity and NAFLD has the potential to aggravate hepatic mitochondrial dysfunction.

Lipid Intake Enhances Muscle Growth But Does Not Influence Glucose Kinetics in 3-Week-Old Low-Birth-Weight Neonatal Pigs.

BACKGROUND: Low-birth-weight (LBWT) neonates grow at a slower rate than their normal-birth-weight (NBWT) counterparts and may develop hypoglycemia postnatally. OBJECTIVE: We investigated whether dietary lipid supplementation would enhance growth and improve glucose production in LBWT neonatal pigs. METHODS: Twelve 3-d-old NBWT (1.606 kg) crossbred pigs were matched to 12 LBWT (1.260 kg) same-sex littermates. At 6 d of age, 6 pigs in each group were fed a low-energy (LE) or a high-energy (HE) isonitrogenous formula containing 5.2% and 7.3% fat, respectively. Body composition was assessed using dual-energy X-ray absorptiometry; plasma glucose and glycerol kinetics were assessed using stable isotope tracers. After killing, weights of skeletal muscles and visceral organs were measured. Data were analyzed by ANOVA for a 2 × 2 factorial design; temporal effects were investigated using repeated-measures analysis. RESULTS: Lipid supplementation did not affect body weight of LBWT or NBWT pigs. However, liver and longissimus dorsi weights as a percentage of body weight were greater for pigs fed an HE diet than for those fed an LE diet (4.3% compared with 3.4% and 1.5% compared with 1.2%, respectively) but remained less for LBWT than for NBWT pigs (3.8% compared with 3.9% and 1.3% compared with 1.5%, respectively) (P < 0.05). In addition, hepatic fat content increased (7.9 compared with 2.6 g) in pigs fed the HE compared with those fed the LE formula (P < 0.05). Lipid supplementation did not influence plasma glucose concentration which remained lower in the LBWT than in the NBWT group (4.1 compared with 4.5 mmol/L) (P < 0.05). CONCLUSIONS: Our data suggest that lipid supplementation modestly improved growth of skeletal muscle and the liver but did not affect glucose homeostasis in all groups, and glucose concentration remained lower in LBWT than in NBWT pigs. These data suggest that the previously reported hyperglycemic effect of lipid supplementation may depend on the route of administration or age of the neonatal pig.

Effect of canagliflozin treatment on hepatic triglyceride content and glucose metabolism in patients with type 2 diabetes.

AIM: To evaluate the impact of the sodium glucose co-transporter 2 inhibitor canagliflozin on intrahepatic triglyceride (IHTG) accumulation and its relationship to changes in body weight and glucose metabolism. MATERIALS AND METHODS: In this double-blind, parallel-group, placebo-controlled, 24-week trial subjects with inadequately controlled type 2 diabetes mellitus (T2DM; HbA1c = 7.7% ± 0.7%) from two centres were randomly assigned (1:1) to canagliflozin 300 mg or placebo. We measured IHTG by proton-magnetic resonance spectroscopy (primary outcome), hepatic/muscle/adipose tissue insulin sensitivity during a 2-step euglycaemic insulin clamp, and beta-cell function during a mixed meal tolerance test. Analyses were per protocol. RESULTS: Between 8 September 2014-13 June 2016, 56 patients were enrolled. Canagliflozin reduced HbA1c (placebo-subtracted change: -0.71% [-1.08; -0.33]) and body weight (-3.4% [-5.4; -1.4]; both P ≤ 0.001). A numerically larger absolute decrease in IHTG occurred with canagliflozin (-4.6% [-6.4; -2.7]) versus placebo (-2.4% [-4.2; -0.6]; P = 0.09). In patients with non-alcoholic fatty liver disease (n = 37), the decrease in IHTG was -6.9% (-9.5; -4.2) versus -3.8% (-6.3; -1.3; P = 0.05), and strongly correlated with the magnitude of weight loss (r = 0.69, P < 0.001). Body weight loss ≥5% with a ≥30% relative reduction in IHTG occurred more often with canagliflozin (38% vs. 7%, P = 0.009). Hepatic insulin sensitivity improved with canagliflozin (P < 0.01), but not muscle or adipose tissue insulin sensitivity. Beta-cell glucose sensitivity, insulin clearance, and disposition index improved more with canagliflozin (P < 0.05). CONCLUSIONS: Canagliflozin improves hepatic insulin sensitivity and insulin secretion and clearance in patients with T2DM. IHTG decreases in proportion to the magnitude of body weight loss, which tended to be greater and occur more often with canagliflozin.

Pioglitazone improves hepatic mitochondrial function in a mouse model of nonalcoholic steatohepatitis.

Pioglitazone is effective in improving insulin resistance and liver histology in patients with nonalcoholic steatohepatitis (NASH). Because dysfunctional mitochondrial metabolism is a central feature of NASH, we hypothesized that an important target of pioglitazone would be alleviating mitochondrial oxidative dysfunction. To this end, we studied hepatic mitochondrial metabolism in mice fed high-fructose high-transfat diet (TFD) supplemented with pioglitazone for 20 wk, using nuclear magnetic resonance-based 13C isotopomer analysis. Pioglitazone improved whole body and adipose insulin sensitivity in TFD-fed mice. Furthermore, pioglitazone reduced intrahepatic triglyceride content and fed plasma ketones and hepatic TCA cycle flux, anaplerosis, and pyruvate cycling in mice with NASH. This was associated with a marked reduction in most intrahepatic diacylglycerol classes and, to a lesser extent, some ceramide species (C22:1, C23:0). Considering the cross-talk between mitochondrial function and branched-chain amino acid (BCAA) metabolism, pioglitazone's impact on plasma BCAA profile was determined in a cohort of human subjects. Pioglitazone improved the plasma BCAA concentration profile in patients with NASH. This appeared to be related to an improvement in BCAA degradation in multiple tissues. These results provide evidence that pioglitazone-induced changes in NASH are related to improvements in hepatic mitochondrial oxidative dysfunction and changes in whole body BCAA metabolism.

Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity.

The hepatic tricarboxylic acid (TCA) cycle is central to integrating macronutrient metabolism and is closely coupled to cellular respiration, free radical generation, and inflammation. Oxidative flux through the TCA cycle is induced during hepatic insulin resistance, in mice and humans with simple steatosis, reflecting early compensatory remodeling of mitochondrial energetics. We hypothesized that progressive severity of hepatic insulin resistance and the onset of nonalcoholic steatohepatitis (NASH) would impair oxidative flux through the hepatic TCA cycle. Mice (C57/BL6) were fed a high-trans-fat high-fructose diet (TFD) for 8 wk to induce simple steatosis and NASH by 24 wk. In vivo fasting hepatic mitochondrial fluxes were determined by(13)C-nuclear magnetic resonance (NMR)-based isotopomer analysis. Hepatic metabolic intermediates were quantified using mass spectrometry-based targeted metabolomics. Hepatic triglyceride accumulation and insulin resistance preceded alterations in mitochondrial metabolism, since TCA cycle fluxes remained normal during simple steatosis. However, mice with NASH had a twofold induction (P< 0.05) of mitochondrial fluxes (µmol/min) through the TCA cycle (2.6 ± 0.5 vs. 5.4 ± 0.6), anaplerosis (9.1 ± 1.2 vs. 16.9 ± 2.2), and pyruvate cycling (4.9 ± 1.0 vs. 11.1 ± 1.9) compared with their age-matched controls. Induction of the TCA cycle activity during NASH was concurrent with blunted ketogenesis and accumulation of hepatic diacylglycerols (DAGs), ceramides (Cer), and long-chain acylcarnitines, suggesting inefficient oxidation and disposal of excess free fatty acids (FFA). Sustained induction of mitochondrial TCA cycle failed to prevent accretion of "lipotoxic" metabolites in the liver and could hasten inflammation and the metabolic transition to NASH.

Cold adaptation increases rates of nutrient flow and metabolic plasticity during cold exposure in Drosophila melanogaster.

Metabolic flexibility is an important component of adaptation to stressful environments, including thermal stress and latitudinal adaptation. A long history of population genetic studies suggest that selection on core metabolic enzymes may shape life histories by altering metabolic flux. However, the direct relationship between selection on thermal stress hardiness and metabolic flux has not previously been tested. We investigated flexibility of nutrient catabolism during cold stress in Drosophila melanogaster artificially selected for fast or slow recovery from chill coma (i.e. cold-hardy or -susceptible), specifically testing the hypothesis that stress adaptation increases metabolic turnover. Using (13)C-labelled glucose, we first showed that cold-hardy flies more rapidly incorporate ingested carbon into amino acids and newly synthesized glucose, permitting rapid synthesis of proline, a compound shown elsewhere to improve survival of cold stress. Second, using glucose and leucine tracers we showed that cold-hardy flies had higher oxidation rates than cold-susceptible flies before cold exposure, similar oxidation rates during cold exposure, and returned to higher oxidation rates during recovery. Additionally, cold-hardy flies transferred compounds among body pools more rapidly during cold exposure and recovery. Increased metabolic turnover may allow cold-adapted flies to better prepare for, resist and repair/tolerate cold damage. This work illustrates for the first time differences in nutrient fluxes associated with cold adaptation, suggesting that metabolic costs associated with cold hardiness could invoke resource-based trade-offs that shape life histories.

Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease.

Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD.

Non-Alcoholic Fatty Liver Disease Induced by Feeding Medium-Chain Fatty Acids Upregulates Cholesterol and Lipid Homeostatic Genes in Skeletal Muscle of Neonatal Pigs.

Non-alcoholic fatty liver disease (NAFLD) is a range of disorders characterized by lipid accumulation in hepatocytes. Although this spectrum of disorders is associated with adult obesity, recent evidence suggests that this condition could also occur independently of obesity, even in children. Previously, we reported that pigs fed a formula containing medium-chain fatty acids (MCFAs) developed hepatic steatosis and weighed less than those fed an isocaloric formula containing long-chain fatty acids (LCFAs). Our objective was to determine the association between NAFLD and the skeletal muscle transcriptome in response to energy and lipid intake. Neonatal pigs were fed one of three formulas: a control formula (CONT, n = 6) or one of two isocaloric high-energy formulas containing either long (LCFA, n = 6) or medium (MCFA, n = 6) chain fatty acids. Pigs were fed for 22 d, and tissues were collected. Body weight at 20 and 22 d was greater for LCFA-fed pigs than their CONT or MCFA counterparts (p < 0.005). Longissimus dorsi weight was greater for LCFA compared with MCFA, while CONT was intermediate (p < 0.05). Lean gain and protein deposition were greater for LCFA than for CONT and MCFA groups (p < 0.01). Transcriptomic analysis revealed 36 differentially expressed genes (DEGs) between MCFA and LCFA, 53 DEGs between MCFA and CONT, and 52 DEGs between LCFA and CONT (FDR < 0.2). Feeding formula high in MCFAs resulted in lower body and muscle weights. Transcriptomics data suggest that the reduction in growth was associated with a disruption in cholesterol metabolism in skeletal muscles.

PEPCK-M expression in mouse liver potentiates, not replaces, PEPCK-C mediated gluconeogenesis.

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.

Elevated TCA cycle function in the pathology of diet-induced hepatic insulin resistance and fatty liver.

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 ß-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.

Remodeling of Hepatocyte Mitochondrial Metabolism and De Novo Lipogenesis During the Embryonic-to-Neonatal Transition in Chickens.

Embryonic-to-neonatal development in chicken is characterized by high rates of lipid oxidation in the late-term embryonic liver and high rates of de novo lipogenesis in the neonatal liver. This rapid remodeling of hepatic mitochondrial and cytoplasmic networks occurs without symptoms of hepatocellular stress. Our objective was to characterize the metabolic phenotype of the embryonic and neonatal liver and explore whether these metabolic signatures are preserved in primary cultured hepatocytes. Plasma and liver metabolites were profiled using mass spectrometry based metabolomics on embryonic day 18 (ed18) and neonatal day 3 (nd3). Hepatocytes from ed18 and nd3 were isolated and cultured, and treated with insulin, glucagon, growth hormone and corticosterone to define hormonal responsiveness and determine their impacts on mitochondrial metabolism and lipogenesis. Metabolic profiling illustrated the clear transition from the embryonic liver relying on lipid oxidation to the neonatal liver upregulating de novo lipogenesis. This metabolic phenotype was conserved in the isolated hepatocytes from the embryos and the neonates. Cultured hepatocytes from the neonatal liver also maintained a robust response to insulin and glucagon, as evidenced by their contradictory effects on lipid oxidation and lipogenesis. In summary, primary hepatocytes from the embryonic and neonatal chicken could be a valuable tool to investigate mechanisms regulating hepatic mitochondrial metabolism and de novo lipogenesis.

Dietary Macronutrient Composition Differentially Modulates the Remodeling of Mitochondrial Oxidative Metabolism during NAFLD.

Diets rich in fats and carbohydrates aggravate non-alcoholic fatty liver disease (NAFLD), of which mitochondrial dysfunction is a central feature. It is not clear whether a high-carbohydrate driven 'lipogenic' diet differentially affects mitochondrial oxidative remodeling compared to a high-fat driven 'oxidative' environment. We hypothesized that the high-fat driven 'oxidative' environment will chronically sustain mitochondrial oxidative function, hastening metabolic dysfunction during NAFLD. Mice (C57BL/6NJ) were reared on a low-fat (LF; 10% fat calories), high-fat (HF; 60% fat calories), or high-fructose/high-fat (HFr/HF; 25% fat and 34.9% fructose calories) diet for 10 weeks. De novo lipogenesis was determined by measuring the incorporation of deuterium from D2O into newly synthesized liver lipids using nuclear magnetic resonance (NMR) spectroscopy. Hepatic mitochondrial metabolism was profiled under fed and fasted states by the incubation of isolated mitochondria with [13C3]pyruvate, targeted metabolomics of tricarboxylic acid (TCA) cycle intermediates, estimates of oxidative phosphorylation (OXPHOS), and hepatic gene and protein expression. De novo lipogenesis was higher in the HFr/HF mice compared to their HF counterparts. Contrary to our expectations, hepatic oxidative function after fasting was induced in the HFr/HF group. This differential induction of mitochondrial oxidative function by the high fructose-driven 'lipogenic' environment could influence the progressive severity of hepatic insulin resistance.

Progressive adaptation of hepatic ketogenesis in mice fed a high-fat diet.

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 beta-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 (micromol/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 PPARalpha(-/-) 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.

Glutamate is the major anaplerotic substrate in the tricarboxylic acid cycle of isolated rumen epithelial and duodenal mucosal cells from beef cattle.

In this study, we aimed to determine the contribution of substrates to tricarboxylic acid (TCA) cycle fluxes in rumen epithelial cells (REC) and duodenal mucosal cells (DMC) isolated from Angus bulls (n = 6) fed either a 75% forage (HF) or 75% concentrate (HC) diet. In separate incubations, [(13)C(6)]glucose, [(13)C(5)]glutamate, [(13)C(5)]glutamine, [(13)C(6)]leucine, or [(13)C(5)]valine were added in increasing concentrations to basal media containing SCFA and a complete mixture of amino acids. Lactate, pyruvate, and TCA cycle intermediates were analyzed by GC-MS followed by (13)C-mass isotopomer distribution analysis. Glucose metabolism accounted for 10-19% of lactate flux in REC from HF-fed bulls compared with 27-39% in REC from HC and in DMC from bulls fed both diets (P < 0.05). For both cell types, as concentration increased, an increasing proportion (3-63%) of alpha-ketoglutarate flux derived from glutamate, whereas glutamine contributed <3% (P < 0.05). Although leucine and valine were catabolized to their respective keto-acids, these were not further metabolized to TCA cycle intermediates. Glucose, glutamine, leucine, and valine catabolism by ruminant gastrointestinal tract cells has been previously demonstrated, but in this study, their catabolism via the TCA cycle was limited. Further, although glutamate's contribution to TCA cycle fluxes was considerable, it was apparent that other substrates available in the media also contributed to the maintenance of TCA fluxes. Lastly, the results suggest that diet composition alters glucose, glutamate, and leucine catabolism by the TCA cycle of REC and DMC.

Delayed Feeding Alters Transcriptional and Post-Transcriptional Regulation of Hepatic Metabolic Pathways in Peri-Hatch Broiler Chicks.

Hepatic fatty acid oxidation of yolk lipoproteins provides the main energy source for chick embryos. Post-hatching these yolk lipids are rapidly exhausted and metabolism switches to a carbohydrate-based energy source. We recently demonstrated that many microRNAs (miRNAs) are key regulators of hepatic metabolic pathways during this metabolic switching. MiRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression in most eukaryotes. To further elucidate the roles of miRNAs in the metabolic switch, we used delayed feeding for 48 h to impede the hepatic metabolic switch. We found that hepatic expression of several miRNAs including miR-33, miR-20b, miR-34a, and miR-454 was affected by delaying feed consumption for 48 h. For example, we found that delayed feeding resulted in increased miR-20b expression and conversely reduced expression of its target FADS1, an enzyme involved in fatty acid synthesis. Interestingly, the expression of a previously identified miR-20b regulator FOXO3 was also higher in delayed fed chicks. FOXO3 also functions in protection of cells from oxidative stress. Delayed fed chicks also had much higher levels of plasma ketone bodies than their normal fed counterparts. This suggests that delayed fed chicks rely almost exclusively on lipid oxidation for energy production and are likely under higher oxidative stress. Thus, it is possible that FOXO3 may function to both limit lipogenesis as well as to help protect against oxidative stress in peri-hatch chicks until the initiation of feed consumption. This is further supported by evidence that the FOXO3-regulated histone deacetylase (HDAC2) was found to recognize the FASN (involved in fatty acid synthesis) chicken promoter in a yeast one-hybrid assay. Expression of FASN mRNA was lower in delayed fed chicks until feed consumption. The present study demonstrated that many transcriptional and post-transcriptional mechanisms, including miRNA, form a complex interconnected regulatory network that is involved in controlling lipid and glucose molecular pathways during the metabolic transition in peri-hatch chicks.

Impact of exenatide on mitochondrial lipid metabolism in mice with nonalcoholic steatohepatitis.

Exenatide (Exe) is a glucagon-like peptide (GLP)-1 receptor agonist that enhances insulin secretion and is associated with induction of satiety with weight loss. As mitochondrial dysfunction and lipotoxicity are central features of nonalcoholic steatohepatitis (NASH), we tested whether Exe improved mitochondrial function in this setting. We studied C57BL/6J mice fed for 24 weeks either a control- or high-fructose, high-trans-fat (TFD)-diet (i.e., a NASH model previously validated by our laboratory). For the final 8 weeks, mice were treated with Exe (30 µg/kg/day) or vehicle. Mitochondrial metabolism was assessed by infusion of [13C3]propionate, [3,4-13C2]glucose and NMR-based 13C-isotopomer analysis. Exenatide significantly decreased fasting plasma glucose, free fatty acids and triglycerides, as well as adipose tissue insulin resistance. Moreover, Exe reduced 23% hepatic glucose production, 15% tri-carboxylic acid (TCA) cycle flux, 20% anaplerosis and 17% pyruvate cycling resulting in a significant 31% decrease in intrahepatic triglyceride content (P = 0.02). Exenatide improved the lipidomic profile and decreased hepatic lipid byproducts associated with insulin resistance and lipotoxicity, such as diacylglycerols (TFD: 111 ± 13 vs Exe: 64 ± 13 µmol/g protein, P = 0.03) and ceramides (TFD: 1.6 ± 0.1 vs Exe: 1.3 ± 0.1 µmol/g protein, P = 0.03). Exenatide lowered expression of hepatic lipogenic genes (Srebp1C, Cd36) and genes involved in inflammation and fibrosis (Tnfa, Timp1). In conclusion, in a diet-induced mouse model of NASH, Exe ameliorates mitochondrial TCA cycle flux and significantly decreases insulin resistance, steatosis and hepatocyte lipotoxicity. This may have significant clinical implications to the potential mechanism of action of GLP-1 receptor agonists in patients with NASH. Future studies should elucidate the relative contribution of direct vs indirect mechanisms at play.