Palmitate Oxidation Research Articles

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

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

Abstract 12876: Post-Transcriptional Regulation of Cardiac Fatty Acid Metabolism by the Tandem Zinc Finger Mrna Binding Protein Tristetraprolin

Introduction: Heart failure (HF) is characterized by a switch from predominantly fatty acid (FA) to glucose metabolism, but the molecular pathways involved in this switch are not totally understood. Identification of novel pathways regulating cellular metabolism could facilitate the development of new therapies. Tristetraprolin (TTP) is a tandem zinc finger protein that binds to AU-rich elements (AREs) in the 3’ untranslated region (UTR) of mRNA molecules and causes their degradation. Its expression is regulated by mTOR, a key protein involved in cellular metabolism. Our preliminary data suggested a link between TTP and FA metabolism in the heart. Thus, we hypothesized that TTP regulates cardiac FA metabolism and that its deletion prevents the development of HF by increasing FA uptake and metabolism in the heart. Results: TTP downregulation in cultured cardiomyocytes resulted in higher palmitate uptake and oxidation, while its overexpression had the opposite effect. Since TTP regulates its targets at the mRNA level, we studied the mRNA levels of ARE-containing genes involved in lipid metabolism, and found that only PPARα mRNA to be significantly increased with TTP downregulation. Furthermore, we demonstrated that TTP physically interacts with PPARα mRNA, and the activity of a luciferase reporter harboring full-length PPARα 3’UTR is increased with TTP downregulation. Additionally, PPARα mRNA is stabilized with TTP knockdown. We then studied the role of TTP in cardiac FA metabolism using mice with cardiac-specific (cs) TTP knockout (KO). Consistent with our in vitro data, cs-TTP KO mice displayed higher fatty acid utilization compared to littermate controls but maintained normal cardiac function at baseline. We also demonstrated a significantly higher TTP levels in failing human and mouse cardiac samples, suggesting that TTP levels are altered in HF. Conclusion: Our results demonstrate that TTP is a novel regulator of cardiac FA metabolism through its effect on PPARα, and that its levels are increased in HF. Thus, modulation of TTP may be a viable therapeutic approach for HF.

Titin truncating variant cardiomyopathy and related sarcomere insufficiency causes high energy demand resulting in mitochondrial dysfunction, autophagosome formation, and apoptosis

Abstract Background/Objectives Titin truncating variants (TTNtv) are the most prevalent genetic cause of dilated cardiomyopathy (DCM), resulting in upregulation of cardiac transcripts of oxidative phosphorylation (1,2). However, the underlying molecular mechanism(s) and cellular consequences of these findings remain unknown. Methods and results To gain insight into the metabolic changes and cellular consequences of a TTNtv, metabolic, mitochondrial, and survival pathways were studied in human TTNtv DCM hearts and isolated cardiomyocytes of TTNtv mice. TTNtv resulted in a significant increase of cardiac transcripts of glycolysis, citric acid cycle, mitochondrial fission, autophagy, and apoptosis when comparing RNAseq in 24 TTNtv and 27 mutation-negative DCM cardiac biopsies. Furthermore, a decrease in the area of myofibrils in human TTNtv hearts (TTNtv vs. mutation-negative DCM: 46%, and 62%, P=0.001), and an increase of mitochondrial (49% and 31%, P=0,001) and autophagosome areas (4% and 2%, P=0.002) was observed using transmission electron microscopy (TEM). Similar patterns of cardiomyocyte disorganization and stress could be seen in TTNtv hearts of mice even without a phenotype. Additionally, observed swollen mitochondria by TEM and decreased quantity of OXPHOS proteins by immunoblotting in murine TTNtv hearts indicate mitochondrial stress. Mitochondrial oxygen consumption at baseline and the maximum respiration in TTNtv cardiomyocytes of mice increased by a factor of 1.8 and 1.5 respectively (both P≤0.05), compared to WT. Furthermore, palmitate oxidation in TTNtv cardiomyocytes increased by 1.3 fold (P=0.005) compared to WT mice, suggestive of increased energy demand in TTNtv. Conclusion Myofibrillar insufficiency in human TTNtv DCM augments the cardiac oxygen and energy consumption, leading to pronounced morphological and functional mitochondrial decompensation. Swelling, damage and fission of mitochondria is further characterized by autophagosome formation and increased apoptosis pathways in TTNtv hearts. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Double-Dose consortium by Dutch Cardiovascular Alliance (DCVA)

Breastfeeding duration modifies the association between maternal weight status and offspring dietary palmitate oxidation

Offspring of obese rodents develop a metabolic phenotype that favors fat deposition. Data regarding the impact of maternal obesity programing of offspring fuel usage in humans is scarce. The objective of this study was to explore the association between maternal weight status and dietary palmitate oxidation (DPO) in 2-y-old offspring, taking into consideration potential confounders and modifiers. Women (n=56) were enrolled by the first trimester of gestation. Maternal physical activity (PA; measured with accelerometers) at enrollment and gestational weight gain (GWG) were measured. Offspring sex, race, and breastfeeding (BF) duration were self-reported. Human milk (HM) composition was determined at 6mo postpartum. At age 2 y, dietary quality [healthy eating index (HEI)] and parental feeding practices [Child Feeding Questionnaire (CFQ)] were assessed. DPO in 2-y-olds (2-yo-DPO) was measured using deuterated palmitic acid. Generalized linear regression analysis was used to model the associations of 2-yo-DPO with maternal weight status [normal weight (NW), BMI <25 (in kg/m2) compared with excessive weight (EW), BMI ≥25]. DPO was higher in offspring of women with EW compared with NW (2.1±1.2%/h compared with 1.4±0.7%/h, P=0.03). Maternal weight status interacted with BF duration in association with 2-yo-DPO (log ß: 0.05, P=0.04). Specifically, 2-yo-DPO was higher in the EW compared with NW group if BF duration was ≥9mo. HM insulin (log ß: 0.35, P=0.002) and HM leptin (log ß: 0.81, P=0.001) concentrations directly associated with 2-yo-DPO. PA (log ß: 0.06, P=0.013), parental feeding restriction (log ß: 0.05, P<0.0001), and male sex (log ß: 0.54, P<0.001) were positively associated with 2-yo-DPO. HEI was negatively associated with 2-yo-DPO (log ß:-0.03, P<0.0001). Higher 2-yo-DPO in offspring of women with EW compared with NW were driven by BF duration. Higher HM insulin and leptin concentrations in women with EW may explain these finding. More studies are needed to confirm these results. This trial was registered at clinicaltrials.gov as NCT03281850.

Obesity elicits a unique metabolomic signature in human airway smooth muscle cells.

Obesity can aggravate asthma by enhancing airway hyperresponsiveness (AHR) and attenuating response to treatment. However, the precise mechanisms linking obesity and asthma remain unknown. Human airway smooth muscle (HASM) cells exhibit amplified excitation-contraction (EC) coupling and force generation in obesity. Therefore, we posit that airway smooth muscle (ASM) cells obtained from obese donors manifest a metabolomic phenotype distinct from that of nonobese donor cells and that a differential metabolic phenotype, at least in part, drives enhanced ASM cell EC coupling. HASM cells derived from age-, sex-, and race-matched nonobese [body mass index (BMI) ≤ 24.9 kg·m-2] and obese (BMI ≥ 29.9 kg·m-2) lung donors were subjected to unbiased metabolomic screening. The unbiased metabolomic screening identified differentially altered metabolites linked to glycolysis and citric acid cycle in obese donor-derived cells compared with nonobese donor cells. The Seahorse assay measured the bioenergetic profile based on glycolysis, mitochondrial respiration, palmitate oxidation, and glutamine oxidation rates in HASM cells. Glycolytic rate and capacity were elevated in obese donor-derived HASM cells, whereas mitochondrial respiration, palmitate oxidation, and glutamine oxidation rates were comparable between obese and nonobese groups. PFKFB3 mRNA and protein expression levels were also elevated in obese donor-derived HASM cells. Furthermore, pharmacological inhibition of PFKFB3 attenuated agonist-induced myosin light chain (MLC) phosphorylation in HASM cells derived from obese and nonobese donors. Our findings identify elevated glycolysis as a signature metabolic phenotype of obesity and inhibition of glycolysis attenuates MLC phosphorylation in HASM cells. These findings identify novel therapeutic targets to mitigate AHR in obesity-associated asthma.

Abstract 3018: Targeting lipid metabolism and diet in advanced prostate cancer

Abstract Lipid metabolism reprogramming provides the energetic needs of prostate cancer cells (PCa) to support growth and cell progress through the cell cycle, sustaining a highly proliferative phenotype by increasing the expression levels of several enzymes involved in fatty acid (FAs) synthesis. Fatty acid synthase (FASN), a key enzyme in de novo fatty acid lipogenesis (DNL), is upregulated in metastatic, castration-resistant prostate cancer (CRPC), and its overexpression is related to carcinogenesis, growth, and metastasis. We previously demonstrated that FASN inhibitors show antitumor activity suppressing PCa cell proliferation and slowing tumor growth, both in vitro and in vivo, via rewiring of lipid cancer metabolism and inducing endoplasmic reticulum stress.We observed that impairment of DNL increases polyunsaturated fatty acids (PUFA) uptake as a compensatory mechanism of survival. Exogenously acquired PUFAs cause the remodeling of the prostate cancer cell lipidome, manifesting an enhanced acyl chain unsaturation across several lipid species. Lipid peroxidation and ROS accumulation are also observed, suggesting cell susceptible to oxidative damage following FASN blockade. Alterations in metabolic pathways cause a higher uptake of acetate and glutamine in PCa cells, as assessed by 14C-nutrient incorporation following FASN inhibition. Mitochondrial dysfunction is induced by reduction in several respiratory parameters, including ATP production and basal respiration. This may be, at least in part, the result of a reduction in phosphatidylglycerol, a cardiolipin precursor, altering mitochondrial membrane composition. Supplementation with the PUFA docosahexaenoic acid (DHA) increases phosphatidylcholine levels and total phospholipid acyl chain unsaturation, which can lead to membrane disorganization in prostate cancer cells. Superoxide anion production is enhanced by DHA treatment, as expected, and increased levels of glucose and palmitate oxidation by mitochondria are observed. Interestingly, AR and AR-V7 protein expression, as well as c-MYC and PSA, is reduced with DHA treatment. We then evaluated how de novo lipid synthesis inhibition combined with manipulation of the exogenous supply of fatty acids could impair tumor cell progression. Due to the obligate cell dependence on PUFA following DNL suppression, we combined FASN and Acetyl-CoA carboxylase (ACC) inhibitors with DHA supplementation and observed a significant cell growth inhibition in CRPC cell lines and prostate cancer organoids.Altogether, our results suggest a novel mechanism to inhibit the growth of castration-resistant prostate cancer, combining lipogenesis inhibition with PUFA-enriched diet to overcome advanced disease. Citation Format: Silvia D. Rodrigues, Caroline Fidalgo Ribeiro, Guilherme H. Tamarindo, Hubert Pakula, Massimo Loda. Targeting lipid metabolism and diet in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3018.

219-LB: The Mitochondrial Calcium Uniporter Regulates Hepatic Lipid Accumulation and Mitochondrial Oxidation

The mitochondrial calcium uniporter (MCU) facilitates mitochondrial calcium influx, and several enzymes that catalyze key steps of the tricarboxylic acid (TCA) cycle require calcium for their activation. As such, it is generally accepted that rates of mitochondrial oxidation correlate with mitochondrial calcium activity. Decreased rates of hepatic mitochondrial oxidation are associated with metabolic dysfunction and ectopic lipid accumulation as seen with type 2 diabetes and nonalcoholic fatty liver disease. Despite this, a role for MCU and mitochondrial calcium in regulating hepatic mitochondrial oxidation is still unclear. To address this question, we generated a liver-specific MCU knockout (MCU KO) mouse model which displayed significantly reduced mitochondrial calcium ([Ca2+]mt, p&amp;lt;0.05) and increased cytosolic calcium ([Ca2+]cyt, p&amp;lt;0.01) in isolated hepatocytes. Despite decreased [Ca2+]mt, we found that deletion of hepatic MCU resulted in increased rates of isocitrate dehydrogenase flux (VIDH, p&amp;lt;0.01) , α-ketoglutarate dehydrogenase flux (VOGDH, p&amp;lt;0.01) , and succinate dehydrogenase flux (VSDH, p&amp;lt;0.05) , using a novel in vivo [13C5]glutamine tracer technique. Further, we report that rates of [14C16]palmitate oxidation (p&amp;lt;0.01) and intrahepatic lipolysis (p&amp;lt;0.05) were significantly increased in precision-cut liver slices collected from MCU KO mice compared to WT, consistent with [Ca2+]cyt activation of CAMKII (p&amp;lt;0.01) and ATGL (p&amp;lt;0.001) in MCU KO mice, and were associated with reductions in hepatic triacylglycerol content in vivo (p&amp;lt;0.01) . Taken together, these data demonstrate that hepatic mitochondrial activity can be dissociated from mitochondrial calcium concentrations and reveal a key role for cytosolic calcium and mitochondrial anaplerosis in the regulation of hepatic mitochondrial fat oxidation. Disclosure T. E. Lamoia: n/a. B. T. Hubbard: None. M. Guerra: None. R. Goodman: None. M. H. Nathanson: None. V. Mootha: Advisory Panel; 5AM Ventures, LLC, Janssen Research &amp; Development, LLC. G. I. Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc. , Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. . Funding F31 DK126362/DK/NIDDK NIH HHST32 GM007324/GM/NIGMS NIH HHS

Feeding Coconut Oil Reduces Fatty Acid Oxidation and Exacerbates Steatosis in the Liver of Neonatal Pigs

Coconut oil is commonly used to enhance the energy content of infant formulas due to its high medium‐chain fatty acid (MCFA) content. Using neonatal pigs as model for the human infant, we previously reported that feeding a formula rich in MCFA led to hepatic fat accumulation compared to an isocaloric long‐chain fatty acid formula (LCFA). In addition, isolated hepatocytes from MCFA fed pigs oxidized more glutamate than LCFA fed pigs, suggesting a shift in liver metabolism. Thus, the objectives of this study were to investigate whether MCFA and LCFA feeding affect hepatic laurate (C12:0), and palmitate (C16:0) oxidation, and determine whether fat type influences the expression of genes related to fatty acid metabolism in the liver. Twenty‐six, 3‐day‐old pigs were allotted one of three sow milk replacer formulas: a control (CONT) formula that provided 80% of caloric requirements, and two isocaloric formulas that provided 120% of caloric requirements with fat either as LCFA or MCFA. Pigs were fed 250 ml∙kg‐1∙day‐1 for 22 days, weights were determined every other day, and formula allowance adjusted accordingly. Body composition was determined using DXA at day ‐1 and 21. On day 22, pigs were euthanized. Muscle and organs were excised, weighed, and sampled. Liver samples were homogenized and incubated with [2‐C14]palmitate or [2‐C14]laurate. Following a 60 min incubation, radio‐labelled enrichment of CO2 and acid‐soluble metabolites were determined. Fat was extracted from liver samples using a modified Bligh and Dyer method, fatty acids were methylated to fatty acid methyl esters, and their concentrations determined by gas chromatography/mass spectrometry. Expression of genes related to fatty acid metabolism was determined using Real‐Time PCR. In addition, citrate synthase (CS) activity was measured in liver homogenates using spectrophotometry. Body weight of pigs fed the LCFA formula was greater (8.3%) than those fed the MCFA and CONT formulas (P&lt; 0.05). In addition, LCFA lean tissue mass was greater (29%) when compared with CONT while MCFA was intermediate (P&lt; 0.05). Liver weight of MCFA pigs was 25 and 77% greater than LCFA and CONT groups (P&lt; 0.05). Liver fat was greater (142%) for MCFA pigs compared with LCFA and CONT counterparts (P&lt; 0.05). Palmitate and laurate oxidation to CO2 was greater (49%) for pigs fed the LCFA and CONT formulas compared with those fed MCFA (P&lt; 0.05). In addition, complete oxidation of palmitate and laurate was greater (112%) for LCFA and CONT pigs (P&lt; 0.05). MCFA fed pigs had greater proportions of laurate, myristate and palmitate in their livers compared with LCFA and CONT groups (P&lt; 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 the MCFA pigs than LCFA and CONT pigs (P&lt; 0.05). However, apolipoprotein‐B (APO‐B) expression was not affected by treatment. These data suggest that feeding a formula rich in MCFA led to hepatic steatosis, and that fatty acid composition in liver did not reflect that of fed formulas. In addition, liver steatosis in pigs fed the MCFA rich formula is correlated with a reduction in the liver’s ability to oxidize fatty acids when compared with pigs fed an isocaloric LCFA based formula.

Hepatocyte-specific eNOS deletion impairs exercise-induced adaptations in hepatic mitochondrial function and autophagy.

Endothelial nitric oxide synthase (eNOS) is a potential mediator of exercise-induced hepatic mitochondrial adaptations. Here, male and female hepatocyte-specific eNOS knockout (eNOShep-/- ) and intact hepatic eNOS (eNOSfl/fl ) mice performed voluntary wheel-running exercise (EX) or remained in sedentary cage conditions for 10 weeks. EX resolved the exacerbated hepatic steatosis in eNOShep-/- male mice. Elevated hydrogen peroxide emission (~50% higher in eNOShep-/- vs. eNOSfl/fl mice) was completely ablated with EX. Interestingly, EX increased [1-14 C] palmitate oxidation in eNOSfl/fl male mice, but this was blunted in the eNOShep-/- male mice. eNOShep-/- mice had lower markers of the energy sensors AMP-activated protein kinase (AMPK)/phospho- (p)AMPK and mammalian target of rapamycin (mTOR) and p-mTOR, as well as the autophagy initiators serine/threonine-protein kinase ULK1 and pULK1, compared with eNOSfl/fl mice. Females showed elevated electron transport chain protein content and markers of mitochondrial biogenesis (transcription factor A, mitochondrial, peroxisome proliferator-activated receptor-gamma coactivator 1α). Collectively, this study demonstrates for the first time, to the authors' knowledge, the requirement of eNOS in hepatocytes in the EX-induced increases in hepatic fatty acid oxidation in male mice. Deletion of eNOS in hepatocytes also appears to impair the energy-sensing ability of the cell and inhibit the activation of the autophagy initiating factor ULK1. These data uncover the important and novel role of hepatocyte eNOS in EX-induced hepatic mitochondrial adaptations.

Oxidation of amino acids, glucose, and fatty acids as metabolic fuels in enterocytes of developing pigs.

Enterocytes of young pigs are known to use glutamine, glutamate, and glucose as major metabolic fuels. However, little is known about the roles of aspartate, alanine, and fatty acids as energy sources for these cells. Therefore, this study simultaneously determined the oxidation of the amino acids and glucose as well as short- and long-chainfatty acids in enterocytes of developing pigs. Jejunal enterocytes were isolated from 0-, 7-, 14- and 21-day-old piglets, and incubated at 37°C for 30min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5mM D-glucose and one of the following: D-[U-14C]glucose, 0.5-5mM L-[U-14C]glutamate, 0.5-5mM L-[U-14C]glutamine, 0.5-5mM L-[U-14C]aspartate, 0.5-5mM L-[U-14C]alanine, 0.5-2mM L-[U-14C]palmitate, 0.5-5mM [U-14C]propionate, and 0.5-5mM [1-14C]butyrate. At the end of the incubation, 14CO2 produced from each 14C-labeled substrate was collected. Rates of oxidation of each substrate by enterocytes from all age groups of piglets increased (P < 0.05) gradually with increasing its extracellular concentrations. The rates of oxidation of glutamate, glutamine, aspartate, and glucose by enterocytes from 0- to 21-day-old pigs and of alanine from newborn pigs were much greater (P < 0.05) than those for the same concentrations of palmitate, propionate, and butyrate. Compared with 0-day-old pigs, the rates of oxidation of glutamate, aspartate, glutamine, alanine, and glucose by enterocytes from 21-day-old pigs decreased (P < 0.05) markedly, without changes in palmitate oxidation. Oxidation of alanine, propionate, butyrate and palmitate by enterocytes of pigs was limited during their postnatal growth. At each postnatal age, the oxidation of glutamate, glutamine, aspartate, and glucose produced much more ATP than alanine, propionate, butyrate and palmitate. The degradation of glutamate was initiated primarily by glutamate-pyruvate and glutamate-oxaloacetatetransaminases. Our results indicated that amino acids (glutamate plus glutamine plus aspartate) are the major metabolic fuels in enterocytes of 0- to 21-day-old pigs.

Oxidation of amino acids, glucose, and fatty acids as metabolic fuels in enterocytes of post-hatching developing chickens.

This study determined the oxidation of amino acids, glucose and fatty acid in enterocytes of developing chickens. Jejunal enterocytes were isolated from 0-, 7-, 21-, and 42-d-old broiler chickens, and incubated at 40°C for 30min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5mM D-glucose and one of the following: 0.5-5mM L-[U-14C]glutamate, 0.5-5mM L-[U-14C]glutamine, 0.5-5mM L-[U-14C]aspartate, 0.5-5mM L-[U-14C]alanine, 0.5-2mM [U-14C]palmitate, D-[U-14C]glucose, 0.5-5mM [U-14C]propionate, and 0.5-5mM [1-14C]butyrate. 14CO2 produced from each 14C-labeled substrate was collected for determination of radioactivity. Among all the substrates studied, glutamate had the greatest rate of oxidation in enterocytes from 0- to 42-d-old chickens. Glutamate transaminases, rather than glutamate dehydrogenase, may be primarily responsible for initiating glutamate degradation. Rates of amino acid and fatty acid oxidation by cells increased (P < 0.05) with increasing their extracellular concentrations from 0.5 to 5mM. Rates of glutamate and glucose oxidation in enterocytes decreased (P < 0.05) with increasing age, and rates of glutamine, aspartate, propionate, and butyrate oxidation were lower (P < 0.05) in 42-d-old chickens than in 0-d-old chickens. By contrast, oxidation of palmitate at 2mM increased (P < 0.05) by 118% in cells from 42-d-old chickens, compared with 0-d-old chickens. Compared with glutamate, oxidation of glutamine, aspartate, alanine, propionate, butyrate, and palmitate was limited in cells from all age groups of chickens. Collectively, these results indicate that glutamate is the major metabolic fuel in enterocytes of 0- to 42-d-old chickens.

No effect of triheptanoin in patients with phosphofructokinase deficiency

Phosphofructokinase deficiency (PFKD) is a rare disorder of glycogen metabolism. The lack of phosphofructokinase activity blocks the oxidative pathway from glucose and glycogen to pyruvate. Patients suffer from myopathy, exercise intolerance, and myoglobinuria. Currently, there is no specific treatment for PFKD. We hypothesized that 2 weeks treatment with triheptanoin could improve oxidative metabolism during exercise by bypassing the blocked pyruvate generation in PFKD. The study was a randomized, double-blind, placebo-controlled crossover study. Three genetically verified patients completed two treatment periods of 14 days each with triheptanoin (0.3–1 g × kg−1 × day−1) or placebo liquid. Primary outcomes were heart rate, fatty acid and total oxidation measured via stable isotope and indirect calorimetry methodology during submaximal exercise. Triheptanoin did not improve the primary outcome heart rate during submaximal exercise compared to placebo. Palmitate oxidation was increased during submaximal exercise in one patient but did not increase in the two other patients during triheptanoin treatment. Palmitate production and palmitate utilization increased during exercise and increased to a greater extent with triheptanoin treatment in all three patients. This study suggests that triheptanoin treatment has no effect on heart rate or exercise performance despite increased palmitate production and utilization in patients with PFKD.

No effect of resveratrol on fatty acid oxidation or exercise capacity in patients with fatty acid oxidation disorders: A randomized clinical cross-over trial.

The objective was to investigate whether resveratrol (RSV) can improve exercise capacity in patients with fatty acid oxidation (FAO) disorders. The study was a randomized, double-blind, cross-over trial. Nine patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency or carnitine palmitoyl transferase (CPT) II deficiency were randomized to receive either 8 weeks of 1000 mg day-1 RSV or placebo (P) followed by a 4-weeks wash-out period and subsequently 8 weeks of the opposite treatment. Primary outcome measures were heart rate and FAO as measured via stable isotope technique during constant workload exercise. Secondary outcome measures included fat and glucose metabolism; perceived exertion; as well as subjective measures of energy expenditure, fatigue, and daily function. Eight participants completed the trial. Heart rate did not differ at the end of exercise after treatment with RSV vs placebo (P=.063). Rate of oxidation of palmitate at end of exercise was not different with 1.5 ± 0.8 (RSV) vs 1.3 ± 0.6 (P) μmol kg-1 min-1 (P=.109). Secondary outcomes did not change except for increased plasma glycerol and decreased plasma glucose levels at the end of exercise after treatment with RSV vs placebo. A daily dose of 1000 mg resveratrol does not improve exercise capacity or FAO during exercise in patients with CPTII or VLCAD deficiencies.

ApoE4 confers mitochondrial substrate oxidation defects that can be bioenergetically compensated by a ketogenic substrate beta-hydroxy butyrate (BHB).

Mitochondria are at the center of neural biogenergetics, and ApoE4 is the single most impactful risk factor for AD. We investigated the impact of ApoE on insulin sensitivity, on mitochondrial substrate utilization and bioenergetics. Persons with ApoE4 have reduced brain carbohydrate metabolism. To test for ApoE4 conferred neural mitochondrial metabolic differences, we constructed a novel stable-ApoE 2,3 and 4 N2a cell model, and tested ApoE's effects on Insulin sensitivity, and mitochondrial glucose, lipid and ketone oxidation. Binding of ApoE isoforms E2, E3 and E4 to Insulin Receptor (IR) was measured by BLI and Co-IP, the impact of ApoE isoforms on mitochondrial glucose and lipid oxidation was measured by Seahorse. ApoE3 was found to sensitize to insulin about 2-fold more potently than ApoE4. ApoE isoforms directly bind Insulin Receptor; the binding constants was in the range 200-300nM. Consistent with the previous insulin-sensitivity finding, ApoE3 caused a significant increase of the glycolytic rate and glucose oxidation relative to ApoE4. As there was no difference in oxidation of TCA cycle intermediates substrates in permeabilized cells, we infer ApoE3's glucose advantage is the result of increased insulin sensitivity. ApoE4 contributed a significant palmitate oxidation defect relative to ApoE2 and ApoE3. As this palmitate oxidation defect was observed in both mitochondria and cells it is likely to occur at or within mitochondria. We observed that the relative defect in ApoE4-dependent glucose and palmitate oxidation can be overcome by 5mM BHB. Thus, at the neural cell level, the metabolic defects contributed by ApoE 4 appear to be rescued by a ketogenic molecule, BHB, that requires neither insulin nor apolipoprotein particle to reach neural mitochondria and provide alternative metabolic support. ApoE4 confers 'double trouble' in mitochondrial glucose and lipid oxidation. ApoE4 confers a defect in mitochondrial lipid oxidation relative to all other isoforms. Simultaneously, ApoE4 lacks the benefit in glucose oxidation conferred by ApoE3, which appears to be driven by the reduced insulin sensitization potency of ApoE4. We also find that BHB can be an alternative source of neural bioenergy that enters mitochondria directly and thus is not affected by ApoE4 'double trouble'.

Glucocorticoids regulate mitochondrial fatty acid oxidation in fetal cardiomyocytes

The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesized that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon a glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In vivo, in neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24h later. Instead, at E17.5, fatty acid oxidation genes were downregulated by dexamethasone, as was GR itself. PGC-1α, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was also downregulated in fetal hearts at E17.5, 24h after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a translational sheep model of preterm birth, both GR and PGC-1α were downregulated in heart. These data suggest that endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by downregulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility. KEY POINTS: Glucocorticoids are steroid hormones that play a vital role in late pregnancy in maturing fetal organs, including the heart. In fetal cardiomyocytes in culture, glucocorticoids promote mitochondrial fatty acid oxidation, suggesting they facilitate the perinatal switch from carbohydrates to fatty acids as the predominant energy substrate. Administration of a synthetic glucocorticoid in late pregnancy in mice downregulates the glucocorticoid receptor and interferes with the normal increase in genes involved in fatty acid metabolism in the heart. In a sheep model of preterm birth, antenatal corticosteroids (synthetic glucocorticoid) downregulates the glucocorticoid receptor and the gene encoding PGC-1α, a master regulator of energy metabolism. These experiments suggest that administration of antenatal corticosteroids in anticipation of preterm delivery may interfere with fetal heart maturation by downregulating the ability to respond to glucocorticoids.

Abstract P364: Liraglutide Protects Against Angiotensin-induced Diastolic Dysfunction And Is Associated With Altered Acetyl-coa Metabolism

Background: The mechanisms underlying diastolic dysfunction remain complicated and poorly understood. Previously implicated mechanisms include increased interstitial fibrosis and oxidative damage, altered calcium handling, and mitochondrial dysfunction. In this study, we investigate changes in the cardiac metabolic profile from mice with Angiotensin-II (Ang)-induced diastolic dysfunction. Because treatment with the GLP-1 agonist liraglutide (Lira) relieves Ang-induced diastolic dysfunction, we examined altered metabolites in Ang and Ang+Lira mice to identify metabolic pathways involved. Methods: 8-wk mice were implanted with Ang pumps (1000 ng/kg/min) +/- Lira (0.2 mg/kg/day) therapy for 4 wks and compared to sham mice. Baseline and 4-wk echocardiography was performed. Hearts were collected for RNA-sequencing, Western blot, histology, radiolabeled palmitate assay, and targeted metabolomics by liquid chromatography-mass spectrometry to assess metabolic changes. Results: After Ang treatment, mice had significant diastolic dysfunction but only mild hypertrophy based on echo and histologic measures and no evidence of systolic change. Compared to sham mice, Ang mice had reduced E/A (Sham, 2.67±0.40; Ang, 1.71±0.11; p&lt;0.05) and peak reverse longitudinal strain rate (rLSR;Sham, 8.48±0.65/s; Ang, 6.081±0.33/s; p&lt;0.05, consistent with diastolic dysfunction. Ang+Lira mice had improved E/A (3.57±0.50, p&lt;0.01 vs. Ang) and rLSR (8.65±0.68/s, p&lt;0.01 vs Ang). Targeted metabolomic analysis of hearts found significantly increased accumulation of Acetyl-CoA in Ang mice (peak area 0.14±0.02) compared to sham (0.04±0.02 ,p&lt;0.05) that is restored by Lira therapy (0.02±0.01 ,p&lt;0.05 vs sham and Ang). A radiolabeled palmitate oxidation assay found decreased palmitate oxidation in both Ang and Ang+Lira mice compared to sham, but interestingly, also demonstrated significantly lower acid soluble metabolite oxidation in the Ang+Lira mice vs. Ang alone. Conclusions: We found that Ang-treated mice accumulate myocardial Acetyl-CoA, suggesting a defect in TCA flux. Lira resolves the increase in Acetyl-CoA and improved measures of diastolic dysfunction. This data may implicate a novel metabolic pathway in the development of diastolic disease.

Abstract 113: Targeting Receptor-interacting Protein 140 (RIP140) To Modulate Energy Metabolism In The Failing Heart

During the development of heart failure (HF), the PPAR/ERR complex becomes deactivated resulting in diminished capacity for mitochondrial fatty acid oxidation (FAO) and ATP production leading to an “energy-starved” state that contributes to progression of HF. Receptor-Interacting protein 140 (RIP140) serves as a co-repressor of PPAR/ERR in some extra-cardiac tissues. We hypothesized that inhibition of RIP140 would re-activate PPAR/ERR enhancing capacity for fuel catabolism and ATP production in the failing heart. Heart and skeletal muscle-specific RIP140 knockout mice (strRIP140KO) were resistant to the development of cardiac hypertrophy and diastolic dysfunction in response to chronic pressure overload that mimicked features of HF with preserved ejection fraction (HFpEF). To further evaluate the role of RIP140 in heart, cardiac-specific (cs) RIP140KO mice were generated. 13 C-substrate NMR studies demonstrated that palmitate oxidation and triglyceride turnover rates were significantly accelerated in isolated perfused csRIP140KO hearts. csRIP140KO were subjected to transverse aortic constriction/apical myocardial infarction surgery (TAC/MI), to produce HF with reduced EF (HFrEF). Compared to controls, csRIP140KO exhibited reduced left ventricular remodeling and systolic dysfunction when subjected to TAC/MI. RNA-sequence analysis demonstrated that many genes involved in FAO, branched-chain amino acid catabolism, oxidative phosphorylation, and adult muscle contraction programs were significantly “protected” (less downregulation) by RIP140 deletion in the context of TAC/MI. To identify candidate cardiac RIP140 targets, “CUT&amp;RUN”-sequencing was conducted on cardiomyocytes (CM) from csRIP140KO and controls to identify changes in enhancer regions. Motif analysis of peaks with increased H3K27ac deposition in the csRIP140KO CM identified ERR, PPAR, myocyte enhancer 2 (MEF2), glucocorticoid receptor (GR), and kruppel-like factor (KLF) binding sites. We conclude that RIP140 serves as a global co-repressor of a network of transcription factors that control cardiac energy metabolic and contractile function, and that inhibition of RIP140 could prove to be a novel therapeutic approach for HF.

A20 deficiency in myeloid cells protects mice from diet-induced obesity and insulin resistance due to increased fatty acid metabolism.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both invitro and invivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

Acetate Does Not Affect Palmitate Oxidation and AMPK Phosphorylation in Human Primary Skeletal Muscle Cells.

Our recent in vivo human studies showed that colonic administration of sodium acetate (SA) resulted in increased circulating acetate levels, which was accompanied by increments in whole-body fat oxidation in overweight-obese men. Since skeletal muscle has a major role in whole-body fat oxidation, we aimed to investigate effects of SA on fat oxidation and underlying mechanisms in human primary skeletal muscle cells (HSkMC). We investigated the dose (0–5 mmol/L) and time (1, 4, 20, and 24 h) effect of SA on complete and incomplete endogenous and exogenous oxidation of 14C-labeled palmitate in HSkMC derived from a lean insulin sensitive male donor. Both physiological (0.1 and 0.25 mmol/L) and supraphysiological (0.5, 1 and 5 mmol/L) concentrations of SA neither increased endogenous nor exogenous fat oxidation over time in HSkMC. In addition, no effect of SA was observed on Thr172-AMPKα phosphorylation. In conclusion, our previously observed in vivo effects of SA on whole-body fat oxidation in men may not be explained via direct effects on HSkMC fat oxidation. Nevertheless, SA-mediated effects on whole-body fat oxidation may be triggered by other mechanisms including gut-derived hormones or may occur in other metabolically active tissues.

Female Mice Are Protected from Metabolic Decline Associated with Lack of Skeletal Muscle HuR.

Simple SummaryMetabolic flexibility describes the ability to adapt to utilization of metabolic fuels such as carbohydrates, lipids, and proteins as they become available. The RNA binding protein HuR controls this flexibility in mouse and human skeletal muscle, but the molecular mechanisms governing this process remain poorly characterized. Additionally, studies from mice indicate that HuR control of metabolic flexibility may be more essential for males than females. This is because males lacking HuR in skeletal muscle develop hallmarks of insulin sensitivity, while females have not been shown to do so. Here we examine this sexual dimorphism in mice lacking HuR in skeletal muscle. Our results reveal that lack of HuR in skeletal muscle drives increased adiposity regardless of sex, but that this increase in adiposity drives the development of insulin resistance in male animals only. Additionally, relative to male mice, the detrimental metabolic phenotype associated with HuR inhibition in skeletal muscle can be corrected by feeding of a diet heavily composed of either lipids or carbohydrates.Male mice lacking HuR in skeletal muscle (HuRm−/−) have been shown to have decreased gastrocnemius lipid oxidation and increased adiposity and insulin resistance. The same consequences have not been documented in female HuRm−/− mice. Here we examine this sexually dimorphic phenotype. HuRm−/− mice have an increased fat mass to lean mass ratio (FM/LM) relative to controls where food intake is similar. Increased body weight for male mice correlates with increased blood glucose during glucose tolerance tests (GTT), suggesting increased fat mass in male HuRm−/− mice as a driver of decreased glucose clearance. However, HuRm−/− female mice show decreased blood glucose levels during GTT relative to controls. HuRm−/− mice display decreased palmitate oxidation in skeletal muscle relative to controls. This difference is more robust for male HuRm−/− mice and more exaggerated for both sexes at high dietary fat. A high-fat diet stimulates expression of Pgc1α in HuRm−/− male skeletal muscle, but not in females. However, the lipid oxidation Pparα pathway remains decreased in HuRm−/− male mice relative to controls regardless of diet. This pathway is only decreased in female HuRm−/− mice fed high fat diet. A decreased capacity for lipid oxidation in skeletal muscle in the absence of HuR may thus be linked to decreased glucose clearance in male but not female mice.