Complete Fatty Acid Oxidation Research Articles

Maternal Exercise Improves Infant Mesenchymal Stem Cell Lipid Storage and Oxidation, and Decreases Infant Adiposity

Rates of pediatric obesity are continuously rising and are likely to translate into high incidence of metabolic disease later in life. Maternal exercise during pregnancy has been established as a useful non-pharmacological intervention to improve infant metabolic health; however, mechanistic insight behind these adaptations remains mostly confined to animal models. Infant mesenchymal stem cells (MSCs) give rise to a multitude of infant peripheral tissue (e.g., skeletal muscle) and remain involved in mature tissue repair and maintenance. Importantly, these cells preserve the phenotype of an offspring donor and represent a model for the investigation of mechanisms behind infant metabolic health improvements. We used undifferentiated MSC collected during a randomized controlled trial to investigate if maternal exercise, regardless of exercise mode, affects infant adiposity and fatty acid oxidation. Compared to control, we observed >160% higher neutral lipid content in Ex-MSCs (p<0.01). However, this finding was coupled with significantly higher complete fatty acid oxidation to CO2 and lower incomplete fat oxidation (ASM production) in Ex-MSCs (p≤0.05). Infants in the exercise group had significantly lower body fat percentage compared to same age infants of controls at one month of age (p≤0.05). There was no difference in lean body mass volume (p>0.05) or infant one-month BMI (p=0.069). Together, these data indicate that maternal exercise exposure in utero is still associated with decreased infant adiposity one month after birth. MSC lipid content was positively associated with maternal fitness and exercise volume (p<0.01) during pregnancy. Finally, an increase in average maternal exercise volume (MET*min/wk; r=-0.4, p≤0.05) and total gestational MET*min (r=-0.36, p≤0.05), but not VO2max (p=0.07) was associated with infant 1-month body fat percentage. Together, these data showcase the influence of maternal exercise exposure on improvements in cellular metabolism and at 1 month of age. Collectively, our data suggests that exposure to maternal exercise could contribute to lower adiposity in early infancy. This project was supported by the American Heart Association (18IPA34150006 to L.E.M.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

E4orf1 Modulates Hepatic Lipid Signaling, Favoring Protection from NAFLD or NASH (P08-092-19)

ObjectivesAdenovirus-36-derived E4orf1 peptide reduces hepatic fat accumulation, fibrosis, and inflammation in high fat fed mice. This property of E4orf1 could be exploited to prevent or treat broader spectrum of fatty liver diseases. Our previous in vitro study suggested that E4orf1 reduces de novo lipogenesis, and increases complete fatty acid oxidation and lipid export in hepatocytes. To better understand the phenomenon, this ex vivo study examined the genes of hepatic lipid metabolism in mice that transgenically express E4orf1. MethodsWe used C57BL/6 mice transgenically expressing E4orf1 in the adipose tissue induced upon doxycycline ingestion (Tg) and wild type C57BL/6 mice (WT) as control. Experiment 1: E4orf1 expression was induced in Tg mice (27 wk old, n = 3) by feeding chow-doxycycline diet for 6 weeks, followed by 10 weeks of 60% high-fat (HF) diet with doxycycline. WT mice (n = 4) also received the same diet for the period. Experiment 2: 50 wk old Tg mice positive or negative for E4orf1 (Tg+, n = 4 and Tg−, n = 5, respectively) received HF-doxycycline diets for 20 weeks. At termination, mice were sacrificed and hepatic RNA was extracted. The expression of key genes associated with hepatic lipid metabolism were determined by RT-PCR. ResultsExperiment 1: The activation of E4orf1 significantly downregulated genes involved in lipogenesis (ChREBP and FASn), lipid import (L-FABP, FATP2, FATP5), and lipid export (ApoB). PPARα and HSD11β1 gene expressions were significantly lower suggesting lower lipid accumulation. Experiment 2: Tg+ mice had significantly lower expressions of SREBP1c, the key promoter of lipogenesis, FASn, and ApoB. E4orf1 did not significantly influence the expressions of key genes of lipid oxidation, such as PGC1α, and CPT1 in both experiments. ConclusionsSimilar to our in vitro data with hepatocytes, this in vivo study indicated the effect of E4orf1 on lipogenesis, but not on lipid oxidation. Overall, the results suggest that E4orf1 reduces de novo lipogenesis and lipid import to the liver, which may lead to reduced hepatic lipid accumulation in mice. This reduced hepatic lipogenesis may protect the E4orf1 expressing mice from progression to hepatic inflammation and fibrosis despite the high fat diet. Funding SourcesNone.

Fetal Hepatic Lipidome Is More Greatly Affected by Maternal Rate of Gain Compared with Vitamin and Mineral Supplementation at day 83 of Gestation.

Herein, we evaluated the hepatic lipid metabolic profiles of bovine fetuses in response to maternal vitamin and mineral supplementation (VMSUP; supplemented (VTM) or not (NoVTM)) and two different rates of gain (GAIN; low gain (LG), 0.28 kg/d, or moderate gain (MG), 0.79 kg/d). Crossbred Angus heifers (n = 35; initial BW = 359.5 ± 7.1 kg) were randomly assigned to a 2 × 2 factorial arrangement, resulting in the following treatment combinations: NoVTM-LG (n = 9), NoVTM-MG (n = 9), VTM-LG (n = 9), and VTM-MG (n = 8). Heifers received their treatments until d 83 of gestation, when they were ovariohysterectomized. Fetuses were harvested and liver samples were analyzed via ultrahigh-performance liquid chromatography-tandem mass spectroscopy to characterize lipid profiles and abundances. We identified 374 biochemicals/metabolites belonging to 57 sub-pathways of the lipid metabolism super-pathway. The majority of the biochemicals/metabolites (n = 152) were significantly affected by the main effect of GAIN. Maternal moderate rates of gain resulted in greater abundances (p ≤ 0.0001) of ω-3 fatty acids (eicosapentaenoate, docosapentaenoate, and docosahexaenoate) and lower abundances (p ≤ 0.0001) of ω-6 fatty acids. Further, MG resulted in the accumulation of several diacylglycerols and depletion of the majority of the monoacylglycerols. Concentrations of nearly all acylcarnitines (p ≤ 0.03) were decreased in VTM-LG fetal livers compared to all other treatment combinations, indicating a greater rate of complete oxidation of fatty acids. Levels of secondary bile acids were impacted by VMSUP, being greater (p ≤ 0.0048) in NoVTM than in VTM fetal livers. Moreover, NoVTM combined with lower rate of gain resulted in greater concentrations of most secondary bile acid biochemicals/metabolites. These data indicate that maternal diet influenced and altered fetal hepatic lipid composition in the first trimester of gestation. Maternal body weight gain exerted a greater influence on fetal lipid profiles than vitamin and mineral supplementation. Specifically, lower rate of gain (0.28 kg/d) resulted in an increased abundance of the majority of the biochemicals/metabolites identified in this study.

Plasma Metabolomic Profiles Are Imbalanced in Adults Thalassemia Patients with Malnutrition and That Imbalance Is Alleviated By Oral Nutritional Supplements: A Prospective Randomized Controlled Trial

Background: Malnutrition is a common condition observed in children with thalassemia. However, the prevalence of malnutrition, alterations of plasma metabolomes, and the impact of nutritional supplements in adults with thalassemia were still unclear. Objectives: To evaluate the prevalence of malnutrition and plasma metabolomic profiles of adult thalassemia when compared with healthy control, as well as to evaluate the effects of oral nutritional supplements (ONS) on nutritional status and metabolomic profiles in those thalassemia patients with malnutrition Methods: Adult thalassemia (n=100) and healthy control (n=20) aged ≥ 18 years old were enrolled in the present study. This study was divided into 2 phases. Phase I of the study: All participants were assessed nutritional status by nutrition assessment 2013 (NT-2013), serum biomarkers (albumin, transferrin), and anthropometry. Plasma metabolomes including amino acids, long-chain free fatty acids, acylcarnitines (C0-18), and Krebs cycle metabolites were measured by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS). All outcomes were compared among 3 groups including thalassemia with malnutrition, thalassemia without malnutrition, and healthy control. Phase II of the study, thalassemia patients with malnutrition, defined as NT-2013 score of 5 or more, were randomized into two subgroups. Each subgroup received either ONS or nutritional advice by a nutritionist for 3 months. ONS consisted of oral complete formula (Blendera-MF®) and/or whey protein based on the assessment by food questionnaire to achieve the target of individual total daily energy expenditure (Harris-Benedict equation multiplied with activity factor) and total protein of 1.3 g/kg/day. NT-2013, anthropometric parameters, serum biomarkers, and plasma metabolomes were re-evaluated after 12 weeks in each subgroup. ONS adherence was assessed every 2 weeks during the study period. Those with less than 80% ONS adherence were excluded from the final analysis. Results: A total of 100 patients were enrolled with a mean age of 34.9 years (range 18-60). Of which, 65.3% were females and 58.1% were transfusion-dependent thalassemia (TDT). The prevalence of malnutrition was 20% (15% mild, 85% moderate degree). Malnutrition was more common in TDT than non-TDT (30.9% vs 6.7%, p=0.003). Twenty age, sex, and body mass index-matched healthy controls were also enrolled. Regarding plasma amino acids, baseline alanine, proline, and tyrosine were significantly increased while arginine was significantly decreased in malnourished patients. Fatty acid-derived acylcarnitines (C12-18) were significantly increased in malnourished patients compared to the control group. Long-chain free fatty acids were not different between groups. Considering Krebs cycle, citrate was significantly decreased, but malate was significantly increased in malnourished patients compared to the control group (Table 1). Three participants had less than 80% ONS adherence. After 12 weeks, the mean NT-2013 score in overall malnourished patients significantly improved, when compared to their baseline (6.0±1.1 to 5.2±1.5, p=0.029). However, there was a trend of more improvement in ONS group than nutritional advice group (6.1±1.0 to 5.0±1.5, p=0.084 vs. 5.9±1.2 to 5.5±1.5, p=0.223). Fatty acid-derived acylcarnitines (C8-18) and malate were significantly decreased after ONS (Table 2). Anthropometric parameters, serum biomarkers, long-chain free fatty acids, and amino acids were not changed. Conclusions: The prevalence of malnutrition in adult thalassemia was 20%. Plasma metabolomic profiles suggested that the malnourished thalassemia had imbalanced protein turnover and incomplete fatty acid oxidation, resulting in the disruption of Krebs' cycle. Among thalassemia patients with malnutrition, both ONS and nutritional advice led to improve nutritional status. ONS for 12 weeks also promoted more complete fatty acid oxidation in malnourished thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD.

NAFLD and its more-advanced form, steatohepatitis (NASH), is associated with obesity and is an independent risk factor for cardiovascular, liver-related, and all-cause mortality. Available human data examining hepatic mitochondrial fatty acid oxidation (FAO) and hepatic mitochondrial turnover in NAFLD and NASH are scant. To investigate this relationship, liver biopsies were obtained from patients with obesity undergoing bariatric surgery and data clustered into four groups based on hepatic histopathological classification: Control (CTRL; no disease); NAFL (steatosis only); Borderline-NASH (steatosis with lobular inflammation or hepatocellular ballooning); and Definite-NASH (D-NASH; steatosis, lobular inflammation, and hepatocellular ballooning). Hepatic mitochondrial complete FAO to CO2 and the rate-limiting enzyme in β-oxidation (β-hydroxyacyl-CoA dehydrogenase activity) were reduced by ~40%-50% with D-NASH compared with CTRL. This corresponded with increased hepatic mitochondrial reactive oxygen species production, as well as dramatic reductions in markers of mitochondrial biogenesis, autophagy, mitophagy, fission, and fusion in NAFL and NASH. These findings suggest that compromised hepatic FAO and mitochondrial turnover are intimately linked to increasing NAFLD severity in patients with obesity.

Apelin-13 Reverses Bupivacaine-Induced Cardiotoxicity via the Adenosine Monophosphate-Activated Protein Kinase Pathway.

Cardiotoxicity can be induced by the commonly used amide local anesthetic, bupivacaine. Bupivacaine can inhibit protein kinase B (AKT) phosphorylation and activated adenosine monophosphate-activated protein kinase alpha (AMPKα). It can decouple mitochondrial oxidative phosphorylation and enhance reactive oxygen species (ROS) production. Apelin enhances the phosphatidylinositol 3-kinase (PI3K)/AKT and AMPK/acetyl-CoA carboxylase (ACC) pathways, promotes the complete fatty acid oxidation in the heart, and reduces the release of ROS. In this study, we examined whether exogenous (Pyr1) apelin-13 could reverse bupivacaine-induced cardiotoxicity. We used the bupivacaine-induced inhibition model in adult male Sprague Dawley (SD) rats (n = 48) and H9c2 cardiomyocyte cell cultures to explore the role of apelin-13 in the reversal of bupivacaine cardiotoxicity, and its possible mechanism of action. AMPKα, ACC, carnitine palmitoyl transferase (CPT), PI3K, AKT, superoxide dismutase 1 (SOD1), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47-phox) were quantified. Changes in mitochondrial ultrastructure were examined, and mitochondrial DNA, cell viability, ROS release, oxygen consumption rate (OCR) were determined. Apelin-13 reduced bupivacaine-induced mitochondrial DNA lesions in SD rats (P < .001), while increasing the expression of AMPKα (P = .007) and PI3K (P = .002). Furthermore, apelin-13 blocked bupivacaine-induced depolarization of the mitochondrial membrane potential (P = .019) and the bupivacaine-induced increases in ROS (P = .001). Also, the AMPK pathway was activated by bupivacaine as well as apelin-13 (P = .002) in H9c2 cardiomyocytes. Additionally, the reduction in the PI3K expression by bupivacaine was mitigated by apelin-13 in H9c2 cardiomyocytes (P = .001). While the aforementioned changes induced by bupivacaine were not abated by apelin-13 after pretreatment with AMPK inhibitor compound C; the bupivacaine-induced changes were still mitigated by apelin-13, even when pretreated with PI3K inhibitor-LY294002. Apelin-13 treatment reduced bupivacaine-induced oxidative stress, attenuated mitochondrial morphological changes and mitochondrial DNA damage, enhanced mitochondrial energy metabolism, and ultimately reversed bupivacaine-induced cardiotoxicity. Our results suggest a role for the AMPK in apelin-13 reversal of bupivacaine-induced cardiotoxicity.

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells

Background and objectiveA number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca2+ driven by sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes). MethodsIn this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively. ResultsBoth acute (4 ​h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid β-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA. ConclusionAltogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.

Postpartum supplementation with fermented ammoniated condensed whey altered nutrient partitioning to support hepatic metabolism

Our previously published paper demonstrated that fermented ammoniated condensed whey (FACW) supplementation improved feed efficiency and metabolic profile in postpartum dairy cows. The objective of this study was to further explore the effects of FACW supplementation on liver triglyceride content, hepatic gene expression and protein abundance, and plasma biomarkers related to liver function, inflammation, and damage. Individually fed multiparous Holstein cows were blocked by calving date and randomly assigned to postpartum (1 to 45 d in milk, DIM) isonitrogenous treatments: control diet (n = 20) or diet supplemented with FACW (2.9% dry matter of diet as GlucoBoost; Fermented Nutrition, Luxemburg, WI, replacing soybean meal; n = 19). Liver biopsies were performed at 14 and 28 DIM for analysis of mRNA expression, protein abundance, and liver triglyceride content. There was marginal evidence for a reduction in liver triglyceride content at 14 DIM in FACW-supplemented cows compared with the control group. Cows supplemented with FACW had greater mRNA expression of glucose-6-phosphatase at 14 DIM relative to control. Supplementation with FACW increased mRNA expression of pyruvate carboxylase (PC), but did not alter cytosolic phosphoenolpyruvate carboxykinase (PCK1), resulting in a 2.4-fold greater PC:PCK1 ratio for FACW-supplemented cows compared with control. There was no evidence for a FACW effect on mRNA expression of propionyl-CoA carboxylase nor on mRNA expression or protein abundance of lactate dehydrogenase A or B. Cows supplemented with FACW had lower plasma urea nitrogen compared with control. Plasma l-lactate was greater for FACW-supplemented cows compared with control at 2 h before feeding time at 21 DIM. There was no evidence for altered expression of IL1B or IL10, or blood biomarkers related to liver function and damage. Greater glucose-6-phosphatase and PC gene expression, together with greater blood glucose and similar milk lactose output, suggests that FACW increased the supply of glucose precursors, resulting in greater gluconeogenesis between 3 and 14 DIM. Greater hepatic PC:PCK1 ratio, together with previously reported decreased plasma β-hydroxybutyrate and the marginal evidence for lower liver triglyceride content at 14 DIM, suggests greater hepatic capacity for complete oxidation of fatty acids in FACW-supplemented cows compared with control. Overall, improvements in metabolite profile and feed efficiency observed with postpartum supplementation of FACW may be attributed to increased gluconeogenic and anaplerotic precursors, most likely propionate, due to modulated rumen fermentation.

2075-P: Insulin Resistance Improved at Six Months following Bariatric Surgery Despite Accelerated Lipolysis and Accumulation of Lipid-Derived Acylcarnitines

Background: Accelerated lipolysis and the accumulation of incomplete products of fatty acid metabolism may contribute to insulin resistance (IR). It remains unclear whether the improvement in IR during the early period following bariatric surgery could be related to changes in lipid metabolism. Aims and Hypothesis: In this study, we compared the rates of lipolysis, lipid-related acylcarnitines (ACs) and IR in morbidly obese individuals at baseline, 6-months after bariatric surgery and with lean controls. Methods: 10 morbidly obese individuals scheduled for sleeve gastrectomy and 14 lean controls were recruited. IR was quantified using OGTT while body composition was measured using the 2H2O technique or DXA scans. Glycerol flux represents whole-body lipolysis and was measured using 2H5-glycerol infusion. Plasma lipid-derived ACs were measured using mass spectrometry as an indicator of complete fatty acid oxidation. Obese subjects were reassessed 6-months after bariatric surgery. Results: Compared to lean controls, obese subjects had significantly higher fat mass (50.7 ± 1.9 vs. 16.8 ± 1.0 kg) and were significantly more IR. They had higher HOMA-IR (6.6 ± 1.0 vs. 1.43 ± 0.31), glucose and insulin AUC after OGTT. Glycerol flux (16098.1 ± 1136.2 vs. 7266.20± 497.66 umol/h), plasma glycerol (117.13 ± 10.96 vs, 46.79± 3.15 umol/L), C14 (0.225 ± 0.079 vs. 0.042± 0.012) and C16 (0.136 ± 0.016 vs. 096 ± 0.003 umol/L) ACs were significantly higher in the obese individuals. 8 subjects returned at 6-months and lost 19% of their total weight, 29% of fat mass and had a 68% decrease in HOMA-IR. Glycerol flux, plasma glycerol, C14 and C18 ACs however did not show any difference compared to baseline and remained significantly higher compared to the lean controls. Conclusion: IR improved 6-months following sleeve gastrecotmy despite accelerated lipolysis and accumulation of byproducts of incomplete fatty acid metabolism. Disclosure H. Tan: None. J.W. Hsu: None. J. Kovalik: None. C. Khoo: None. S. Chacko: None. S. Ganguly: None. E. Tai: Advisory Panel; Self; Amgen Inc., BASF. O. Lai: None. E. Lim: None. F. Jahoor: None.

Increasing Acyl CoA thioesterase activity alters phospholipid profile without effect on insulin action in skeletal muscle of rats

Increased lipid metabolism in muscle is associated with insulin resistance and therefore, many strategies have been employed to alter fatty acid metabolism and study the impact on insulin action. Metabolism of fatty acid requires activation to fatty acyl CoA by Acyl CoA synthases (ACSL) and fatty acyl CoA can be hydrolysed by Acyl CoA thioesterases (Acot). Thioesterase activity is low in muscle, so we overexpressed Acot7 in muscle of chow and high-fat diet (HFD) rats and investigated effects on insulin action. Acot7 overexpression modified specific phosphatidylcholine and phosphatidylethanolamine species in tibialis muscle of chow rats to levels similar to those observed in control HFD muscle. The changes in phospholipid species did not alter glucose uptake in tibialis muscle under hyperinsulinaemic/euglycaemic clamped conditions. Acot7 overexpression in white extensor digitorum longus (EDL) muscle increased complete fatty acid oxidation ex-vivo but was not associated with any changes in glucose uptake in-vivo, however overexpression of Acot7 in red EDL reduced insulin-stimulated glucose uptake in-vivo which correlated with increased incomplete fatty acid oxidation ex-vivo. In summary, although overexpression of Acot7 in muscle altered some aspects of lipid profile and metabolism in muscle, this had no major effect on insulin-stimulated glucose uptake.

Lower Concentrations of Circulating Medium and Long-Chain Acylcarnitines Characterize Insulin Resistance in Persons with HIV.

In human immunodeficiency virus (HIV)-negative individuals, a plasma metabolite profile, characterized by higher levels of branched-chain amino acids (BCAA), aromatic amino acids, and C3/C5 acylcarnitines, is associated with insulin resistance and increased risk of diabetes. We sought to characterize the metabolite profile accompanying insulin resistance in HIV-positive persons to assess whether the same or different bioenergetics pathways might be implicated. We performed an observational cohort study of 70 nondiabetic, HIV-positive individuals (50% with body mass index ≥30 kg/m2) on efavirenz, tenofovir, and emtricitabine with suppressed HIV-1 RNA levels (<50 copies/mL) for at least 2 years and a CD4+ count over 350 cells/μL. We measured fasting insulin resistance using the homeostatic model assessment 2, plasma free fatty acids (FFA) using gas chromatography, and amino acids, acylcarnitines, and organic acids using liquid chromatography/mass spectrometry. We assessed the relationship of plasma metabolites with insulin resistance using multivariable linear regression. The median age was 45 years, median CD4+ count was 701 cells/μL, and median hemoglobin A1c was 5.2%. Insulin resistance was associated with higher plasma C3 acylcarnitines (p = .01), but not BCAA or C5 acylcarnitines. However, insulin resistance was associated with lower plasma levels of C18, C16, C12, and C2 acylcarnitines (p ≤ .03 for all), and lower C18 and C16 acylcarnitine:FFA ratios (p = .002, and p = .03, respectively). In HIV-positive persons, lower levels of plasma acylcarnitines, including the C2 product of complete fatty acid oxidation, are a more prominent feature of insulin resistance than changes in BCAA, suggesting impaired fatty acid uptake and/or mitochondrial oxidation is a central aspect of glucose intolerance in this population.

Aerobic capacity mediates susceptibility for the transition from steatosis to steatohepatitis.

Low intrinsic aerobic capacity is associated with increased all-cause and liver-related mortality in humans. Low intrinsic aerobic capacity in the low capacity runner (LCR) rat increases susceptibility to acute and chronic high-fat/high-sucrose diet-induced steatosis, without observed increases in liver inflammation. Addition of excess cholesterol to a high-fat/high-sucrose diet produced greater steatosis in LCR and high capacity runner (HCR) rats. However, the LCR rat demonstrated greater susceptibility to increased liver inflammatory and apoptotic markers compared to the HCR rat. The progressive non-alcoholic fatty liver disease observed in the LCR rats following western diet feeding was associated with further declines in liver fatty acid oxidation and mitochondrial respiratory capacity compared to HCR rats. Low aerobic capacity increases risk for non-alcoholic fatty liver disease and liver-related disease mortality, but mechanisms mediating these effects remain unknown. We recently reported that rats bred for low aerobic capacity (low capacity runner; LCR) displayed susceptibility to high fat diet-induced steatosis in association with reduced hepatic mitochondrial fatty acid oxidation (FAO) and respiratory capacity compared to high aerobic capacity (high capacity runner; HCR) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16-week 'western diet' (WD) high in fat (45%kcal), cholesterol (1%w/w) and sucrose (15%kcal). Unlike previously with a diet high in fat and sucrose alone, the inclusion of cholesterol in the WD induced hepatomegaly and steatosis in both HCR and LCR rats, while producing greater cholesterol ester accumulation in LCR compared to HCR rats. Importantly, WD-fed low-fitness LCR rats displayed greater inflammatory cell infiltration, serum alanine transaminase, expression of hepatic inflammatory markers (F4/80, MCP-1, TLR4, TLR2 and IL-1β) and effector caspase (caspase 3 and 7) activation compared to HCR rats. Further, LCR rats had greater WD-induced decreases in complete FAO and mitochondrial respiratory capacity. Intrinsic aerobic capacity had no impact on WD-induced hepatic steatosis; however, rats bred for low aerobic capacity developed greater hepatic inflammation, which was associated with reduced hepatic mitochondrial FAO and respiratory capacity and increased accumulation of cholesterol esters. These results confirm epidemiological reports that aerobic capacity impacts progression of liver disease and suggest that these effects are mediated through alterations in hepatic mitochondrial function.

Aerobic capacity and hepatic mitochondrial lipid oxidation alters susceptibility for chronic high-fat diet-induced hepatic steatosis.

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.

Apelin Enhances Brown Adipogenesis and Browning of White Adipocytes

Brown adipose tissue expends energy in the form of heat via the mitochondrial uncoupling protein UCP1. Recent studies showed that brown adipose tissue is present in adult humans and may be exploited for its anti-obesity and anti-diabetes actions. Apelin is an adipocyte-derived hormone that plays important roles in energy metabolism. Here, we report that apelin-APJ signaling promotes brown adipocyte differentiation by increasing the expressions of brown adipogenic and thermogenic transcriptional factors via the PI3K/Akt and AMPK signaling pathways. It is also found that apelin relieves the TNFα inhibition on brown adipogenesis. In addition, apelin increases the basal activity of brown adipocytes, as evidenced by the increased PGC1α and UCP1 expressions, mitochondrial biogenesis, and oxygen consumption. Finally, we provide both in vitro and in vivo evidence that apelin is able to increase the brown-like characteristics in white adipocytes. This study, for the first time, reveals the brown adipogenic and browning effects of apelin and suggests a potential therapeutic route to combat obesity and related metabolic disorders.

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease.

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance—ROS and PKCε activity—were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.

Sex differences in biomarkers associated with insulin resistance in obese adolescents: metabolomic profiling and principal components analysis.

Obesity and insulin resistance (IR) predispose to type 2 diabetes mellitus. Yet only half of obese adolescents have IR and far fewer progress to type 2 diabetes mellitus. We hypothesized that amino acid and fatty acid metabolites may serve as biomarkers or determinants of IR in obese teens. Fasting blood samples were analyzed by tandem mass spectrometry in 82 obese adolescents. A principal components analysis and multiple linear regression models were used to correlate metabolic components with surrogate measures of IR: homeostasis model assessment index of insulin resistance (HOMA-IR), adiponectin, and triglyceride (TG) to high-density lipoprotein (HDL) ratio. Branched-chain amino acid (BCAA) levels and products of BCAA catabolism were higher (P < .01) in males than females with comparable body mass index (BMI) z-score. In multivariate analyses, HOMA-IR in males correlated positively with BMI z-score and a metabolic signature containing BCAA, uric acid, and long-chain acylcarnitines and negatively with byproducts of complete fatty acid oxidation (R(2) = 0.659, P < .0001). In contrast, only BMI z-score correlated with HOMA-IR in females. Adiponectin correlated inversely with BCAA and uric acid (R(2) = 0.268, P = .0212) in males but not females. TG to HDL ratio correlated with BMI z-score and the BCAA signature in females but not males. BCAA levels and byproducts of BCAA catabolism are higher in obese teenage boys than girls of comparable BMI z-score. A metabolic signature comprising BCAA and uric acid correlates positively with HOMA-IR in males and TG to HDL ratio in females and inversely with adiponectin in males but not females. Likewise, byproducts of fatty acid oxidation associate inversely with HOMA-IR in males but not females. Our findings underscore the roles of sex differences in metabolic function and outcomes in pediatric obesity.

Abstract 1426: Fumarate hydratase and deuterium depletion control oncogenesis via NADPH-dependent reductive synthesis: mitochondrial matrix water, DNA deuteration and epigenetic events

Abstract Clear cell kidney tumors become exclusively oxidative pentose cycle, hence cytoplasmic free water dependent by fumarate hydratase mutations, which disrupts the use of low deuterium containing metabolic water of the mitochondrial matrix and consumes pentose cycle-derived NADPH for reductive carboxylation [Yang Y., et al. PLoS One 8: e72179 (2013); Mullen AR, et al. Nature 481: 385-8, (2011)]. Metabolic water by complete fat oxidation, coupled with cytochrome-c, contains low average ∼115 ppm (parts per million) deuterium due to deuterium discrimination by plant lipogenic enzymes during photosynthesis. We herein report that mono-deuterated, 100 ppm, 50 ppm and 25 ppm extracellular (free) water treatment significantly decrease nucleotide and nuclear membrane behenic- and lignoceric acid synthesis in comparison with natural 150 ppm deuterium containing water via the oxidative branch of the pentose cycle in breast (MCF7) and lung (H441) cancer cell cultures, which recapitulates metabolism after genetically restored mitochondrial fumarate hydratase function in clear cell kidney tumors. Targeted [1,2-13C2]-D-glucose to [1-13C1]-D-ribose and 13C-glutamate fate associations indicate that the serine synthesis, one-carbon (folate) metabolism and the glycine cleavage (SOGC) pathway [Tedeschi PM, et al. Cell Death Dis 4: e877, (2013)] also mediates the metabolic control of deuterium depletion in MIA-PaCa pancreatic adenocarcinoma cells. We conclude that impaired mitochondria are involved in cell transformation by limiting the low natural deuterium containing complete fatty acid oxidation product, metabolic water, to enter nuclear membranes and nucleotides via reductive synthesis. In turn, “heavy” natural water and sugar dependent NADPH production taken over by the oxidative branch of the pentose cycle, as well as the SOGC-pathway, are deuterium loading “ticking time bombs” with a strong isotope effect and thus oncogenic epigenetic events that include unstable hydrogen bonds, aneuploidy in DNA structures and chemically altered methylation sites with severely disrupted gene expression patterns. Nevertheless, extra-mitochondrial NADPH synthesis opens a therapeutic window for deuterium depleted water to maintain and/or restore normal cellular functions. Our study provides a novel mechanism regarding lipid based ketogenic diets in the presence of hyperbaric oxygen treatment to decrease metastasis formation by producing low deuterium metabolic water via complete oxidation to prevent DNA, histone and nuclear membrane deuteration during NADPH dependent reductive synthesis. Citation Format: László G. Boros, Emmanuelle J. Meuillet, Ildikó Somlyai, Gábor Jancsó, György Jákli, Krisztina Krempels, László G. Puskás, István L. Nagy, Miklós Molnár, Keith R. Laderoute, Patricia A. Thompson, Gábor Somlyai. Fumarate hydratase and deuterium depletion control oncogenesis via NADPH-dependent reductive synthesis: mitochondrial matrix water, DNA deuteration and epigenetic events. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1426. doi:10.1158/1538-7445.AM2014-1426

Cilostazol induces mitochondrial fatty acid β-oxidation in C2C12 myotubes

Cilostazol is a drug licensed for the treatment of intermittent claudication. Its main action is to elevate intracellular levels of cyclic monophosphate (cAMP) by inhibiting the activity of type III phosphodiesterase, a cAMP-degrading enzyme. The effects of cilostazol on fatty acid oxidation (FAO) are as yet unknown. In this study, we report that cilostazol can elevate complete FAO and decrease both triacylglycerol (TAG) accumulation and TAG secretion. This use of cilostazol treatment increases expression of PGC-1α and, subsequently, its target genes, such as ERRα, NOR1, CD36, CPT1, MCAD, and ACO. Expression of these factors is linked to fatty acid β-oxidation but this effect is inhibited by H-89, a specific inhibitor of the PKA/CREB pathway. Importantly, knockdown of PGC-1α using siRNA abolished the effects of cilostazol in fatty acid oxidation (FAO) and TAG metabolism. These findings suggested that the PKA/CREB/PGC-1α pathway plays a critical role in cilostazol-induced fatty acid oxidation and TAG metabolism.

Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β‐oxidation

Exercise substantially improves metabolic health, making the elicited mechanisms important targets for novel therapeutic strategies. Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein highly selectively expressed in skeletal muscle. Here we report that moderate UCP3 overexpression (roughly 3-fold) in muscles of UCP3 transgenic (UCP3 Tg) mice acts as an exercise mimetic in many ways. UCP3 overexpression increased spontaneous activity (∼40%) and energy expenditure (∼5-10%) and decreased oxidative stress (∼15-20%), similar to exercise training in wild-type (WT) mice. The increase in complete fatty acid oxidation (FAO; ∼30% for WT and ∼70% for UCP3 Tg) and energy expenditure (∼8% for WT and 15% for UCP3 Tg) in response to endurance training was higher in UCP3 Tg than in WT mice, showing an additive effect of UCP3 and endurance training on these two parameters. Moreover, increases in circulating short-chain acylcarnitines in response to acute exercise in untrained WT mice were absent with training or in UCP3 Tg mice. UCP3 overexpression had the same effect as training in decreasing long-chain acylcarnitines. Outcomes coincided with a reduction in muscle carnitine acetyltransferase activity that catalyzes the formation of acylcarnitines. Overall, results are consistent with the conclusions that circulating acylcarnitines could be used as a marker of incomplete muscle FAO and that UCP3 is a potential target for the treatment of prevalent metabolic diseases in which muscle FAO is affected.

Activation of type 2 cannabinoid receptors (CB2R) promotes fatty acid oxidation through the SIRT1/PGC-1α pathway

Abnormal fatty acid oxidation has been associated with obesity and type 2 diabetes. At the transcriptional level, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) has been reported to strongly increase the ability of hormone nuclear receptors PPARα and ERRα to drive transcription of fatty acid oxidation enzymes. In this study, we report that a specific agonist of the type 2 cannabinoid receptor (CB2R) can lead to fatty acid oxidation through the PGC-1α pathway. We have found that CB2R is expressed in differentiated C2C12 myotubes, and that use of the specific agonist trans-caryophyllene (TC) stimulates sirtuin 1 (SIRT1) deacetylase activity by increasing the phosphorylation of cAMP response element-binding protein (CREB), thus leading to increased levels of PGC-1α deacetylation. This use of TC treatment increases the expression of genes linked to the fatty acid oxidation pathway in a SIRT1/PGC-1α-dependent mechanism and also drastically accelerates the rate of complete fatty acid oxidation in C2C12 myotubes, neither of which occur when CB2R mRNA is knocked down using siRNA. These results reveal that activation of CB2R by a selective agonist promotes lipid oxidation through a signaling/transcriptional pathway. Our findings imply that pharmacological manipulation of CB2R may provide therapeutic possibilities to treat metabolic diseases associated with lipid dysregulation.