Mitochondrial Palmitate Oxidation Research Articles

Relationship between serum β-hydroxybutyrate and hepatic fatty acid oxidation in individuals with obesity and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation that can progress to inflammation (nonalcoholic steatohepatitis, NASH), and fibrosis. Serum β-hydroxybutyrate (β-HB), a product of the ketogenic pathway, is commonly used as a surrogate marker for hepatic fatty acid oxidation (FAO). However, it remains uncertain whether this relationship holds true in the context of NAFLD in humans. We compared fasting serum β-HB levels with direct measurement of liver mitochondrial palmitate oxidation in humans stratified based on NAFLD severity (n = 142). Patients were stratified based on NAFLD activity score (NAS): NAS = 0 (no disease), NAS = 1-2 (mild), NAS = 3-4 (moderate), and NAS ≥ 5 (advanced). Moderate and advanced NAFLD is associated with reductions in liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), serum β-HB, but not 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) mRNA, relative to no disease. Worsening liver mitochondrial complete palmitate oxidation corresponded with lower HMGCS2 mRNA but not total (complete + incomplete) palmitate oxidation. Interestingly, we found that liver HMGCS2 mRNA and serum β-HB correlated with liver mitochondrial β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity and CPT1A mRNA. Also, lower mitochondrial mass and markers of mitochondrial turnover positively correlated with lower HMGCS2 in the liver. These data suggest that liver ketogenesis and FAO occur at comparable rates in individuals with NAFLD. Our findings support the utility of serum β-HB to serve as a marker of liver injury and hepatic FAO in the context of NAFLD.NEW & NOTEWORTHY Serum β-hydroxybutyrate (β-HB) is frequently utilized as a surrogate marker for hepatic fatty acid oxidation; however, few studies have investigated this relationship during states of liver disease. We found that the progression of nonalcoholic fatty liver disease (NAFLD) is associated with reductions in circulating β-HB and liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). As well, decreased rates of hepatic fatty acid oxidation correlated with liver HMGCS2 mRNA and serum β-HB. Our work supports serum β-HB as a potential marker for hepatic fatty acid oxidation and liver injury during NAFLD.

Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets.

Dietary n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are known to influence glucose homeostasis. We recently showed that Elovl2 expression in beta cells, which regulates synthesis of endogenous DHA, was associated with glucose tolerance and played a key role in insulin secretion. The present study aimed to examine the role of the very long chain fatty acid elongase 2 (ELOVL2)/DHA axis on the adverse effects of palmitate with high glucose, a condition defined as glucolipotoxicity, on beta cells. We detected ELOVL2 in INS-1 beta cells and mouse and human islets using quantitative PCR and western blotting. Downregulation and adenoviral overexpression of Elovl2 was carried out in beta cells. Ceramide and diacylglycerol levels were determined by radio-enzymatic assay and lipidomics. Apoptosis was quantified using caspase-3 assays and poly (ADP-ribose) polymerase cleavage. Palmitate oxidation and esterification were determined by [U-14C]palmitate labelling. We found that glucolipotoxicity decreased ELOVL2 content in rodent and human beta cells. Downregulation of ELOVL2 drastically potentiated beta cell apoptosis induced by glucolipotoxicity, whereas adenoviral Elovl2 overexpression and supplementation with DHA partially inhibited glucolipotoxicity-induced cell death in rodent and human beta cells. Inhibition of beta cell apoptosis by the ELOVL2/DHA axis was associated with a decrease in ceramide accumulation. However, the ELOVL2/DHA axis was unable to directly alter ceramide synthesis or metabolism. By contrast, DHA increased palmitate oxidation but did not affect its esterification. Pharmacological inhibition of AMP-activated protein kinase and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid β-oxidation, attenuated the protective effect of the ELOVL2/DHA axis during glucolipotoxicity. Downregulation of CPT1 also counteracted the anti-apoptotic action of the ELOVL2/DHA axis. By contrast, a mutated active form of Cpt1 inhibited glucolipotoxicity-induced beta cell apoptosis when ELOVL2 was downregulated. Our results identify ELOVL2 as a critical pro-survival enzyme for preventing beta cell death and dysfunction induced by glucolipotoxicity, notably by favouring palmitate oxidation in mitochondria through a CPT1-dependent mechanism.

ENOS Deficiency Increases Susceptibility to Western Diet Induced Hepatic Mitochondrial Dysfunction

Nitric oxide produced by endothelial nitric oxide synthase (eNOS) can influence mitochondrial biogenesis and electron transport chain activity. Hepatic mitochondrial dysfunction is a hallmark feature of nonalcoholic fatty liver disease (NAFLD), yet the underlying mechanisms remain elusive. Here, we tested the hypothesis that eNOS plays an important role in sustaining hepatic mitochondrial function in NAFLD. Wild‐type (WT) and eNOS knockout (eNOS‐/‐) mice received either control (CON; 10% fat, 3.5% sucrose) or a western diet high in fat (45%), sucrose (17%), and cholesterol (1%; HFC) for 18‐weeks. HFC diet feeding induced histological NAFLD and increased weight gain and % body fat in both genotypes compared to CON diet. Complete hepatic mitochondrial palmitate oxidation to CO2 was elevated in both eNOS‐/‐ groups compared with WT mice(p<0.05), while incomplete acid‐soluble metabolite production was elevated only in eNOS‐/‐‐CON compared to other groups. In addition, maximal FCCP‐uncoupled mitochondrial respiration tended to be elevated in eNOS‐/‐‐CON compared to WT‐CON (+24%; p=0.08) but was significantly reduced with HFC feeding in eNOS‐/‐ mice (‐37% vs eNOS‐/‐‐CON; p<0.05). Finally, HFC feeding dramatically reduced hepatic mRNA expression of the putative control for mitochondrial biogenesis, PGC‐1α, in WT (‐33%) and eNOS‐/‐ (‐58%) compared with WT‐CON (p<0.05). These results support the hypothesis that eNOS limits susceptibility to western diet‐induced hepatic mitochondrial dysfunction and highlights a potential role for eNOS in hepatic mitochondrial biogenesis. Supported by:HL036088 (MHL) and VHA‐CDA2 1299‐03(RSR).

Combining metformin and aerobic exercise training in the treatment of type 2 diabetes and NAFLD in OLETF rats

Here, we sought to compare the efficacy of combining exercise and metformin for the treatment of type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) in hyperphagic, obese, type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (age: 20 wk, hyperglycemic and hyperinsulinemic; n = 10/group) were randomly assigned to sedentary (O-SED), SED plus metformin (O-SED + M; 300 mg·kg(-1)·day(-1)), moderate-intensity exercise training (O-EndEx; 20 m/min, 60 min/day, 5 days/wk treadmill running), or O-EndEx + M groups for 12 wk. Long-Evans Tokushima Otsuka (L-SED) rats served as nonhyperphagic controls. O-SED + M, O-EndEx, and O-EndEx + M were effective in the management of type 2 diabetes, and all three treatments lowered hepatic steatosis and serum markers of liver injury; however, O-EndEx lowered liver triglyceride content and fasting hyperglycemia more than O-SED + M. In addition, exercise elicited greater improvements compared with metformin alone on postchallenge glycemic control, liver diacylglycerol content, hepatic mitochondrial palmitate oxidation, citrate synthase, and β-HAD activities and in the attenuation of markers of hepatic fatty acid uptake and de novo fatty acid synthesis. Surprisingly, combining metformin and aerobic exercise training offered little added benefit to these outcomes, and in fact, metformin actually blunted exercise-induced increases in complete mitochondrial palmitate oxidation and β-HAD activity. In conclusion, aerobic exercise training was more effective than metformin administration in the management of type 2 diabetes and NAFLD outcomes in obese hyperphagic OLETF rats. Combining therapies offered little additional benefit beyond exercise alone, and findings suggest that metformin potentially impairs exercise-induced hepatic mitochondrial adaptations.

258 THE SEVERITY OF ISCHEMIA REPERFUSION DAMAGE IS DETERMINED BY THE CHANGES IN METABOLISM PATTERN OF ENERGY SUBSTRATES IN MITOCHONDRIA

Healthy heart is able to switch appropriately between glucose and fatty acids (FAs) to meet the energy requirements. Due to rapid energy turnover and low energy storage capacity, myocardial functioning relies on energy substrate levels in blood plasma. Availability and utilization of energy substrates can determine the outcome of cardiovascular events, particularly ischemia related, where stimulated glucose metabolism could be preferable. We hypothesize that changes in metabolism pattern of glucose and FAs in mitochondria determine the outcome of ischemia-reperfusion injury in the fed and fasted states. Male Wistar rats were randomly separated into two experimental groups, fed (n=18) and fasted (n=18). Rats in the fed group had unlimited access to food, whereas those in the fasted group were deprived of food for 18 h prior to the start of the experiment. The myocardial infarction experiment in isolated hearts from fed (n=10) and fasted (n=10) rats was performed. To determine the accumulation of glucose and palmitate metabolites in mitochondria, hearts were perfused with radiolabelled glucose and palmitate. The preference of substrates in isolated cardiac mitochondria was determined using radiolabelled substrates. Lactate and pyruvate oxidation rates were measured in the presence or absence of different concentrations of palmitoyl-carnitine, while palmitate oxidation rate was measured in the presence or absence of lactate or pyruvate. Permeabilized cardiac fibers were used to measure mitochondrial respiration on pyruvate+malate and palmitoyl-CoA. Infarct size in the fed state was 2.7-fold smaller than in the fasted state. Mitochondrial respiration was increased by 20% in the fed state when pyruvate+malate were used as substrates, but no difference was observed when palmitoyl-CoA was used as a respiratory substrate. The content of FA metabolites in mitochondria in the fasted state was 3.5-fold higher than in the fed state. Meanwhile no changes in glucose metabolites were observed in fed and fasted states. Measurements of pyruvate, lactate and palmitate oxidation in mitochondria showed that the oxidation rates are highly dependent on the concentration for these three substrates. Palmitoyl-carnitine effectively reduced the rates of pyruvate and lactate oxidation in mitochondria in a dose-dependent manner. In contrast, only pyruvate, but not lactate, significantly reduced the palmitate oxidation rate in mitochondria. Our results demonstrate that cardiac recovery from ischemia-reperfusion injury is improved in the fed state due to enhanced lactate and pyruvate (glucose) metabolism. Altogether our results suggest that the activated long chain FAs content within the mitochondria determines the rates of pyruvate and lactate metabolism, particularly in the fasted state.

Hepatic steatosis development with four weeks of physical inactivity in previously active, hyperphagic OLETF rats

Physical activity-induced prevention of hepatic steatosis is maintained during short-term (7-day) transitions to an inactive state; however, whether these protective effects are present under a longer duration of physical inactivity is largely unknown. Here, we sought to determine whether previous physical activity had protective effects on hepatic steatosis and metabolic health following 4 wk of physical inactivity. Four-week old, hyperphagic, male Otsuka Long-Evans Tokushima fatty (OLETF) rats were randomly assigned to either a sedentary group for 16 wk (OLETF-SED), given access to running wheels for 16 wk with wheels locked 5 h (OLETF-WL5hr) or given access to running wheels for 12 wk with wheels locked 4 wk (OLETF-WL4wk) prior to death. Four weeks of physical inactivity caused hepatic steatosis development, but liver triglycerides remained 60% lower than OLETF-SED (P < 0.01), and this was associated with only a partial loss in activity-induced improvements in body composition, serum lipids, and glycemic control. Total hepatic mitochondrial palmitate oxidation, citrate synthase, and β-HAD activity returned to SED levels following 4 wk of inactivity, whereas markers of fatty acid uptake and lipogenesis remained relatively suppressed following 4 wk of inactivity. In addition, 4 wk of inactivity caused a complete loss of activity-induced increases in serum IL-6 and reductions in regulated upon activation, normal T-cell expressed, and secreted (RANTES), and a partial loss in reductions in leptin, monocyte chemoattractant protein-1, and TNF-α. In conclusion, 4 wk of physical inactivity does not result in a complete loss in physical activity-induced benefits but does cause deterioration in the liver phenotype and overall metabolic health in hyperphagic OLETF rats.

High-intensity interval training increases intrinsic rates of mitochondrial fatty acid oxidation in rat red and white skeletal muscle

High-intensity interval training (HIIT) can increase mitochondrial volume in skeletal muscle. However, it is unclear whether HIIT alters the intrinsic capacity of mitochondrial fatty acid oxidation, or whether such changes are associated with changes in mitochondrial FAT/CD36, a regulator of fatty acid oxidation, or with reciprocal changes in the nuclear receptor coactivator (peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α)) and the corepressor (receptor-interacting protein 140 (RIP140)). We examined whether HIIT alters fatty acid oxidation rates in the isolated subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria of red and white skeletal muscle and (or) induces changes in muscle PGC-1α and RIP140 proteins and mitochondrial FAT/CD36 protein content. Rats were divided into untrained or HIIT-trained groups. HIIT animals performed 10 bouts of 1-min high-intensity treadmill running (30-55 m·min(-1)), separated by 2 min of rest, for 5 days a week for 4 weeks. As expected, after the training period, HIIT increased mitochondrial enzymes (citrate synthase, COXIV, and β-hydroxyacyl CoA dehydrogenase) in red and white muscle, indicating that muscle mitochondrial volume had increased. HIIT also increased the rates of palmitate oxidation in mitochondria of red (37% for SS and 19% for IMF) and white (36% for SS and 12% for IMF) muscle. No changes occurred in SS and IMF mitochondrial FAT/CD36 proteins, despite increasing FAT/CD36 at the whole-muscle level (27% for red and 22% for white). Concurrently, muscle PGC-1α protein was increased in red (22%) and white (16%) muscle, but RIP140 was not altered. These results indicate that increases in SS and IMF mitochondrial fatty acid oxidation induced by HIIT are accompanied by an increase in PGC-1α, but not RIP140 or FAT/CD36.

Altered Hepatic Lipid Metabolism Contributes to Nonalcoholic Fatty Liver Disease in Leptin-Deficient Ob/Ob Mice

Nonalcoholic fatty liver disease (NAFLD) is strongly linked to obesity, insulin resistance, and abnormal hepatic lipid metabolism; however, the precise regulation of these processes remains poorly understood. Here we examined genes and proteins involved in hepatic oxidation and lipogenesis in 14-week-old leptin-deficient Ob/Ob mice, a commonly studied model of obesity and hepatic steatosis. Obese Ob/Ob mice had increased fasting glucose, insulin, and calculated HOMA-IR as compared with lean wild-type (WT) mice. Ob/Ob mice also had greater liver weights, hepatic triglyceride (TG) content, and markers of de novo lipogenesis, including increased hepatic gene expression and protein content of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD-1), as well as elevated gene expression of PPARγ and SREBP-1c compared with WT mice. While hepatic mRNA levels for PGC-1α, PPARα, and TFAM were elevated in Ob/Ob mice, measures of mitochondrial function (β-HAD activity and complete (to CO2) and total mitochondrial palmitate oxidation) and mitochondrial OXPHOS protein subunits I, III, and V content were significantly reduced compared with WT animals. In summary, reduced hepatic mitochondrial content and function and an upregulation in de novo lipogenesis contribute to obesity-associated NAFLD in the leptin-deficient Ob/Ob mouse.

Caffeine‐stimulated fatty acid oxidation is blunted in CD36 null mice

The increase in skeletal muscle fatty acid metabolism during exercise has been associated with the release of calcium. We examined whether this increase in fatty acid oxidation was attributable to a calcium-induced translocation of the fatty acid transporter CD36 to the sarcolemma, thereby providing an enhanced influx of fatty acids to increase their oxidation. Calcium release was triggered by caffeine (3 mm) to examine fatty acid oxidation in intact soleus muscles of WT and CD36-KO mice, while fatty acid transport and mitochondrial fatty acid oxidation were examined in giant vesicles and isolated mitochondria, respectively, from caffeine-perfused hindlimb muscles of WT and CD36-KO mice. Western blotting was used to examine calcium-induced signalling. In WT, caffeine stimulated muscle palmitate oxidation (+136%), but this was blunted in CD36-KO mice (-70%). Dantrolene inhibited (WT) or abolished (CD36-KO) caffeine-induced palmitate oxidation. In muscle, caffeine-stimulated palmitate oxidation was not attributable to altered mitochondrial palmitate oxidation. Instead, in WT, caffeine increased palmitate transport (+55%) and the translocation of fatty acid transporters CD36, FABPpm, FATP1 and FATP4 (26-70%) to the sarcolemma. In CD36-KO mice, caffeine-stimulated FABPpm, and FATP1 and 4 translocations were normal, but palmitate transport was blunted (-70%), comparable to the reductions in muscle palmitate oxidation. Caffeine did not alter the calcium-/calmodulin-dependent protein kinase II phosphorylation but did increase the phosphorylation of AMPK and acetyl-CoA carboxylase comparably in WT and CD36-KO. These studies indicate that sarcolemmal CD36-mediated fatty acid transport is a primary mediator of the calcium-induced increase in muscle fatty acid oxidation.

Unsaturation of Mitochondrial Membrane Lipids is Related to Palmitate Oxidation in Subsarcolemmal and Intermyofibrillar Mitochondria

Membrane lipid composition is thought to influence the function of integral membrane proteins; however, the potential for lipid composition to influence overall mitochondrial long-chain fatty acids (LCFA) oxidation is currently unknown. Therefore, the naturally occurring variability of LCFA oxidation rates within subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in muscles with varying oxidative potentials (heart→red→white) was utilized to examine this relationship. To this end, SS and IMF mitochondria were isolated and palmitate oxidation rates were compared to membrane phospholipid composition. Among tissues, rates of palmitate oxidation in mitochondria displayed a 2.5-fold range, creating the required range to determine potential relationships with membrane lipid composition. In general, the percent mole fraction of phospholipid head groups and major fatty acid subclasses were similar in all mitochondria studied. However, rates of palmitate oxidation were positively correlated with both the unsaturation index and relative abundance of cardiolipin within mitochondria (r=0.57 and 0.49, respectively; p<0.05). Thus, these results suggest that mitochondrial LCFA oxidation may be significantly influenced by the total unsaturation and percent mole fraction of cardiolipin of the mitochondrial membrane, whereas other indices of membrane structure (e.g., percent mole fraction of other predominant membrane phospholipids, chain length, and ratio of phosphatidylcholine to phosphatidylethanolamine) were not significantly correlated.

FAT/CD36-null mice reveal that mitochondrial FAT/CD36 is required to upregulate mitochondrial fatty acid oxidation in contracting muscle

The plasma membrane fatty acid transport protein FAT/CD36 is also present at the mitochondria, where it may contribute to the regulation of fatty acid oxidation, although this has been challenged. Therefore, we have compared enzyme activities and rates of mitochondrial palmitate oxidation in muscles of wild-type (WT) and FAT/CD36 knockout (KO) mice, at rest and after muscle contraction. In WT and KO mice, carnitine palmitoyltransferase-I, citrate synthase, and beta-hydroxyacyl-CoA dehydrogenase activities did not differ in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria of WT and FAT/CD36 KO mice. Basal palmitate oxidation rates were lower (P < 0.05) in KO mice (SS -18%; IMF -13%). Muscle contraction increased fatty acid oxidation (+18%) and mitochondrial FAT/CD36 protein (+16%) in WT IMF but not in WT SS, or in either mitochondrial subpopulation in KO mice. This revealed that the difference in IMF mitochondrial fatty acid oxidation between WT and KO mice can be increased approximately 2.5-fold from 13% under basal conditions to 35% during muscle contraction. The FAT/CD36 inhibitor sulfo-N-succinimidyl oleate (SSO), inhibited palmitate transport across the plasma membrane in WT, but not in KO mice. In contrast, SSO bound to mitochondrial membranes and reduced palmitate oxidation rates to a similar extent in both WT and KO mitochondria ( approximately 80%; P < 0.05). In addition, SSO reduced state III respiration with succinate as a substrate, without altering mitochondrial coupling (P/O ratios). Thus, while SSO inhibits FAT/CD36-mediated palmitate transport at the plasma membrane, SSO has undefined effects on mitochondria. Nevertheless, the KO animals reveal that FAT/CD36 contributes to the regulation of mitochondrial fatty acid oxidation, which is especially important for meeting the increased metabolic demands during muscle contraction.

In obese rat muscle transport of palmitate is increased and is channeled to triacylglycerol storage despite an increase in mitochondrial palmitate oxidation

Intramuscular triacylglycerol (IMTG) accumulation in obesity has been attributed to increased fatty acid transport and/or to alterations in mitochondrial fatty acid oxidation. Alternatively, an imbalance in these two processes may channel fatty acids into storage. Therefore, in red and white muscles of lean and obese Zucker rats, we examined whether the increase in IMTG accumulation was attributable to an increased rate of fatty acid transport rather than alterations in subsarcolemmal (SS) or intermyofibrillar (IMF) mitochondrial fatty acid oxidation. In obese animals selected parameters were upregulated, including palmitate transport (red: +100%; white: +51%), plasmalemmal FAT/CD36 (red: +116%; white: +115%; not plasmalemmal FABPpm, FATP1, or FATP4), IMTG concentrations (red: approximately 2-fold; white: approximately 4-fold), and mitochondrial content (red +30%). Selected mitochondrial parameters were also greater in obese animals, namely, palmitate oxidation (SS red: +91%; SS white: +26%; not IMF mitochondria), FAT/CD36 (SS: +65%; IMF: +65%), citrate synthase (SS: +19%), and beta-hydroxyacyl-CoA dehydrogenase activities (SS: +20%); carnitine palmitoyltransferase-I activity did not differ. A comparison of lean and obese rat muscles revealed that the rate of change in IMTG concentration was eightfold greater than that of fatty acid oxidation (SS mitochondria), when both parameters were expressed relative to fatty transport. Thus fatty acid transport, esterification, and oxidation (SS mitochondria) are upregulated in muscles of obese Zucker rats, with these effects being most pronounced in red muscle. The additional fatty acid taken up is channeled primarily to esterification, suggesting that upregulation in fatty acid transport as opposed to altered fatty acid oxidation is the major determinant of intramuscular lipid accumulation.

The role of protein-mediated transport in regulating mitochondrial long-chain fatty acid oxidation

This thesis is an investigation of the role of fatty acid translocase (FAT/CD36), plasma membrane associated fatty acid binding protein (FABPpm), and carnitine palmitoyltransferase I (CPTI) in transporting long-chain fatty acids (LCFAs) across mitochondrial membranes. Maximal CPTI activity, as well as the sensitivity of CPTI for its substrate palmitoyl-CoA (P-CoA) and its inhibitor malonyl-CoA (M-CoA), were measured in mitochondria isolated from human vastus lateralis muscles at rest and following muscle contraction. Exercise did not alter maximal CPTI activity or the sensitivity of CPTI for P-CoA. In contrast, exercise progressively attenuated the ability of M-CoA to inhibit CPTI activity. Mitochondrial FAT/CD36 protein content was also measured at rest, during, and following 2 h of cycling at ~60% maximal oxygen uptake. Exercise progressively increased the content of mitochondrial FAT/CD36 (+59%), which was significantly (p &lt; 0.05) correlated with palmitate oxidation during exercise (r = 0.52), while palmitate oxidation was inhibited ~80% by the administration of a specific FAT/CD36 inhibitor. These data suggest that alterations in CPTI M-CoA sensitivity and increases in mitochondrial FAT/CD36 coordinate exercise-induced increases in fatty acid oxidation. FABPpm, another plasma membrane transport protein, has identical amino acid sequence to mitochondrial aspartate aminotransferase (mAspAT). Since FABPpm contributes to plasma membrane fatty acid transport, the role of FABPpm with respect to mitochondrial LCFA transport was investigated. However, unlike FAT/CD36, muscle contraction did not induce an increase in mitochondrial FABPpm protein in rat or human skeletal muscle. In addition, electrotransfecting FABPpm cDNA into rat skeletal muscle upregulated this protein in mitochondria by 80% without altering mitochondrial palmitate oxidation. In contrast, electrotransfection increased mAspAT activity by 90%, and this was correlated (r = 0.75; p &lt; 0.01) with FABPpm protein. These data suggest that FABPpm does not contribute to the regulation of mitochondrial LCFA transport. Previously, it has been suggested that mitochondria from obese individuals contain an inherent dysfunction to oxidize LCFAs. In age-matched lean (BMI = 23.3 ± 0.7 kg·m–2) and obese (BMI = 37.6 ± 2.2 kg·m–2) individuals, isolated mitochondrial palmitate oxidation was not altered. In addition, mitochondrial FAT/CD36 content was not different in lean and obese individuals. In contrast, citrate synthase and β-hydroxyacyl-CoA dehydrogenase, common markers of total mitochondrial content, were decreased with obesity. Therefore, the decrease in mitochondrial content appeared to account for the observed reductions in whole-muscle LCFA oxidation.

Fatty acid binding protein facilitates sarcolemmal fatty acid transport but not mitochondrial oxidation in rat and human skeletal muscle

The transport of long-chain fatty acids (LCFAs) across mitochondrial membranes is regulated by carnitine palmitoyltransferase I (CPTI) activity. However, it appears that additional fatty acid transport proteins, such as fatty acid translocase (FAT)/CD36, influence not only LCFA transport across the plasma membrane, but also LCFA transport into mitochondria. Plasma membrane-associated fatty acid binding protein (FABPpm) is also known to be involved in sacrolemmal LCFA transport, and it is also present on the mitochondria. At this location, it has been identified as mitochondrial aspartate amino transferase (mAspAT), despite being structurally identical to FABPpm. Whether this protein is also involved in mitochondrial LCFA transport and oxidation remains unknown. Therefore, we have examined the ability of FABPpm/mAspAT to alter mitochondrial fatty acid oxidation. Muscle contraction increased (P < 0.05) the mitochondrial FAT/CD36 content in rat (+22%) and human skeletal muscle (+33%). By contrast, muscle contraction did not alter the content of mitochondrial FABPpm/mAspAT protein in either rat or human muscles. Electrotransfecting rat soleus muscles, in vivo, with FABPpm cDNA increased FABPpm protein in whole muscle (+150%; P < 0.05), at the plasma membrane (+117%; P < 0.05) and in mitochondria (+80%; P < 0.05). In these FABPpm-transfected muscles, palmitate transport into giant vesicles was increased by +73% (P < 0.05), and fatty acid oxidation in intact muscle was increased by +18% (P < 0.05). By contrast, despite the marked increase in mitochondrial FABPpm/mAspAT protein content (+80%), the rate of mitochondrial palmitate oxidation was not altered (P > 0.05). However, electrotransfection increased mAspAT activity by +70% (P < 0.05), and the mitochondrial FABPpm/mAspAT protein content was significantly correlated with mAspAT activity (r = 0.75). It is concluded that FABPpm has two distinct functions depending on its subcellular location: (a) it contributes to increasing sarcolemmal LCFA transport while not contributing directly to LCFA transport into mitochondria; and (b) its primary role at the mitochondria level is to transport reducing equivalents into the matrix.

PPAR‐α agonism improves whole body and muscle mitochondrial fat oxidation, but does not alter intracellular fat concentrations in burn trauma children

Insulin resistance is often associated with increased levels of intracellular triglycerides, diacylglycerol and decreased fat β-oxidation. It was unknown if this relationship was present in patients with acute trauma induced insulin resistance. A double blind placebo controlled trial was conducted in 18 children with severe burn injury. Metabolic studies to assess whole body palmitate oxidation and insulin sensitivity, muscle biopsies for mitochondrial palmitate oxidation, diacylglycerol, fatty acyl Co-A and carnitine concentrations, and magnetic resonance spectroscopy for muscle and liver triglycerides were compared before and after two weeks of placebo or PPAR-α agonist treatment. Insulin sensitivity and basal whole body palmitate oxidation as measured with an isotope tracer increased significantly (P=0.003 and P=0.004, respectively) after PPAR-α agonist treatment compared to placebo. Mitochondrial palmitate oxidation rates in muscle samples increased significantly after PPAR-α treatment (P=0.002). However, the concentrations of muscle triglyceride, diacylglycerol, fatty acyl CoA and carnitine and liver triglycerides did not change with either treatment. PKC-𝛉 activation during hyper-insulinemia decreased significantly following PPAR-α treatment. Impaired palmitate oxidation and increased PKC activity are related to reduced insulin sensitivity in acute trauma, but a relation between these two responses cannot be attributed to alterations in intracellular lipid concentrations.

PPAR-α agonism improves whole body and muscle mitochondrial fat oxidation, but does not alter intracellular fat concentrations in burn trauma children in a randomized controlled trial

BackgroundInsulin resistance is often associated with increased levels of intracellular triglycerides, diacylglycerol and decreased fat β-oxidation. It was unknown if this relationship was present in patients with acute insulin resistance induced by trauma.MethodsA double blind placebo controlled trial was conducted in 18 children with severe burn injury. Metabolic studies to assess whole body palmitate oxidation and insulin sensitivity, muscle biopsies for mitochondrial palmitate oxidation, diacylglycerol, fatty acyl Co-A and fatty acyl carnitine concentrations, and magnetic resonance spectroscopy for muscle and liver triglycerides were compared before and after two weeks of placebo or PPAR-α agonist treatment.ResultsInsulin sensitivity and basal whole body palmitate oxidation as measured with an isotope tracer increased significantly (P = 0.003 and P = 0.004, respectively) after PPAR-α agonist treatment compared to placebo. Mitochondrial palmitate oxidation rates in muscle samples increased significantly after PPAR-α treatment (P = 0.002). However, the concentrations of muscle triglyceride, diacylglycerol, fatty acyl CoA, fatty acyl carnitine, and liver triglycerides did not change with either treatment. PKC-θ activation during hyper-insulinemia decreased significantly following PPAR-α treatment.ConclusionPPAR-α agonist treatment increases palmitate oxidation and decreases PKC activity along with reduced insulin sensitivity in acute trauma, However, a direct link between these responses cannot be attributed to alterations in intracellular lipid concentrations.

Upregulation of liver VLDL receptor and FAT/CD36 expression in LDLR−/− apoB100/100 mice fed trans-10,cis-12 conjugated linoleic acid

This study explores the mechanisms responsible for the fatty liver setup in mice fed trans-10,cis-12 conjugated linoleic acid (t10c12 CLA), hypothesizing that an induction of low density lipoprotein receptor (LDLR) expression is associated with lipid accumulation. To this end, the effects of t10c12 CLA treatment on lipid parameters, serum lipoproteins, and expression of liver lipid receptors were measured in LDLR(-/-) apoB(100/100) mice as a model of human familial hypercholesterolemia itself depleted of LDLR. Mice were fed t10c12 CLA over 2 or 4 weeks. We first observed that the treatment induced liver steatosis, even in the absence of LDLR. Mice treated for 2 weeks exhibited hypertriglyceridemia with high levels of VLDL and HDL, whereas a 4 week treatment inversely induced a reduction of serum triglycerides (TGs), essentially through a decrease in VLDL levels. In the absence of LDLR, the mRNA levels of other proteins, such as VLDL receptor, lipoprotein lipase, and fatty acid translocase, usually not expressed in the liver, were upregulated, suggesting their involvement in the steatosis setup and lipoprotein clearance. The data also suggest that the TG-lowering effect induced by t10c12 CLA treatment was attributable to both the reduction of circulating free fatty acids in response to the severe lipoatrophy and the high capacity of liver to clear off plasma lipids.

Isolated Mitochondrial Palmitate Oxidation is not Impaired with Obesity

PURPOSE: Numerous studies have suggested that obesity is associated with skeletal muscle mitochondrial dysregulation, reducing the capacity to oxidize fatty acids and ultimately shifting the fate of fatty acids to favor storage. Elevations in the content of intramuscular triacylglycerides (IMTG), and the associated increases in diacylglycerides (DAG) and ceramides, have been associated with an attenuation in insulin signaling, and subsequently been shown to be strong predictors of insulin resistance. While previous research has focused on the ability of whole muscle to oxidize fatty acids, the purpose of the present study was to ascertain the ability of skeletal muscle mitochondria isolated from obese subjects to oxidize fatty acids. METHODS: Rectus abdominus muscle was sampled from nine lean (age; 47±3 yrs, mass; 59.7±2.0 kg, BMI; 23.3±0.7 kg/m2) and nine obese (age; 45±3 yrs, mass; 99.2±6.8 kg, BMI; 37.6±2.2 kg/m2) women. A portion of muscle was homogenized and used to determine maximal citrate synthase (CS) and β-hydroxyacyl-CoA dehydrogenase (β-HAD) activities. The majority of the muscle was then used to isolated viable mitochondria, and to determine palmitate oxidation rates and fatty acid transport protein contents through Western analysis. RESULTS: Obesity decreased (P<0.05) CS and β-HAD activity by 34% and 17%, respectively. CS significantly correlated with BMI (r=−0.53), however β-HAD did not (r=−0.31). Palmitate oxidation did not differ between lean and obese individuals (59±4 vs. 69±9, nmol/mg protein/hour), nor did the content of mitochondrial fatty acid translocase (FAT)/CD36 (100±16 vs 120±17, arbitrary units) or fatty acid binding protein (FABP) (100±17 vs. 93±8, arbitrary units). However, regardless of BMI classification, palmitate oxidation significantly (P<0.01) correlated with FAT/CD36 content (r=0.67), but did not with FABP content (r=0.42). CONCLUSIONS: It is concluded that reductions in fatty acid oxidation that have been reported with obesity result from decreases in mitochondrial volume, and not from an inability of remaining mitochondria to oxidize fatty acids. This research was supported by NSERC and CIHR.

Mitochondrial LCFA Oxidation, FAT/CD36 Content and CPTI Activity in Human Skeletal Muscle During Aerobic Exercise

PURPOSE: Mitochondrial fatty acid transport is a rate-limiting step in long chain fatty acid (LCFA) oxidation, and traditionally was believed to be solely regulated by carnitine palmitoyltransferase I (CPTI). Recently, it has been proposed that mitochondrial LCFA transport may involve fatty acid translocase (FAT)/CD36 in addition to CPTI, however mitochondrial FAT/CD36 has not been studied in human skeletal muscle during exercise. The present study investigated the ability of malonyl-CoA (M-CoA) to inhibit CPTI, and the presence and functional significance of FAT/ CD36 on mitochondria. We hypothesized that exercise induced increases in fatty acid oxidation would occur as a result of decreases in the sensitivity of CPTI to M-CoA and increased mitochondrial FAT/CD36 protein. METHODS: Participants cycled at ∼60% VO2peak for 120 min, and blood and ventilatory data was collected at rest and every 30 minutes during exercise. Purcutaneous needle biopsies were taken from the vastus lateralis at rest, 30 min and following 120 min. Mitochondria were immediately isolated, and used for CPTI activity measurements, palmitate oxidation rates and Western blots for FAT/CD36 protein content (n=15). RESULTS: Whole body fat oxidation rates progressively increased during exercise (p<0.05), and concomitantly M-CoA inhibition of CPTI was progressively attenuated. Compared to rest, 120 min of cycling reduced (p<0.05) the inhibition of 0.7, 2, 5 and 10 uMM-CoAby 16%, 21%, 30% and 34%, respectively. However, exercise did not alter the maximal CPTI activity. Palmitate oxidation rates in isolated mitochondria progressively increased (p<0.05) during exercise, and was positively correlated with whole body fat oxidation rates (r=0.78). Associated with the exercise induced increase in mitochondrial palmitate oxidation was a 63% increase (p<0.05) in mitochondrial FAT/CD36 protein content. Importantly, the addition of sulfo-N-succimidyl oleate, a specific inhibitor of FAT/CD36, reduced mitochondrial palmitate oxidation by ∼80%. CONCLUSIONS: The data suggests that exercise induced increases in fatty acid oxidation occur as a result of decreases in the sensitivity of CPTI to the inhibitor M-CoA and increased mitochondrial FAT/CD36 protein. It is proposed that translocation from an intracellular depot is a feasible explanation. We conclude that these two novel mechanisms are not mutually exclusive, but rather interact to influence LCFA oxidation.

Mitochondrial long chain fatty acid oxidation, fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activity in human skeletal muscle during aerobic exercise

Mitochondrial fatty acid transport is a rate-limiting step in long chain fatty acid (LCFA) oxidation. In rat skeletal muscle, the transport of LCFA at the level of mitochondria is regulated by carnitine palmitoyltransferase I (CPTI) activity and the content of malonyl-CoA (M-CoA); however, this relationship is not consistently observed in humans. Recently, fatty acid translocase (FAT)/CD36 was identified on mitochondria isolated from rat and human skeletal muscle and found to be involved in LCFA oxidation. The present study investigated the effects of exercise (120 min of cycling at approximately 60% V(O2peak)) on CPTI palmitoyl-CoA and M-CoA kinetics, and on the presence and functional significance of FAT/CD36 on skeletal muscle mitochondria. Whole body fat oxidation rates progressively increased during exercise (P < 0.05), and concomitantly M-CoA inhibition of CPTI was progressively attenuated. Compared to rest, 120 min of cycling reduced (P < 0.05) the inhibition of 0.7, 2, 5 and 10 microM M-CoA by 16%, 21%, 30% and 34%, respectively. Whole body fat oxidation and palmitate oxidation rates in isolated mitochondria progressively increased (P < 0.05) during exercise, and were positively correlated (r = 0.78). Mitochondrial FAT/CD36 protein increased by 63% (P < 0.05) during exercise and was significantly (P < 0.05) correlated with mitochondrial palmitate oxidation rates at all time points (r= 0.41). However, the strongest (P < 0.05) correlation was observed following 120 min of cycling (r = 0.63). Importantly, the addition of sulfo-N-succimidyloleate, a specific inhibitor of FAT/CD36, reduced mitochondrial palmitate oxidation to approximately 20%, indicating FAT/CD36 is functionally significant with respect to LCFA oxidation. We hypothesize that exercise-induced increases in fatty acid oxidation occur as a result of an increased ability to transport LCFA into mitochondria. We further suggest that decreased CPTI M-CoA sensitivity and increased mitochondrial FAT/CD36 protein are both important for increasing whole body fatty acid oxidation during prolonged exercise.