Muscle fatty acid (FA) metabolism is impaired in obesity and insulin resistance, reflected by reduced rates of FA oxidation and accumulation of lipids. It has been suggested that interventions that increase FA oxidation may enhance insulin action by reducing these lipid pools. Here, we examined the effect of endurance training on rates of mitochondrial FA oxidation, the activity of carnitine palmitoyltransferase I (CPT I), and the lipid content in muscle of obese individuals and related these to measures of glucose tolerance. Nine obese subjects completed 8 wk of moderate-intensity endurance training, and muscle biopsies were obtained before and after training. Training significantly improved glucose tolerance, with a reduction in the area under the curve for glucose (P < 0.05) and insulin (P = 0.01) during an oral glucose tolerance test. CPT I activity increased 250% (P = 0.001) with training and became less sensitive to inhibition by malonyl-CoA. This was associated with an increase in mitochondrial FA oxidation (+120%, P < 0.001). Training had no effect on muscle triacylglycerol content; however, there was a trend for training to reduce both the total diacylglcyerol (DAG) content (-15%, P = 0.06) and the saturated DAG-FA species (-27%, P = 0.06). Training reduced both total ceramide content (-42%, P = 0.01) and the saturated ceramide species (-32%, P < 0.05). These findings suggest that the improved capacity for mitochondrial FA uptake and oxidation leads not only to a reduction in muscle lipid content but also a to change in the saturation status of lipids, which may, at least in part, provide a mechanism for the enhanced insulin action observed with endurance training in obese individuals.

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 ∼60%V O 2 peak ) 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 μM 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 ∼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.

Lipolysis is the catabolic pathway by which triglycerides are hydrolyzed into fatty acids. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) have the capacity to hydrolyze in vitro the first ester bond of triglycerides, but their respective contributions to whole cell lipolysis in human adipocytes is unclear. Here, we have investigated the roles of HSL, ATGL, and its coactivator CGI-58 in basal and forskolin-stimulated lipolysis in a human white adipocyte model, the hMADS cells. The hMADS adipocytes express the various components of fatty acid metabolism and show lipolytic capacity similar to primary cultured adipocytes. We show that lipolysis and fatty acid esterification are tightly coupled except in conditions of stimulated lipolysis. Immunocytochemistry experiments revealed that acute forskolin treatment promotes HSL translocation from the cytosol to small lipid droplets and redistribution of ATGL from the cytosol and large lipid droplets to small lipid droplets, resulting in enriched colocalization of the two lipases. HSL or ATGL overexpression resulted in increased triglyceride-specific hydrolase capacity, but only ATGL overexpression increased whole cell lipolysis. HSL silencing had no effect on basal lipolysis and only partially reduced forskolin-stimulated lipolysis. Conversely, silencing of ATGL or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Altogether, these results suggest that ATGL/CGI-58 acts independently of HSL and precedes its action in the sequential hydrolysis of triglycerides in human hMADS adipocytes.Adipose tissue fat stores in humans are mainly dependent upon fatty acid (FA) 2 supply, FA esterification to triglycerides (TG), and TG breakdown, or lipolysis. Adipose tissue lipolysis is governed by three lipases. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) both have the capacity to initiate TG degradation by cleaving the first ester bond, but HSL is unique in its capacity to break down the second ester bond, converting diglycerides (DG) to monoglycerides (1-3). The non-rate-limiting monoglyceride lipase completes lipolysis by cleaving the last ester bond from a monoglyceride molecule, leading to glycerol release (4). Adipose tissue lipolysis has received much attention over the past 10 years because of its altered regulation in obesity (5).HSL resides freely in the cytosol and can associate with lipid droplets (LD). It is regulated by hormones such as catecholamines, insulin, and natriuretic peptides. Catecholamines bind to ␤-adrenoceptors on adipocyte cell membranes and activate cyclic AMP-dependent protein kinase. Similarly, natriuretic peptides bind to type A receptors and activate cyclic GMPdependent protein kinase (6). The protein kinase action in stimulated lipolysis is 2-fold: 1) phosphorylation of HSL, leading to its translocation from the cytosol to LD (7,8), and 2) phosphorylation of perilipin A (6, 9, 10), the predominant perilipin isoform in adipocytes, enhancing interaction be...

Partial inhibition of adipose tissue lipolysis does not increase fat mass but improves glucose metabolism and insulin sensitivity through modulation of fatty acid turnover and induction of fat cell de novo lipogenesis.

) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 M) palmitate oxidation was decreased by 95% (P Ͻ 0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P Ͻ 0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation. fatty acid translocase; long-chain fatty acid ACCUMULATION OF LONG-CHAIN fatty acids (LCFA) in muscle cells occurs when cellular uptake rates exceed mitochondrial oxidation rates. It has been suggested that mitochondrial transport, and not oxidation per ser, is rate-limiting (18); thus, proper understanding of LCFA transport across the mitochondrial membrane is required. The carnitine palmitoyltransferase (CPT) system has long been viewed as the primary mode of LCFA-CoA transport across the mitochondrial membranes. The CPT complex is comprised of the regulated enzyme CPT I (EC 2.3.1.21), the carnitine-acylcarnitine translocase (CAT), and the latent CPT II (35). CPT I spans the mitochondrial outer membrane (MOM), with its catalytic site facing the cytosol (21). CAT and CPT II are located on the outer and inner leaflets of the mitochondrial inner membrane (MIM), respectively [see McGarry and Brown for review (35)]. CPT I catalyzes the trans-esterification of LCFA-CoA to LCFAcarnitine. CAT accepts LCFA-carnitine in exchange for carnitine, the latter being recycled in the intermembrane space (IMS). LCFA-carnitine is then translocated to the MIM where it is reconverted to LCFA-CoA by CPT II. Taken altogether, this system translocates LCFA-CoA from the cytosol into the mitochondrial matrix.Evidence for regulation of the CPT system was first identified in rat skeletal muscle (53, 54). Briefly, malonyl-CoA (M-CoA), the product of the acetyl-CoA carboxylase (ACC) reaction, inhibits CPT I activity at rest. When aerobic exercise duration (53, 54) and/or intensity (40) increases in ra...

Both acute and chronic apelin treatment have been shown to improve insulin sensitivity in mice. However, the effects of apelin on fatty acid oxidation (FAO) during obesity-related insulin resistance have not yet been addressed. Thus, the aim of the current study was to determine the impact of chronic treatment on lipid use, especially in skeletal muscles. High-fat diet (HFD)-induced obese and insulin-resistant mice treated by an apelin injection (0.1 μmol/kg/day i.p.) during 4 weeks had decreased fat mass, glycemia, and plasma levels of triglycerides and were protected from hyperinsulinemia compared with HFD PBS-treated mice. Indirect calorimetry experiments showed that apelin-treated mice had a better use of lipids. The complete FAO, the oxidative capacity, and mitochondrial biogenesis were increased in soleus of apelin-treated mice. The action of apelin was AMP-activated protein kinase (AMPK) dependent since all the effects studied were abrogated in HFD apelin-treated mice with muscle-specific inactive AMPK. Finally, the apelin-stimulated improvement of oxidative capacity led to decreased levels of acylcarnitines and enhanced insulin-stimulated glucose uptake in soleus. Thus, by promoting complete lipid use in muscle of insulin-resistant mice through mitochondrial biogenesis and tighter matching between FAO and the tricarboxylic acid cycle, apelin treatment could contribute to insulin sensitivity improvement.

Uncoupling protein 3 (UCP3) expression is directly correlated to fatty acid oxidation in skeletal muscle. UCP3 has been hypothesized to facilitate high rates of fatty acid oxidation, but evidence thus far is lacking. Our aim was to investigate the effects of UCP3 overexpression and ablation on fatty acid uptake and metabolism in muscle of mice having congenic backgrounds. In mice constitutively expressing the UCP3 protein (human form) at levels just over twofold higher than normal (230% of wild-type levels), indirect calorimetry demonstrated no differences in total energy expenditure (VO2), but a shift toward increased fat oxidation compared with wild-type (WT) mice. Metabolic efficiency (gram weight gain/kcal ingested) was similar between Ucp3 overexpressors, WT and Ucp3 (-/-) mice. In muscle of Ucp3-tg mice, plasma membrane fatty acid binding protein (FABPpm) content was increased compared with WT mice. Although hormone-sensitive lipase activity was unchanged across the genotypes, there were increases in carnitine palmitoyltransferase I, beta-hydroxyacylCoA dehydrogenase, and citrate synthase activities and decreases in intramuscular triacylglycerol in muscle of Ucp3-tg mice. There were no differences in muscle mitochondrial content. High-energy phosphates and total muscle carnitine and CoA were also greater in Ucp3-tg compared with WT mice. Taken together, the findings demonstrate an increased capacity for fat oxidation in the absence of significant increases in thermogenesis in Ucp3-tg mice. Findings from Ucp3 (-/-) mice revealed few differences compared with WT mice, consistent with the possibility of compensatory mechanisms. In conjunction with our observed increases in CoA and carnitine in muscle of Ucp3 overexpressors, the findings support the hypothesized role for Ucp3 in facilitating fatty acid oxidation in muscle.

Weight loss in response to caloric restriction is variable. Because skeletal muscle mitochondrial proton leak may account for a large proportion of resting metabolic rate, we compared proton leak in diet-resistant and dietresponsive overweight women and compared the expression and gene characteristics of uncoupling protein (UCP)2 and UCP3. Of 1,129 overweight women who completed the University of Ottawa Weight Management Clinic program, 353 met compliance criteria and were free of medical conditions that could affect weight loss. Subjects were ranked according to percent body weight loss during the first 6 weeks of a 900-kcal meal replacement protocol. The highest and lowest quintiles of weight loss were defined as diet responsive and diet resistant, respectively. After body weight had been stable for at least 10 weeks, 12 of 70 subjects from each group consented to muscle biopsy and blood sampling for determinations of proton leak, UCP mRNA expression, and genetic studies. Despite similar baseline weight and age, weight loss was 43% greater, mitochondrial proton leak-dependent (state 4) respiration was 51% higher (P ‫؍‬ 0.0062), and expression of UCP3 mRNA abundance was 25% greater (P < 0.001) in diet-responsive than in diet-resistant subjects. There were no differences in UCP2 mRNA abundance. None of the known polymorphisms in UCP3 or its 5 flanking sequence were associated with weight loss or UCP3 mRNA abundance. Thus, proton leak and the expression of UCP3 correlate with weight loss success and may be candidates for pharmacological regulation of fat oxidation in obese diet-resistant subjects. Diabetes 51: 2459 -2466, 2002 A t the Weight Management Program at the University of Ottawa, we have documented a 10-fold variation in the rate of weight loss in 353 highly compliant women on a standard exercise program and standard 900-kcal meal replacement protocol. These women were ranked according to percent body weight loss, and highest and lowest quintiles were defined as diet responsive and diet resistant, respectively. Regression analyses demonstrated that the known variables regulating energy requirements, including initial weight, age, and plasma free triiodothyronine (T3) concentrations, accounted for only half of this variability (1), leading us to search for novel molecular determinants of weight loss success.Variable responses to overfeeding have been reported. Rodent studies have demonstrated that genetic factors not only regulate weight gain in response to high-fat highcalorie diets but also determine the susceptibility to obesity when energy intake is controlled (2). In response to the ingestion of hypercaloric diets, the majority of subjects gain less weight than anticipated, and a process of adaptive thermogenesis appears to play a role in the defense against obesity (3). Several studies have demonstrated marked interindividual variability in the susceptibility to weight gain in response to overfeeding (4), and identical twins show marked similarity in this regard, suggesting an important genetic cont...