Evidence of a malonyl-CoA-insensitive carnitine palmitoyltransferase I activity in red skeletal muscle

Carnitine palmitoyltransferase I (CPT I), which is expressed as two distinct isoforms in liver (alpha) and muscle (beta), catalyzes the rate-limiting step in the transport of fatty acid into the mitochondria. Malonyl-CoA, a potent inhibitor of CPT I, is considered a key regulator of fatty acid oxidation in both tissues. Still unanswered is how muscle beta-oxidation proceeds despite malonyl-CoA concentrations that exceed the IC(50) for CPT Ibeta. We evaluated malonyl-CoA-suppressible [(14)C]palmitate oxidation and CPT I activity in homogenates of red (RG) and white (WG) gastrocnemius, soleus (SOL), and extensor digitorum longus (EDL) muscles. Adding 10 microM malonyl-CoA inhibited palmitate oxidation by 29, 39, 60, and 89% in RG, SOL, EDL, and WG, respectively. Thus malonyl-CoA resistance, which correlated strongly (0.678) with absolute oxidation rates (RG > SOL > EDL > WG), was greater in red than in white muscles. Similarly, malonyl-CoA-resistant palmitate oxidation and CPT I activity were greater in mitochondria from RG compared with WG. Ribonuclease protection assays were performed to evaluate whether our data might be explained by differential expression of CPT I splice variants. We detected the presence of two CPT Ibeta splice variants that were more abundant in red compared with white muscle, but the relative expression of the two mRNA species was unrelated to malonyl-CoA resistance. These results provide evidence of a malonyl-CoA-insensitive CPT I activity in red muscle, suggesting fiber type-specific expression of distinct CPT I isoforms and/or posttranslational modulations that have yet to be elucidated.     

Regulation of CPT I activity in intermyofibrillar and subsarcolemmal mitochondria from human and rat skeletal muscle

Carnitine palmitoyltransferase I (CPT I) is considered the rate-limiting enzyme in the transfer of long-chain fatty acids (LCFA) into the mitochondria and is reversibly inhibited by malonyl-CoA (M-CoA) in vitro. In rat skeletal muscle, M-CoA levels decrease during exercise, releasing the inhibition of CPT I and increasing LCFA oxidation. However, in human skeletal muscle, M-CoA levels do not change during moderate-intensity exercise despite large increases in fat oxidation, suggesting that M-CoA is not the sole regulator of increased CPT I activity during exercise. In the present study, we measured CPT I activity in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria isolated from human vastus lateralis (VL), rat soleus (Sol), and red gastrocnemius (RG) muscles. We tested whether exercise-related levels ( approximately 65% maximal O2 uptake) of calcium and adenylate charge metabolites (free AMP, ADP, and Pi) could override the M-CoA-induced inhibition of CPT I activity and explain the increased CPT I flux during exercise. Protein content was approximately 25-40% higher in IMF than in SS mitochondria in all muscles. Maximal CPT I activity was similar in IMF and SS mitochondria in all muscles (VL: 282 +/- 46 vs. 280 +/- 51; Sol: 390 +/- 81 vs. 368 +/- 82; RG: 252 +/- 71 vs. 278 +/- 44 nmol.min-1.mg protein-1). Sensitivity to M-CoA did not differ between IMF and SS mitochondria in all muscles (25-31% inhibition in VL, 52-70% in Sol and RG). Calcium and adenylate charge metabolites did not override the M-CoA-induced inhibition of CPT I activity in mitochondria isolated from VL, Sol, and RG muscles. Decreasing pH from 7.1 to 6.8 reduced CPT I activity by approximately 34-40% in both VL mitochondrial fractions. In summary, this study reports no differences in CPT I activity or sensitivity to M-CoA between IMF and SS mitochondria isolated from human and rat skeletal muscles. Exercise-induced increases in calcium and adenylate charge metabolites do not appear responsible for upregulating CPT I activity in human or rat skeletal muscle during moderate aerobic exercise.     

Subsarcolemmal and intermyofibrillar mitochondria play distinct roles in regulating skeletal muscle fatty acid metabolism

Skeletal muscle contains two populations of mitochondria that appear to be differentially affected by disease and exercise training. It remains unclear how these mitochondrial subpopulations contribute to fiber type-related and/or training-induced changes in fatty acid oxidation and regulation of carnitine palmitoyltransferase-1 (CPT1), the enzyme that controls mitochondrial fatty acid uptake in skeletal muscle. To this end, we found that fatty acid oxidation rates were 8.9-fold higher in subsarcolemmal mitochondria (SS) and 5.3-fold higher in intermyofibrillar mitochondria (IMF) that were isolated from red gastrocnemius (RG) compared with white gastrocnemius (WG) muscle, respectively. Malonyl-CoA (10 µM), a potent inhibitor of CPT1, completely abolished fatty acid oxidation in SS and IMF mitochondria from WG, whereas oxidation rates in the corresponding fractions from RG were inhibited only 89% and 60%, respectively. Endurance training also elicited mitochondrial adaptations that resulted in enhanced fatty acid oxidation capacity. Ten weeks of treadmill running differentially increased palmitate oxidation rates 100% and 46% in SS and IMF mitochondria, respectively. In SS mitochondria, elevated fatty acid oxidation rates were accompanied by a 48% increase in citrate synthase activity but no change in CPT1 activity. Nonlinear regression analyses of mitochondrial fatty acid oxidation rates in the presence of 0–100 µM malonyl-CoA indicated that IC₅₀ values were neither dependent on mitochondrial subpopulation nor affected by exercise training. However, in IMF mitochondria, training reduced the Hill coefficient (P < 0.05), suggesting altered CPT1 kinetics. These results demonstrate that endurance exercise provokes subpopulation-specific changes in mitochondrial function that are characterized by enhanced fatty acid oxidation and modified CPT1-malonyl-CoA dynamics. endurance exercise training; CPT-1; fiber type; rat; mitochondrial subpopulations     

Intestinal absorption, blood transport and hepatic and muscle metabolism of fatty acids in preruminant and ruminant animals

Current research on lipid metabolism in ruminants aims to improve the growth and health of the animals and the muscle characteristics associated with meat quality. This review, therefore, focuses on fatty acid (FA) metabolism from absorption to partitioning between tissues and metabolic pathways. In young calves, which were given high-fat milk diets, lipid absorption is delayed because the coagulation of milk caseins results in the retention of dietary fat as an insoluble clot in the abomasum. After weaning, the calves were fed forage- and cereal-based diets containing low levels of long-chain fatty acids (LCFA) but leading to high levels of volatile fatty acid (VFA) production by the rumen microflora. Such differences in dietary FA affect: i) the lipid transport system via the production of lipoproteins by the intestine and the liver, and (ii) the subsequent metabolism of lipids and FA by tissues. In preruminant calves, high-fat feed stimulates the secretion of triacylglycerols (TG)-rich lipoproteins (chylomicrons, very-low density lipoproteins (VLDL)). Diets rich in polyunsaturated FA (PUFA) stimulate the production of chylomicrons by the intestine (at peak lipid absorption) and of high density lipoproteins by the liver, leading to high blood concentrations of cholesterol. High levels of non-esterified FA (NEFA) uptake by the liver in high-yielding dairy cows in early lactation leads to TG infiltration of the hepatocytes (fatty liver). This is due to the low chronic capacity of the liver to synthesise and secrete VLDL particles. This abnormality in hepatic FA metabolism involves defects in apolipoprotein B synthesis and low availability of apolipoproteins and lipids for VLDL packaging. Fatty liver in calves is also caused by milk containing either soybean oil (rich in n-6 PUFA), or coconut oil (rich in C12:0 and C14:0). The ability of muscle tissue to use FA as an energy source depends on its mitochondrial content and, hence, on many physiological factors. The uptake and partitioning of LCFA between oxidation and storage in muscle is regulated by the activity of key intracellular enzymes and binding proteins. One such protein, carnitine palmitoyltransferase I (CPT I) controls the transport of LCFA into mitochondria. Metabolites derived from LCFA inhibit glucose oxidation, decrease the activity of CPT I and decrease the efficiency of ATP production by mitochondria. Most research on tissue lipid metabolism in ruminants is focused on: i) the partitioning of FA oxidation between intracellular peroxisomes and mitochondria in the liver and in muscles; (ii) the regulation of lipid metabolism by leptin, a recently discovered hormone secreted by mature adipocytes; and iii) the effects of activation of the nuclear receptors (PPARs and RXR) by LCFA or by phytol metabolites derived from chlorophyll.     

Overexpression of carnitine palmitoyltransferase I in skeletal muscle in vivo increases fatty acid oxidation and reduces triacylglycerol esterification

A key regulatory point in the control of fatty acid (FA) oxidation is thought to be transport of FAs across the mitochondrial membrane by carnitine palmitoyltransferase I (CPT I). To investigate the role of CPT I in FA metabolism, we used in vivo electrotransfer (IVE) to locally overexpress CPT I in muscle of rodents. A vector expressing the human muscle isoform of CPT I was electrotransferred into the right lateral muscles of the distal hindlimb [tibialis cranialis (TC) and extensor digitorum longus (EDL)] of rats, and a control vector expressing GFP was electrotransferred into the left muscles. Initial studies showed that CPT I protein expression peaked 7 days after IVE (+104%, P<0.01). This was associated with an increase in maximal CPT I activity (+30%, P < 0.001) and a similar increase in palmitoyl-CoA oxidation (+24%; P<0.001) in isolated mitochondria from the TC. Importantly, oxidation of the medium-chain FA octanoyl-CoA and CPT I sensitivity to inhibition by malonyl-CoA were not altered by CPT I overexpression. FA oxidation in isolated EDL muscle strips was increased with CPT I overexpression (+28%, P<0.01), whereas FA incorporation into the muscle triacylglycerol (TAG) pool was reduced (-17%, P<0.01). As a result, intramyocellular TAG content was decreased with CPT I overexpression in both the TC (-25%, P<0.05) and the EDL (-45%, P<0.05). These studies demonstrate that acute overexpression of CPT I in muscle leads to a repartitioning of FAs away from esterification and toward oxidation and highlight the importance of CPT I in regulating muscle FA metabolism.     

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) 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 muM) palmitate oxidation was decreased by 95% (P<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<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.     

Hyperthyroidism facilitates cardiac fatty acid oxidation through altered regulation of cardiac carnitine palmitoyltransferase: studies in vivo and with cardiac myocytes.

The aim was to establish whether increased cardiac fatty acid oxidation in hyperthyroidism is due to direct alterations in cardiac metabolism which favour fatty acid oxidation and/or whether normal regulatory links between changes in glucose supply and fatty acid oxidation are dysfunctional. Euthyroid rats were sampled in the absorptive state or after 48 h starvation. Rats were rendered hyperthyroid by injection of tri-iodothyronine (1000 microg/kg body wt. per day; 3 days). We evaluated the regulatory significance of direct effects of hyperthyroidism by measuring rates of palmitate oxidation in the absence or presence of glucose using cardiac myocytes. The results were examined in relation to the activity/regulatory characteristics of cardiac carnitine palmitoyltransferase (CPT) estimated by measuring rates of [3H]palmitoylcarnitine formation from [3H]carnitine and palmitoyl-CoA by isolated mitochondria. To define the involvement of other hormones, we examined whether hyperthyroidism altered basal or agonist-stimulated cardiac cAMP concentrations in cardiac myocytes and whether the effects of hyperthyroidism could be reversed by 24 h exposure to insulin infused subcutaneously (2 i. u. per day; Alzet osmotic pumps). Rates of 14C-palmitate oxidation (to 14CO2) by cardiac myocytes were significantly increased (1.6 fold; P< 0.05) by hyperthyroidism, whereas the percentage suppression of palmitate oxidation by glucose was greatly diminished. Cardiac CPT activities in mitochondria from hyperthyroid rats were 2-fold higher and the susceptibility of cardiac CPT activity to inhibition by malonyl-CoA was decreased. These effects were not mimicked by 48 h starvation. The decreased susceptibility of cardiac CPT activities to malonyl-CoA inhibition in hyperthyroid rats was normalised by 24 h exposure to elevated insulin concentration. Acute insulin addition did not influence the response to glucose in cardiac myocytes from euthyroid or hyperthyroid rats and basal and agonist-stimulated cAMP concentrations were unaffected by hyperthyroidism in vivo. The data provide insight into possible mechanisms by which hyperthyroidism facilitates fatty acid oxidation by the myocardium, identifying changes in cardiac CPT activity and malonyl-CoA sensitivity that would be predicted to render cardiac fatty acid oxidation less sensitive to external factors influencing malonyl-CoA content, and thereby to favour fatty acid oxidation. The increased CPT activity observed in response to hyperthyroidism may be a consequence of an impaired action of insulin but occurs through a cAMP-independent mechanism.     

Interrelation between long-chain fatty acid oxidation rate and carnitine palmitoyltransferase 1 activity with different isoforms in rat tissues

This study examined the interrelation between the long-chain fatty acid (LCFA) oxidation rate and the carnitine palmitoyltransferase (CPT) 1 activity in various tissues containing L-CPT1 or M-CPT1. The Liver, kidney, heart, white and red gastrocnemius muscles, and white and brown adipose tissues obtained from Sprague-Dawley rats were examined. In the tissues containing L-CPT1 the liver showed a significantly higher (P<0.01) palmitate oxidation rate and CPT1 activity than the kidney. Among the tissues containing M-CPT1, the brown adipose tissue showed the highest palmitate oxidation rate and CPT1 activity. The tissues containing M-CPT1 (r2=0.907, p<0.001) showed a strong positive correlation between the palmitate oxidation rate and the CPT1 activity. The ratios of the palmitate oxidation rate to the CPT1 activity were calculated. The ratio in the liver was highest and the ratio in the kidney was lowest among the tissues. The ratios of the tissues containing M-CPT1 were similar. These results showed that the LCFA oxidation rates in the tissues containing M-CPT1 were directly proportional to the CPT1 activity, but not similarly proportional to the CPT1 activity in the tissues containing L-CPT1. In conclusion, CPT1 activity seems very important factor for LCFA oxidation, but it might be not the only rate-limiting step in LCFA oxidation.     

Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for β-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.     

Regulation of carnitine palmitoyltransferase (CPT) I during fasting in rainbow trout (Oncorhynchus mykiss) promotes increased mitochondrial fatty acid oxidation

Periods of fasting, in most animals, are fueled principally by fatty acids, and changes in the regulation of fatty acid oxidation must exist to meet this change in metabolic substrate use. We examined the regulation of carnitine palmitoyltransferase (CPT) I, to help explain changes in mitochondrial fatty acid oxidation with fasting. After fasting rainbow trout (Oncorhynchus mykiss) for 5 wk, the mitochondria were isolated from red muscle and liver to determine (1) mitochondrial fatty acid oxidation rate, (2) CPT I activity and the concentration of malonyl-CoA needed to inhibit this activity by 50% (IC(50)), (3) mitochondrial membrane fluidity, and (4) CPT I (all five known isoforms) and peroxisome proliferator-activated receptor (PPARα and PPARβ) mRNA levels. Fatty acid oxidation in isolated mitochondria increased during fasting by 2.5- and 1.75-fold in liver and red muscle, respectively. Fasting also decreased sensitivity of CPT I to malonyl-CoA (increased IC(50)), by two and eight times in red muscle and liver, respectively, suggesting it facilitates the rate of fatty acid oxidation. In the liver, there was also a significant increase CPT I activity per milligram mitochondrial protein and in whole-tissue PPARα and PPARβ mRNA levels. However, there were no changes in mitochondrial membrane fluidity in either tissue, indicating that the decrease in CPT I sensitivity to malonyl-CoA is not due to bulk fluidity changes in the membrane. However, there were significant differences in CPT I mRNA levels during fasting. Overall, these data indicate some important changes in the regulation of CPT I that promote the increased mitochondrial fatty acid oxidation that occurs during fasting in trout.     

Peroxisomal-mitochondrial oxidation in a rodent model of obesity-associated insulin resistance

Peroxisomal oxidation yields metabolites that are more efficiently utilized by mitochondria. This is of potential clinical importance because reduced fatty acid oxidation is suspected to promote excess lipid accumulation in obesity-associated insulin resistance. Our purpose was to assess peroxisomal contributions to mitochondrial oxidation in mixed gastrocnemius (MG), liver, and left ventricle (LV) homogenates from lean and fatty (fa/fa) Zucker rats. Results indicate that complete mitochondrial oxidation (CO(2) production) using various lipid substrates was increased approximately twofold in MG, unaltered in LV, and diminished approximately 50% in liver of fa/fa rats. In isolated mitochondria, malonyl-CoA inhibited CO(2) production from palmitate 78%, whereas adding isolated peroxisomes reduced inhibition to 21%. These data demonstrate that peroxisomal products may enter mitochondria independently of CPT I, thus providing a route to maintain lipid disposal under conditions where malonyl-CoA levels are elevated, such as in insulin-resistant tissues. Peroxisomal metabolism of lignoceric acid in fa/fa rats was elevated in both liver and MG (LV unaltered), but peroxisomal product distribution varied. A threefold elevation in incomplete oxidation was solely responsible for increased hepatic peroxisomal oxidation (CO(2) unaltered). Alternatively, only CO(2) was detected in MG, indicating that peroxisomal products were exclusively partitioned to mitochondria for complete lipid disposal. These data suggest tissue-specific destinations for peroxisome-derived products and emphasize a potential role for peroxisomes in skeletal muscle lipid metabolism in the obese, insulin-resistant state.     

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.     

Metabolic challenges reveal impaired fatty acid metabolism and translocation of FAT/CD36 but not FABPpm in obese Zucker rat muscle

We examined, in muscle of lean and obese Zucker rats, basal, insulin-induced, and contraction-induced fatty acid transporter translocation and fatty acid uptake, esterification, and oxidation. In lean rats, insulin and contraction induced the translocation of the fatty acid transporter FAT/CD36 (43 and 41%, respectively) and plasma membrane-associated fatty acid binding protein (FABPpm; 19 and 60%) and increased fatty acid uptake (63 and 40%, respectively). Insulin and contraction increased lean muscle palmitate esterification and oxidation 72 and 61%, respectively. In obese rat muscle, basal levels of sarcolemmal FAT/CD36 (+33%) and FABPpm (+14%) and fatty acid uptake (+30%) and esterification (+32%) were increased, whereas fatty acid oxidation was reduced (-28%). Insulin stimulation of obese rat muscle increased plasmalemmal FABPpm (+15%) but not plasmalemmal FAT/CD36, blunted fatty acid uptake and esterification, and failed to reduce fatty acid oxidation. In contracting obese rat muscle, the increases in fatty acid uptake and esterification and FABPpm translocation were normal, but FAT/CD36 translocation was impaired and fatty acid oxidation was blunted. There was no relationship between plasmalemmal fatty acid transporters and palmitate partitioning. In conclusion, fatty acid metabolism is impaired at several levels in muscles of obese Zucker rats; specifically, they are 1) insulin resistant with respect to FAT/CD36 translocation and fatty acid uptake, esterification, and oxidation and 2) contraction resistant with respect to fatty acid oxidation and FAT/CD36 translocation, but, conversely, 3) obese muscles are neither insulin nor contraction resistant at the level of FABPpm. Finally, 4) there is no evidence that plasmalemmal fatty acid transporters contribute to the channeling of fatty acids to specific metabolic destinations within the muscle.     

Stearoyl-CoA desaturase-1 deficiency reduces ceramide synthesis by downregulating serine palmitoyltransferase and increasing beta-oxidation in skeletal muscle

Stearoyl-CoA desaturase (SCD) has recently been shown to be a critical control point of lipid partitioning and body weight regulation. Lack of SCD1 function significantly increases insulin sensitivity in skeletal muscles and corrects the hypometabolic phenotype of leptin-deficient ob/ob mice, indicating the direct antilipotoxic action of SCD1 deficiency. The mechanism underlying the metabolic effects of SCD1 mutation is currently unknown. Here we show that SCD1 deficiency reduced the total ceramide content in oxidative skeletal muscles (soleus and red gastrocnemius) by approximately 40%. The mRNA levels and activity of serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, as well as the incorporation of [14C]palmitate into ceramide were decreased by approximately 50% in red muscles of SCD1-/- mice. The content of fatty acyl-CoAs, which contribute to de novo ceramide synthesis, was also reduced. The activity and mRNA levels of carnitine palmitoyltransferase I (CPT I) and the rate of beta-oxidation were increased in oxidative muscles of SCD1-/- mice. Furthermore, SCD1 deficiency increased phosphorylation of AMP-activated protein kinase (AMPK), suggesting that AMPK activation may be partially responsible for the increased fatty acid oxidation and decreased ceramide synthesis in red muscles of SCD1-/- mice. SCD1 deficiency also reduced SPT activity and ceramide content and increased AMPK phosphorylation and CPT I activity in muscles of ob/ob mice. Taken together, these results indicate that SCD1 deficiency reduces ceramide synthesis by decreasing SPT expression and increasing the rate of beta-oxidation in oxidative muscles.     

Novel effect of C75 on carnitine palmitoyltransferase I activity and palmitate oxidation

C75 is a potential drug for the treatment of obesity. It was first identified as a competitive, irreversible inhibitor of fatty acid synthase (FAS). It has also been described as a malonyl-CoA analogue that antagonizes the allosteric inhibitory effect of malonyl-CoA on carnitine palmitoyltransferase I (CPT I), the main regulatory enzyme involved in fatty acid oxidation. On the basis of MALDI-TOF analysis, we now provide evidence that C75 can be transformed to its C75-CoA derivative. Unlike the activation produced by C75, the CoA derivative is a potent competitive inhibitor that binds tightly but reversibly to CPT I. IC50 values for yeast-overexpressed L- or M-CPT I isoforms, as well as for purified mitochondria from rat liver and muscle, were within the same range as those observed for etomoxiryl-CoA, a potent inhibitor of CPT I. When a pancreatic INS(823/13), muscle L6E9, or kidney HEK293 cell line was incubated directly with C75, fatty acid oxidation was inhibited. This suggests that C75 could be transformed in the cell to its C75-CoA derivative, inhibiting CPT I activity and consequently fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 in mice produced short-term inhibition of CPT I activity in mitochondria from the liver, soleus, and pancreas, indicating that C75 could be transformed to its C75-CoA derivative in these tissues. Finally, in silico molecular docking studies showed that C75-CoA occupies the same pocket in CPT I as palmitoyl-CoA, suggesting an inhibiting mechanism based on mutual exclusion. Overall, our results describe a novel role for C75 in CPT I activity, highlighting the inhibitory effect of its C75-CoA derivative.     

A novel function for fatty acid translocase (FAT)/CD36: involvement in long chain fatty acid transfer into the mitochondria

Fatty acid translocase (FAT)/CD36 is a long chain fatty acid transporter present at the plasma membrane, as well as in intracellular pools of skeletal muscle. In this study, we assessed the unexpected presence of FAT/CD36 in both subsarcolemmal and intermyofibril fractions of highly purified mitochondria. Functional assessments demonstrated that the mitochondria could bind (14)C-labeled palmitate, but could only oxidize it in the presence of carnitine. However, the addition of sulfo-N-succinimidyl oleate, a known inhibitor of FAT/CD36, resulted in an 87 and 85% reduction of palmitate oxidation in subsarcolemmal and intermyofibril fractions, respectively. Further studies revealed that maximal carnitine palmitoyltransferase I (CPTI) activity in vitro was inhibited by succinimidyl oleate (42 and 48% reduction). Interestingly, CPTI immunoprecipitated with FAT/CD36, indicating a physical pairing. Tissue differences in mitochondrial FAT/CD36 protein follow the same pattern as the capacity for fatty acid oxidation (heart > red muscle > white muscle). Additionally, chronic stimulation of hindlimb muscles (7 days) increased FAT/CD36 expression and also resulted in a concomitant increase in mitochondrial FAT/CD36 content (46 and 47% increase). Interestingly, with acute electrical stimulation of hindlimb muscles (30 min), FAT/CD36 expression was not altered, but there was an increase in the mitochondrial content of FAT/CD36 compared with the non-stimulated control limb (35 and 37% increase). Together, these data suggest a role for FAT/CD36 in mitochondrial long chain fatty acid uptake and demonstrate system flexibility to match FAT/CD36 mitochondrial content with an increased capacity for fatty acid oxidation, possibly involving translocation of FAT/CD36 to the mitochondria.     

Endurance training increases FFA oxidation and reduces triacylglycerol utilization in contracting rat soleus

We examined the effects of 8 wk of intense endurance training on free fatty acid (FFA) transporters and metabolism in resting and contracting soleus muscle using pulse-chase procedures. Endurance training increased maximal citrate synthase activity in red muscles (+54 to +91%; P 相似文献   

Direct effects of fructose metabolism on fatty acid oxidation in a recombined rat liver mitochondria-hish speed supernatant system.

The effects of fructose on the oxidation of [1-(14)C]palmitate in a rat liver mitochondria-high speed supernatant system have been investigated. This model system permitted study of the direct effects of fructose and the metabolism of fructose on fatty acid oxidation in the near absence of fatty acid esterification. Fructose inhibited the utilization of albumin-bound [1-(14)C] palmitate in the mitochondria-supernatant system, but did not affect fatty acid utilization by isolated liver mitochondria. Although fructose decreased the ATP content in the mitochondrial-supernatant system, the level of ATP throughout the incubation period was sufficient for maximal fatty acid activation. Fructose decreased the conversion of [1-(14)C]palmitate to 14CO2 and depressed the formation of total labeled oxidation products (14CO2 + 14C-labeled ketone bodies) in this system. The results suggest that fructose metabolism inhibited fatty acid oxidation in the mitochondria-supernatant system by competitive substrate oxidation and thereby decreased utilization of the added [1-(14)C]palmitate. The ihibition of L-[L-(14)C]palmitoylcarnitine oxidation, fructose was in all respects similar to its inhibition of palmitate oxidation, indicating that the site of fructose interaction was within the beta-oxidation sequence. These observations support the concept (Ontko, J.A. [1972] J. Biol. Chem. 247, 1788-1800) that the reciprocal changes in esterification and oxidation of palmitate caused by fructose in liver cells are primarily mediated via inhibitory effects on long-chain fatty acid oxidation.     

Fatty acid oxidation and related gene expression in heart depleted of carnitine by mildronate treatment in the rat

The metabolic and genic effects induced by a 20-fold lowering of carnitine content in the heart were studied in mildronate-treated rats. In the perfused heart, the proportion of palmitate taken up then oxidized was 5-10% lower, while the triacylglycerol (TAG) formation was 100% greater than in controls. The treatment was shown to increase the maximal capacity of heart homogenates to oxidize palmitate, the mRNA level of carnitine palmitoyltransferase I (CPT-I) isoforms, the specific activity of CPT-I in subsarcolemmal mitochondria and the total carnitine content of isolated mitochondria. Concomitantly, the increased mRNA expression of lipoprotein lipase, fatty acid translocase and enzymes of TAG synthesis was associated with a 5- and 2-times increase in serum TAG and free fatty acid contents, respectively. The compartmentation of carnitine at its main functional location was expected to allow the increased CPT-I activity to ensure in vivo correct fatty acid oxidation rates. All the inductions related to fatty acid transport, oxidation and esterification most likely stem from the abundance of blood lipids providing cardiomyocytes with more fatty acids.