Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate''s protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.     

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.     

Evidence for an impaired long-chain fatty acid oxidation and ketogenesis in Fao hepatoma cells.

Fatty acid metabolism has been studied in Fao rat hepatoma cells. In basal conditions of culture, [1-14C]oleate is mainly esterified (85% of oleate uptake) in Fao cells, phospholipids being the most important esterified products (60% of oleate esterified). Addition of N6,O2'-dibutyryl-adenosine 3',5'-monophosphate (0.1 mM) in Fao cells does not change the metabolic fate of oleate whereas it induces gluconeogenesis and phosphoenolpyruvate carboxykinase mRNA accumulation. It is shown that the limitation of oleate oxidation is located at the level of the entry into mitochondria since octanoate is actively oxidized in Fao cells. Neither the activities of carnitine palmitoyltransferase (CPT) I and II nor the CPT II protein amount are affected by cAMP addition. The limitation of oleate oxidation in Fao cells results from (a) a high rate of lipogenesis and a high malonyl-CoA concentration, (b) a CPT I very sensitive to malonyl-CoA inhibition. The presence of an active oleate oxidation in mitochondria isolated from Fao cells confirms that CPT I is the limiting step of oleate oxidation. Moreover, Fao cells are unable to perform ketogenesis. This particular feature results from a specific deficiency in mitochondrial hydroxymethylglutaryl-CoA synthase protein, activity and gene expression. The metabolic characteristics observed in Fao cells could be a common feature in hepatoma cell lines with regard to the low capacity for long-chain fatty acid oxidation and ketone body production observed in the rat H4IIE and the human HepG2 cells.     

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.     

Effects of carnitine palmitoyltransferases on cancer cellular senescence

The carnitine palmitoyltransferase (CPT) family is essential for fatty acid oxidation. Recently, we found that CPT1C, one of the CPT1 isoforms, plays a vital role in cancer cellular senescence. However, it is unclear whether other isoforms (CPT1A, CPT1B, and CPT2) have the same effect on cellular senescence. This study illustrates the different effects of CPT knockdown on PANC-1 cell proliferation and senescence and MDA-MB-231 cell proliferation and senescence, as demonstrated by cell cycle kinetics, Bromodeoxyuridine incorporation, senescence-associated β-galactosidase activity, colony formation, and messenger RNA (mRNA) expression of key senescence-associated secretory phenotype factors. CPT1C exhibits the most substantial effect on cell senescence. Lipidomics analysis was performed to further reveal that the knockdown of CPTs changed the contents of lipids involved in mitochondrial function, and lipid accumulation was induced. Moreover, the different effects of the isoform deficiencies on mitochondrial function were measured and compared by the level of radical oxygen species, mitochondrial transmembrane potential, and the respiratory capacity, and the expression of the genes involved in mitochondrial function were determined at the mRNA level. In summary, CPT1C exerts the most significant effect on mitochondrial dysfunction-associated tumor cellular senescence among the members of the CPT family, which further supports the crucial role of CPT1C in cellular senescence and suggests that inhibition of CPT1C may represent as a new strategy for cancer treatment through the induction of tumor senescence.     

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     

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.     

Aging results in paradoxical susceptibility of fat cell progenitors to lipotoxicity

Aging is associated with metabolic syndrome, tissue damage by cytotoxic lipids, and altered fatty acid handling. Fat tissue dysfunction may contribute to these processes. This could result, in part, from age-related changes in preadipocytes, since they give rise to new fat cells throughout life. To test this hypothesis, preadipocytes cultured from rats of different ages were exposed to oleic acid, the most abundant fatty acyl moiety in fat tissue and the diet. At fatty acid concentrations at which preadipocytes from young animals remained viable, cells from old animals accumulated lipid in multiple small lipid droplets and died, with increased apoptotic index, caspase activity, BAX, and p53. Rather than inducing apoptosis, oleic acid promoted adipogenesis in preadipocytes from young animals, with appearance of large lipid droplets. CCAAT/enhancer-binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) increased to a greater extent in cells from young than old animals after oleate exposure. Oleic acid, but not glucose, oxidation was impaired in preadipocytes and fat cells from old animals. Expression of carnitine palmitoyltransferase (CPT)-1, which catalyzes the rate-limiting step in fatty acid beta-oxidation, was not reduced in preadipocytes from old animals. At lower fatty acid levels, constitutively active CPT I expression enhanced beta-oxidation. At higher levels, CPT I was not as effective in enhancing beta-oxidation in preadipocytes from old as young animals, suggesting that mitochondrial dysfunction may contribute. Consistent with this, medium-chain acyl-CoA dehydrogenase expression was reduced in preadipocytes from old animals. Thus preadipocyte fatty acid handling changes with aging, with increased susceptibly to lipotoxicity and impaired fatty acid-induced adipogenesis and beta-oxidation.