No abstract

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD ؉͞؊ mice yielded an abnormally low number of LCAD ؉͞؊ and ؊͞؊ offspring, indicating frequent gestational loss. LCAD ؊͞؊ mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD ؊͞؊ males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD ؊͞؊ mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.Mitochondrial fatty acid oxidation (FAO) is the primary means by which energy is derived from metabolism of fatty acids. This process is important during periods of fasting or prolonged strenuous activity, providing as much as 80 to 90% of fatty acid-derived energy for heart and liver function (1). Mitochondrial FAO also provides acetyl-CoA for hepatic ketogenesis and the energy required for nonshivering thermogenesis by brown adipose tissue (2). The initial step in mitochondrial FAO is the ␣-␤ dehydrogenation of the acyl-CoA ester by a family of four closely related, chain length-specific enzymes, the acyl-CoA dehydrogenases, which include verylong-chain, long-chain, medium-chain, and short-chain acylCoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD, respectively). These enzymes catalyze the same type of reaction but differ in specificity according to the chain length of their fatty acid (acyl-CoA) substrates.

Alterations in mitochondrial function have been implicated in the pathogenesis of insulin resistance and type 2 diabetes. However, it is unclear whether the reduced mitochondrial function is a primary or acquired defect in this process. To determine whether primary defects in mitochondrial ␤-oxidation can cause insulin resistance, we studied mice with a deficiency of long-chain acylCoA dehydrogenase (LCAD), a key enzyme in mitochondrial fatty acid oxidation. Here, we show that LCAD knockout mice develop hepatic steatosis, which is associated with hepatic insulin resistance, as reflected by reduced insulin suppression of hepatic glucose production during a hyperinsulinemic-euglycemic clamp. The defects in insulin action were associated with an Ϸ40% reduction in insulin-stimulated insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity and an Ϸ50% decrease in Akt2 activation. These changes were associated with increased PKC activity and an aberrant 4-fold increase in diacylglycerol content after insulin stimulation. The increase in diacylglycerol concentration was found to be caused by de novo synthesis of diacylglycerol from medium-chain acyl-CoA after insulin stimulation. These data demonstrate that primary defects in mitochondrial fatty acid oxidation capacity can lead to diacylglycerol accumulation, PKC activation, and hepatic insulin resistance.diacylglycerol ͉ mitochondria ͉ nonalcoholic fatty liver disease ͉ PKC R ecent studies have implicated alterations in mitochondrial function in the pathogenesis of insulin resistance and type 2 diabetes mellitus (1-8). It has been proposed that decreased mitochondrial fatty acid oxidation can result in insulin resistance by promoting increased intracellular diacylglycerol content, which in turn leads to activation of novel PKCs in liver and skeletal muscle and decreased insulin signaling and action in these tissues (9). However, it remains to be determined whether reduced mitochondrial function plays a primary role in causing the insulin resistance or whether it is a result of the increase in intracellular lipid content or other acquired factors (6, 10). To address this question, we examined insulin action in liver and skeletal muscle, using the hyperinsulinemic-euglycemic clamp, in long-chain acyl-CoA dehydrogenase (LCAD)-deficient (LCAD Ϫ/Ϫ ) mice, a genetic model of defective fatty acid oxidation. LCAD is a mitochondrial matrix enzyme catalyzing the first step for the oxidation of long-chain fatty acyl-CoAs. LCAD Ϫ/Ϫ mice are known to have impaired fatty acid oxidation and develop a disease similar to other disorders of mitochondrial fatty acid oxidation (11-12). We also examined the impact of LCAD deficiency on whole-body glucose and fatty acid oxidation in these mice, using indirect calorimetry. Results Metabolic Profile of the LCAD ؊/؊ Mice. LCADϪ/Ϫ mice, fed a standard rodent diet, had similar body weights but a 60% increase in whole-body fat content compared with their WT littermates (Table 1). However, they ate 11% less of the standard r...

Background Carnitine palmitoyltransferase 1(CPT1) is a rate-limiting step of mitochondrial β-oxidation by controlling the mitochondrial uptake of long-chain acyl-CoAs. The muscle isoform, CPT1b, is the predominant isoform expressed in the heart. It has been suggested that inhibiting CPT-1 activity by specific CPT-1 inhibitors exerts protective effects against cardiac hypertrophy and heart failure. However, clinical and animal studies have shown mixed results, thereby posting concerns on the safety of this class of drugs. Preclinical studies using genetically modified animal models should provide a better understanding of targeting CPT1 in order to evaluate it as a safe and effective therapeutic approach. Methods and Results Heterozygous CPT1b knockout mice (CPT1b+/−) were subjected to transverse aorta constriction (TAC)-induced pressure-overload. These mice showed overtly normal cardiac structure/function under the basal condition. Under a severe pressure-overload condition induced by two weeks of transverse aorta constriction (TAC), CPT1b+/− mice were susceptible to premature death with congestive heart failure. Under a milder pressure-overload condition, CPT1b+/− mice exhibited exacerbated cardiac hypertrophy and remodeling compared with that in wild-type littermates. There were more pronounced impairments of cardiac contraction with greater eccentric cardiac hypertrophy in CPT1b+/− than in controlled mice. Moreover, the CPT1b+/− heart exhibited exacerbated mitochondrial abnormalities and myocardial lipid accumulation with elevated triglycerides and ceramide content, leading to greater cardiomyocytes apoptosis. Conclusions We conclude that CPT1b deficiency can cause lipotoxicity in the heart under pathological stress, leading to exacerbation of cardiac pathology. Therefore, caution should be applied in the clinical use of CPT-1 inhibitors.