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...

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation in humans. To better understand the pathogenesis of this disease, we developed a mouse model for MCAD deficiency (MCAD−/−) by gene targeting in embryonic stem (ES) cells. The MCAD−/− mice developed an organic aciduria and fatty liver, and showed profound cold intolerance at 4 °C with prior fasting. The sporadic cardiac lesions seen in MCAD−/− mice have not been reported in human MCAD patients. There was significant neonatal mortality of MCAD−/− pups demonstrating similarities to patterns of clinical episodes and mortality in MCAD-deficient patients. The MCAD-deficient mouse reproduced important aspects of human MCAD deficiency and is a valuable model for further analysis of the roles of fatty acid oxidation and pathogenesis of human diseases involving fatty acid oxidation.

In the present paper, we describe a novel method which enables the analysis of tissue acylcarnitines and carnitine biosynthesis intermediates in the same sample. This method was used to investigate the carnitine and fatty acid metabolism in wild-type and LCAD-/- (long-chain acyl-CoA dehydrogenase-deficient) mice. In agreement with previous results in plasma and bile, we found accumulation of the characteristic C14:1-acylcarnitine in all investigated tissues from LCAD-/- mice. Surprisingly, quantitatively relevant levels of 3-hydroxyacylcarnitines were found to be present in heart, muscle and brain in wild-type mice, suggesting that, in these tissues, long-chain 3-hydroxyacyl-CoA dehydrogenase is rate-limiting for mitochondrial beta-oxidation. The 3-hydroxyacylcarnitines were absent in LCAD-/- tissues, indicating that, in this situation, the beta-oxidation flux is limited by the LCAD deficiency. A profound deficiency of acetylcarnitine was observed in LCAD-/- hearts, which most likely corresponds with low cardiac levels of acetyl-CoA. Since there was no carnitine deficiency and only a marginal elevation of potentially cardiotoxic acylcarnitines, we conclude from these data that the cardiomyopathy in the LCAD-/- mouse is caused primarily by a severe energy deficiency in the heart, stressing the important role of LCAD in cardiac fatty acid metabolism in the mouse.

Mitochondrial fatty acid oxidation provides an important energy source for cellular metabolism and decreased mitochondrial fatty acid oxidation has been implicated in the pathogenesis of type 2 diabetes. Paradoxically, mice with an inherited deficiency of the mitochondrial fatty acid oxidation enzyme, very long chain acyl-CoA dehydrogenase (VLCAD), manifested increased fatty acid oxidation in liver, muscle and brown adipose tissue and a lower whole body respiratory quotient compared to WT mice. Moreover, VLCAD−/− mice were protected from fat-induced liver and muscle insulin resistance, which was associated with reduced intracellular diacylglycerol content and decreased activity of protein kinase Cε and protein kinase Cθ in liver and muscle respectively. The increased insulin sensitivity in the VLCAD−/− mice was associated with increased liver and muscle AMPK activity and increased PPARα expression in muscle and brown adipose tissue. Taken together these data suggest that VLCAD−/− mice were protected from diet-induced obesity and insulin resistance due to chronic activation of AMPK (liver and muscle) and PPARα (muscle and BAT) activity resulting in increased fatty acid oxidation and decreased intramyocellular and hepatocellular diacylglycerol content. Furthermore these data demonstrate that mitochondrial dysfunction can paradoxically result in increased insulin sensitivity due to these compensatory mechanisms.

To better understand carnitine palmitoyltransferase 1a (liver isoform, gene=Cpt-1a, protein=CPT-1a) deficiency in human disease, we developed a gene knockout mouse model. We used a replacement gene targeting strategy in ES cells that resulted in the deletion of exons 11-18, thus producing a null allele. Homozygous deficient mice (CPT-1a -/-) were not viable. There were no CPT-1a -/- pups, embryos or fetuses detected from day 10 of gestation to term. FISH analysis demonstrated targeting vector recombination at the expected single locus on chromosome 19. The inheritance pattern from heterozygous matings was skewed in both C57BL/6NTac, 129S6/SvEvTac (B6;129 mixed) and 129S6/SvEvTac (129 coisogenic) genetic backgrounds biased toward CPT-1a +/- mice (>80%). There was no sex preference with regard to germ-line transmission of the mutant allele. CPT-1a +/- mice had decreased Cpt-1a mRNA expression in liver, heart, brain, testis, kidney, and white fat. This resulted in 54.7% CPT-1 activity in liver from CPT-1a +/- males but no significant difference in females as compared to CPT-1a +/+ controls. CPT-1a +/- mice showed no fatty change in liver and were cold tolerant. Fasting free fatty acid concentrations were significantly elevated, while blood glucose concentrations were significantly lower in 6-week-old CPT-1a +/- mice compared to controls. Although the homozygous mutants were not viable, we did find some aspects of haploinsufficiency in the CPT-1a +/- mutants, which will make them an important mouse model for studying the role of CPT-1a in human disease.

Cardiac hypertrophy is a common finding in human patients with inborn errors of long-chain fatty acid oxidation. Mice with either very long-chain acyl-CoA dehydrogenase deficiency (VLCAD−/−) or long-chain acyl-CoA dehydrogenase deficiency (LCAD−/−) develop cardiac hypertrophy. Cardiac hypertrophy, initially measured using heart/body weight ratios, was manifested most severely in LCAD−/− male mice. VLCAD−/− mice, as a group, showed a mild increase in normalized cardiac mass (8.8% hypertrophy compared to all wild-type [WT] mice). In contrast, LCAD−/− mice as a group showed more severe cardiac hypertrophy (32.2% increase compared to all WT mice). Based on a clear male predilection, we investigated the role of dietary plant estrogenic compounds commonly found in mouse diets due to soy or alfalfa components providing natural phytoestrogens or isoflavones in cardioprotection of LCAD−/− mice. Male LCAD−/− mice fed an isoflavone-free test diet had more severe cardiac hypertrophy (58.1% hypertrophy compared to WT mice fed the same diet. There were no significant differences in the female groups fed any of the diets. Echocardiography measurement performed on male LCAD deficient mice fed a standard diet at ~3 months of age confirmed the substantial cardiac hypertrophy in these mice compared with WT controls. Left ventricular wall thickness of interventricular septum and posterior wall was remarkably increased in LCAD−/− mice compared with that of WT controls. Accordingly, the calculated LV mass after normalization to body weight was increased about 40% in the LCAD−/− mice compared with WT mice. In summary, we found that metabolic cardiomyopathy, expressed as hypertrophy, developed in mice due to either VLCAD deficiency or LCAD deficiency; however, LCAD deficiency was the most profound and appeared to be attenuated either by endogenous estrogen in females or phytoestrogens in the diet as isoflavones in males.

We investigated the effects of the HIV protease inhibitor ritonavir on body composition, serum lipids, and gene expression in C57BL/6 mice. Dual-energy X-ray absorptiometry measurements in ritonavir-treated male mice revealed whole-body lipoatrophy. In female mice fat reduction was restricted to the gonadal depot. A histopathological analysis showed no visible abnormalities in liver or adipose tissue from ritonavir-treated mice, although adipocytes were significantly smaller in diameter. Serum triglyceride levels were increased in ritonavir-treated male mice. Ritonavir was coadministered with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist gemfibrozil and the PPARgamma agonist rosiglitazone for 8 weeks. Neither drug alleviated the hypertriglyceridemia or lipoatrophy in ritonavir-treated male mice. Rather, gemfibrozil exacerbated the lipoatrophy. Ritonavir reduced basal expression of two PPARalpha target genes in liver, as well as the PPARgamma target gene phosphoenolpyruvate carboxykinase (PEPCK) in adipose tissues. Ritonavir partially inhibited induction of PPAR target genes by gemfibrozil and rosiglitazone. Gemfibrozil induced expression of fatty acid oxidation genes in liver, and this induction was less substantial when ritonavir was coadministered. Similarly, rosiglitazone induced expression of uncoupling protein-1, uncoupling protein-2, and PEPCK in adipose tissues, and this effect was partially inhibited by ritonavir. Thus, the effects of ritonavir on serum triglycerides and body composition may be due, at least in part, to an inhibition of PPAR function.