Here we investigated the biological functions of adiponectin/ACRP30, a fat-derived hormone, by disrupting the gene that encodes it in mice. Adiponectin/ACRP30-knockout (KO) mice showed delayed clearance of free fatty acid in plasma, low levels of fatty-acid transport protein 1 (FATP-1) mRNA in muscle, high levels of tumor necrosis factor-alpha (TNF-alpha) mRNA in adipose tissue and high plasma TNF-alpha concentrations. The KO mice exhibited severe diet-induced insulin resistance with reduced insulin-receptor substrate 1 (IRS-1)-associated phosphatidylinositol 3 kinase (PI3-kinase) activity in muscle. Viral mediated adiponectin/ACRP30 expression in KO mice reversed the reduction of FATP-1 mRNA, the increase of adipose TNF-alpha mRNA and the diet-induced insulin resistance. In cultured myocytes, TNF-alpha decreased FATP-1 mRNA, IRS-1-associated PI3-kinase activity and glucose uptake, whereas adiponectin increased these parameters. Our results indicate that adiponectin/ACRP30 deficiency and high TNF-alpha levels in KO mice reduced muscle FATP-1 mRNA and IRS-1-mediated insulin signaling, resulting in severe diet-induced insulin resistance.

Peroxisome proliferator-activated receptor ␣ (PPAR␣) is a member of the steroid/nuclear receptor superfamily and mediates the biological and toxicological effects of peroxisome proliferators. To determine the physiological role of PPAR␣ in fatty acid metabolism, levels of peroxisomal and mitochondrial fatty acid metabolizing enzymes were determined in the PPAR␣ null mouse. Constitutive liver ␤-oxidation of the long chain fatty acid, palmitic acid, was lower in the PPAR␣ null mice as compared with wild type mice, indicating defective mitochondrial fatty acid catabolism. In contrast, constitutive oxidation of the very long chain fatty acid, lignoceric acid, was not different between wild type and PPAR␣ null mice, suggesting that constitutive expression of enzymes involved in peroxisomal ␤-oxidation is independent of PPAR␣. Indeed, the PPAR␣ null mice had normal levels of the peroxisomal acyl-CoA oxidase, bifunctional protein (hydratase ؉ 3-hydroxyacyl-CoA dehydrogenase), and thiolase but lower constitutive expression of the D-type bifunctional protein (hydratase ؉ 3-hydroxyacyl-CoA dehydrogenase). Several mitochondrial fatty acid metabolizing enzymes including very long chain acyl-CoA dehydrogenase, long chain acyl-CoA dehydrogenase, short chain-specific 3-ketoacyl-CoA thiolase, and long chain acylCoA synthetase are also expressed at lower levels in the untreated PPAR␣ null mice, whereas other fatty acid metabolizing enzymes were not different between the untreated null mice and wild type mice. A lower constitutive expression of mRNAs encoding these enzymes was also found, suggesting that the effect was due to altered gene expression. In wild type mice, both peroxisomal and mitochondrial enzymes were induced by the peroxisome proliferator Wy-14,643; induction was not observed in the PPAR␣ null animals. These data indicate that PPAR␣ modulates constitutive expression of genes encoding several mitochondrial fatty acid-catabolizing enzymes in addition to mediating inducible mitochondrial and peroxisomal fatty acid ␤-oxidation, thus establishing a role for the receptor in fatty acid homeostasis.

We have generated a transgenic mouse with no white fat tissue throughout life. These mice express a dominant-negative protein, termed A-ZIP/F, under the control of the adipose-specific aP2 enhancer/promoter. This protein prevents the DNA binding of B-ZIP transcription factors of both the C/EBP and Jun families. The transgenic mice (named A-ZIP/F-1) have no white adipose tissue and dramatically reduced amounts of brown adipose tissue, which is inactive. They are initially growth delayed, but by week 12, surpass their littermates in weight. The mice eat, drink, and urinate copiously, have decreased fecundity, premature death, and frequently die after anesthesia. The physiological consequences of having no white fat tissue are profound. White adipose tissue (WAT) is the major organ for regulated storage of triglycerides for use as metabolic energy. WAT helps control energy homeostasis, including food intake, metabolic efficiency, and energy expenditure, via its secreted hormone, leptin, and possibly additional unknown hormones. The quantity of body fat varies widely in mammals, ranging from 2% to >50% of body mass, typically from 10% to 20% in mice and humans. Much of this variability can be observed within a single individual, highlighting the delicate balance of factors controlling fat deposition. The huge variation in fat mass is unlike that of any other organ in the body and is determined by both an individual's genetic background and environmental factors including diet and physical activity (Comuzzie and Allison 1998; Hill and Peters 1998). Excess body fat, or obesity, is a major health problem, particularly in America, increasing the risk of diabetes, hypertension, and coronary artery disease (Thomas 1995). The mechanisms by which obesity causes these diseases, however, are unclear. To understand better the contribution of adipose tissue to diabetes and metabolism, it would be valuable to examine a mouse with no adipose tissue. To this end, we produced a transgenic mouse with essentially no white adipose tissue and examined the contribution of WAT to energy metabolism, reproductive function, and disease susceptibility.Mutant mice with either increased or decreased levels of WAT have been reported. For example, two mutations that disrupt signaling between WAT and the brain (ob/ ob and db/db, affecting leptin and its receptor, respectively) cause an increase in WAT amount leading to diabetes (Coleman 1978;Zhang et al. 1994; Chen et al. 1996). These mice have increased food intake and decreased physical and sympathetic nerve activity, all contributing to obesity. Adipose-specific expression of a diphtheria toxigene resulted in mice with either a severe phenotype including neonatal death or a mild phenotype, characterized by resistance to induced obesity or delayed loss of WAT at 10 months (Ross et al. 1993;Burant et al. 1997). These results suggest that WAT may be an essential organ for life. At present, there are no mice, from either knockout or transgene technologies that are devoid of WAT throughout develop...

Previous kinetic studies have identified a high-affinity (S)-warfarin 7-hydroxylase present in human liver microsomes which appears to be responsible for the termination of warfarin's biological activity. Inhibition of the formation of (S)-7-hydroxywarfarin, the inactive, major metabolite of racemic warfarin in humans, is known to be the cause of several of the drug interactions experienced clinically upon coadministration of warfarin with other therapeutic agents. In order to identify the specific form(s) of human liver cytochrome P-450 involved in this particular toxicity, we have determined the metabolic profiles of 11 human cytochrome P-450 forms expressed in HepG2 cells toward both (R)- and (S)-warfarin. Of the 11 forms examined only 2C9 displayed the regioselectivity and stereoselectivity appropriate for the high-affinity human liver microsomal (S)-7-hydroxylase. We further compared Michaelis-Menten and sulfaphenazole inhibition constants for (S)-warfarin 7-hydroxylation catalyzed by cDNA-expressed 2C9 and by human liver microsomes. Similar kinetic constants were obtained for each enzyme source. It is concluded that 2C9 is likely to be a principal form of human liver P-450 which modulates the in vivo anticoagulant activity of the drug. It is further concluded that those drug interactions with warfarin that arise as a result of decreased clearance of the biologically more potent S-enantiomer may have as their common basis the inhibition of P-450 2C9.

The peroxisome proliferator-activated receptor ␣ (PPAR␣) is a member of the nuclear receptor superfamily and mediates the biological effects of peroxisome proliferators. To determine the physiological role of PPAR␣ in cardiac fatty acid metabolism, we examined the regulation of expression of cardiac fatty acid-metabolizing proteins using PPAR␣-null mice. The capacity for constitutive myocardial ␤-oxidation of the medium and long chain fatty acids, octanoic acid and palmitic acid, was markedly reduced in the PPAR␣-null mice as compared with the wild-type mice, indicating that mitochondrial fatty acid catabolism is impaired in the absence of PPAR␣. In contrast, constitutive ␤-oxidation of the very long chain fatty acid, lignoceric acid, did not differ between the mice, suggesting that the constitutive expression of enzymes involved in peroxisomal ␤-oxidation is independent of PPAR␣ . Indeed, PPAR␣-null mice had normal levels of the peroxisomal ␤-oxidation enzymes except the D-type bifunctional protein. At least seven mitochondrial fatty acid-metabolizing enzymes were expressed at much lower levels in the PPAR␣-null mice, whereas other fatty acid-metabolizing enzymes were present at similar or slightly lower levels in the PPAR␣-null, as compared with wild-type mice. Additionally, lower constitutive mRNA expression levels of fatty acid transporters were found in the PPAR␣-null mice, suggesting a role for PPAR␣ in fatty acid transport and catabolism. Indeed, in fatty acid metabolism experiments in vivo, myocardial uptake of iodophenyl 9-methylpentadecanoic acid and its conversion to 3-methylnonanoic acid were reduced in the PPAR␣-null mice. Interestingly, a decreased ATP concentration after exposure to stress, abnormal cristae of the mitochondria, abnormal caveolae, and fibrosis were observed only in the myocardium of the PPAR␣-null mice. These cardiac abnormalities appeared to proceed in an age-dependent manner. Taken together, the results presented here indicate that PPAR␣ controls constitutive fatty acid oxidation, thus establishing a role for the receptor in cardiac fatty acid homeostasis. Furthermore, altered expression of fatty acid-metabolizing proteins seems to lead to myocardial damage and fibrosis, as inflammation and abnormal cell growth control can cause these conditions.