Reddy, Janardan K., and M. Sambasiva Rao. Lipid Metabolism and Liver Inflammation. II. Fatty liver disease and fatty acid oxidation.

Peroxisome proliferator activated-receptor (PPAR) isoforms, ␣ and ␥, function as important coregulators of energy (lipid) homeostasis. PPAR␣ regulates fatty acid oxidation primarily in liver and to a lesser extent in adipose tissue, whereas PPAR␥ serves as a key regulator of adipocyte differentiation and lipid storage. Of the two PPAR␥ isoforms, PPAR␥1 and PPAR␥2 generated by alternative splicing, PPAR␥1 isoform is expressed in liver and other tissues, whereas PPAR␥2 isoform is expressed exclusively in adipose tissue where it regulates adipogenesis and lipogenesis. Since the function of PPAR␥1 in liver is not clear, we have, in this study, investigated the biological impact of overexpression of PPAR␥1 in mouse liver. Adenovirus-PPAR␥1 injected into the tail vein induced hepatic steatosis in PPAR␣ ؊/؊ mice. Northern blotting and gene expression profiling results showed that adipocyte-specific genes and lipogenesis-related genes are highly induced in PPAR␣ ؊/؊ livers with PPAR␥1 overexpression. These include adipsin, adiponectin, aP2, caveolin-1, fasting-induced adipose factor, fat-specific gene 27 (FSP27), CD36, ⌬ 9 desaturase, and malic enzyme among others, implying adipogenic transformation of hepatocytes. Of interest is that hepatic steatosis per se, induced either by feeding a diet deficient in choline or developing in fasted PPAR␣ ؊/؊ mice, failed to induce the expression of these PPAR␥-regulated adipogenesis-related genes in steatotic liver. These results suggest that a high level of PPAR␥ in mouse liver is sufficient for the induction of adipogenic transformation of hepatocytes with adipose tissue-specific gene expression and lipid accumulation. We conclude that excess PPAR␥ activity can lead to the development of a novel type of adipogenic hepatic steatosis.

To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR 'y (mPPARy) gene. This gene extends > 105 kb and gives rise to two mRNAs (mPPARy1 and mPPARy2) that differ at their 5' ends. The mPPARy2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPARyl and reveals a different 5' untranslated sequence.We show that mPPARy1 mRNA is encoded by eight exons, whereas the mPPARy2 mRNA is encoded by seven exons.Most of the 5' untranslated sequence of mPPARy1 mRNA is encoded by two exons, whereas the 5' untranslated sequence and the extra 30 N-terminal amino acids of mPPARy2 are encoded by one exon, which is located between the second and third exons coding for mPPARyl. The last six exons of mPPARy gene code for identical sequences in mPPARyl and mPPARy2 isoforms. The mPPARyl and mPPARy2 isoforms are transcribed from different promoters. The mPPARy gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase the diversity of ligand and tissue-specific transcriptional responses.

Fasting causes lipolysis in adipose tissue leading to the release of large quantities of free fatty acids into circulation that reach the liver where they are metabolized to generate ketone bodies to serve as fuels for other tissues. Since fatty acid-metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor ␣ (PPAR␣), we investigated the role of PPAR␣ in the induction of these enzymes in response to fasting and their relationship to the development of hepatic steatosis in mice deficient in PPAR␣ (PPAR␣ ؊/؊ ), peroxisomal fatty acyl-CoA oxidase (AOX ؊/؊ ), and in both PPAR␣ and AOX (double knockout (DKO)). Fasting for 48 -72 h caused profound impairment of fatty acid oxidation in both PPAR␣؊/؊ and DKO mice, and DKO mice revealed a greater degree of hepatic steatosis when compared with PPAR␣ ؊/؊ mice. The absence of PPAR␣ in both PPAR␣ ؊/؊ and DKO mice impairs the induction of mitochondrial ␤-oxidation in liver following fasting which contributes to hypoketonemia and hepatic steatosis. Pronounced steatosis in DKO mouse livers is due to the added deficiency of peroxisomal ␤-oxidation system in these animals due to the absence of AOX. In mice deficient in AOX alone, the sustained hyperactivation of PPAR␣ and up-regulation of mitochondrial ␤-oxidation and microsomal -oxidation systems as well as the regenerative nature of a majority of hepatocytes containing numerous spontaneously proliferated peroxisomes, which appear refractory to store triglycerides, blunt the steatotic response to fasting. Starvation for 72 h caused a decrease in PPAR␣ hepatic mRNA levels in wild type mice, with no perceptible compensatory increases in PPAR␥ and PPAR␦ mRNA levels. PPAR␥ and PPAR␦ hepatic mRNA levels were lower in fed PPAR␣ ؊/؊ and DKO mice when compared with wild type mice, and fasting caused a slight increase only in PPAR␥ levels and a decrease in PPAR␦ levels. Fasting did not change the PPAR isoform levels in AOX ؊/؊ mouse liver. These observations point to the critical importance of PPAR␣ in the transcriptional regulatory responses to fasting and in determining the severity of hepatic steatosis.

Peroxisomal ␤-oxidation system consists of four consecutive reactions to preferentially metabolize very long chain fatty acids. The first step of this system, catalyzed by acyl-CoA oxidase (AOX), converts fatty acylCoA to 2-trans-enoyl-CoA. Herein, we show that mice deficient in AOX exhibit steatohepatitis, increased hepatic H 2 O 2 levels, and hepatocellular regeneration, leading to a complete reversal of fatty change by 6 to 8 months of age. The liver of AOX؊/؊ mice with regenerated hepatocytes displays profound generalized spontaneous peroxisome proliferation and increased mRNA levels of genes that are regulated by peroxisome proliferator-activated receptor ␣ (PPAR␣). Hepatic adenomas and carcinomas develop in AOX؊/؊ mice by 15 months of age due to sustained activation of PPAR␣. These observations implicate acyl-CoA and other putative substrates for AOX, as biological ligands for PPAR␣; thus, a normal AOX gene is indispensable for the physiological regulation of PPAR␣.