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␣.

Using DNA sequences 5′ to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5′ DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org).

The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

Peroxisomal genetic disorders, such as Zellweger syndrome, are characterized by defects in one or more enzymes involved in the peroxisomal ␤-oxidation of very long chain fatty acids and are associated with defective peroxisomal biogenesis. The biologic role of peroxisomal ␤-oxidation system, which consists of three enzymes: fatty acyl-CoA oxidase (ACOX), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), and thiolase, has been examined in mice by disrupting ACOX gene, which encodes the first and rate-limiting enzyme of this system. Homozygous (ACOX ؊/؊) mice lacked the expression of ACOX protein and accumulate very long chain fatty acids in blood. However, these homozygous mice are viable, but growth-retarded and infertile. During the first 3-4 months of age, the livers of ACOX ؊/؊ mice reveal severe microvesicular fatty metamorphosis of hepatocytes. In such steatotic cells, peroxisome assembly is markedly defective; as a result, they contain few or no peroxisomes. Few hepatocytes in 1-3-monthold ACOX ؊/؊ mice contain numerous peroxisomes, and these peroxisome-rich hepatocytes show no fatty change. At this stage, the basal mRNA levels of HD, thiolase, and other peroxisome proliferator-induced target genes were elevated in ACOX ؊/؊ mouse liver, but these mice, when treated with a peroxisome proliferator, showed no increases in the number of hepatic peroxisomes and in the mRNAs levels of these target genes. Between 4 and 5 months of age, severe steatosis resulted in scattered cell death, steatohepatitis, formation of lipogranulomas, and focal hepatocellular regeneration. In 6 -7-month-old animals, the newly emerging hepatocytes, which progressively replaced steatotic cells, revealed spontaneous peroxisome proliferation. These livers showed marked increases in the mRNA levels of the remaining two genes of the ␤-oxidation system, suggesting that ACOX gene disruption leads to increased endogenous ligand-mediated transcription levels. These observations demonstrate links among peroxisomal ␤-oxidation, development of severe microvesicular fatty liver, peroxisome assembly, cell death, and cell proliferation in liver.