Peroxisome proliferator chemicals, acting via the peroxisome proliferator-activated receptor-␣ (Ppar␣), are potent hepatic mitogens and carcinogens in mice and rats. To test whether Ppar␣ is required for hepatic growth in response to other stimuli, we studied liver regeneration and hepatic gene expression following partial hepatectomy (PH) of wild-type and Ppar␣-null mice. Ppar␣-null mice had a 12-to 24-hour delay in liver regeneration associated with a delayed onset and lower peak magnitude of hepatocellular DNA synthesis. Furthermore, these mice had a 24-hour lag in the hepatic expression of the G 1 /S checkpoint regulator genes Ccnd1 and cMyc and increased expression of the IL-1␤ cytokine gene. Hepatic expression of Ccnd1, cMyc, IL-1r1, and IL-6r was induced in wild-type mice, but not Ppar␣-null mice, after acute exposure to the potent Ppar␣ agonist Wy-14,643, indicating a role for Ppar␣ in regulating the expression of these genes. Expression of the fatty acid -hydroxylase gene Cyp4a14, a commonly used indicator gene for Ppar␣ activation, was strongly induced in wild-type mice after hepatectomy, suggesting that altered hepatocyte lipid processing may also contribute to the impaired regeneration in mice lacking the Ppar␣ gene. In conclusion, liver regeneration in Ppar␣-null mice is transiently impaired and is associated with altered expression of genes involved in cell cycle control, cytokine signaling, and fat metabolism. (HEPATOLOGY 2002;36:544-554.) P eroxisome proliferator (PP) xenobiotics are potent hepatic mitogens and carcinogens in mice and rats. 1 Increases in hepatocyte replication and tumor formation after PP exposure require activation of the peroxisome proliferator-activated receptor-␣ (Ppar␣). 2 Many transcriptional targets of Ppar␣ have been identified, but none have direct roles in regulating cell proliferation or apoptosis. One of the most effective models for studying hepatocellular proliferation is liver regeneration following hepatocellular loss because of partial hepatectomy (PH) or chemical damage. 3 In the original technique for PH described by Higgins and Anderson in 1931, 4 resection of 70% of the hepatic mass of a rat results in 95% of the remaining hepatocytes rapidly entering the cell cycle. DNA synthesis follows about 12 to 14 hours after resection and reaches a maximum activation at 24 hours. These events occur about 20 hours later in mice. The original mass of the liver is restored within 7 days, with most of the recovery occurring by 3 days. 4 The signals that stimulate and maintain this process are not entirely clear but include the transcriptional activation of several groups of genes in a distinct temporal order. 3,[5][6][7] First, hepatocytes must be primed, presumably by cytokines, to respond to various growth factors. After priming, transition from G 0 to G 1 phases of the cell cycle requires induction of immediate-early class genes, which begins at about 30 minutes post-PH and lasts for about 4 hours. Progression of hepatocytes in vivo through late G 1 phase requires gr...

The obesity epidemic in industrialized countries is associated with increases in cardiovascular disease (CVD) and certain types of cancer. In animal models, caloric restriction (CR) suppresses these diseases as well as chemical-induced tissue damage. These beneficial effects of CR overlap with those altered by agonists of nuclear receptors (NR) under control of the fastingresponsive transcriptional co-activator, peroxisome proliferator-activated co-activator 1␣ (PGC-1␣). In a screen for compounds that mimic CR effects in the liver, we found statistically significant overlaps between the CR transcript profile in wild-type mice and the profiles altered by agonists of lipid-activated NR, including peroxisome proliferator-activated receptor ␣ (PPAR␣), liver X receptor, and their obligate heterodimer partner, retinoid X receptor. The overlapping genes included those involved in CVD (lipid metabolism and inflammation) and cancer (cell fate). Based on this overlap, we hypothesized that some effects of CR are mediated by PPAR␣. As determined by transcript profiling, 19% of all gene expression changes in wild-type mice were dependent on PPAR␣, including Cyp4a10 and Cyp4a14, involved in fatty acid -oxidation, acute phase response genes, and epidermal growth factor receptor but not increases in PGC-1␣. CR protected the livers of wild-type mice from damage induced by thioacetamide, a liver toxicant and hepatocarcinogen. CR protection was lost in PPAR␣-null mice due to inadequate tissue repair. These results demonstrate that PPAR␣ mediates some of the effects of CR and indicate that a pharmacological approach to mimicking many of the beneficial effects of CR may be possible.

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Fibrates, such as ciprofibrate, fenofibrate, and clofibrate, are peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists that have been in clinical use for many decades for treatment of dyslipidemia. When mice and rats are given PPARalpha agonists, these drugs cause hepatic peroxisome proliferation, hypertrophy, hyperplasia, and eventually hepatocarcinogenesis. Importantly, primates are relatively refractory to these effects; however, the mechanisms for the species differences are not clearly understood. Cynomolgus monkeys were exposed to ciprofibrate at various dose levels for either 4 or 15 days, and the liver transcriptional profiles were examined using Affymetrix human GeneChips. Strong upregulation of many genes relating to fatty acid metabolism and mitochondrial oxidative phosphorylation was observed; this reflects the known pharmacology and activity of the fibrates. In addition, (1) many genes related to ribosome and proteasome biosynthesis were upregulated, (2) a large number of genes downregulated were in the complement and coagulation cascades, (3) a number of key regulatory genes, including members of the JUN, MYC, and NFkappaB families were downregulated, which appears to be in contrast to the rodent, where JUN and MYC are reported to upregulated after PPARalpha agonist treatment, (4) no transcriptional signal for DNA damage or oxidative stress was observed, and (5) transcriptional signals consistent with an anti-proliferative and a pro-apoptotic effect were seen. We also compared the primate data to literature reports of hepatic transcriptional profiling in PPARalpha-treated rodents, which showed that the magnitude of induction in beta-oxidation pathways was substantially greater in the rodent than the primate.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.

Lipid homeostasis is controlled in part by the nuclear receptors peroxisome proliferator (PP)-activated receptor ␣ (PPAR␣) and liver X receptor (LXR) through regulation of genes involved in fatty acid and cholesterol metabolism. Exposure to agonists of retinoid X receptor (RXR), the obligate heterodimer partner of PPAR␣, and LXR results in responses that partially overlap with those of PP. To better understand the gene networks regulated by these nuclear receptors, transcript profiles were generated from the livers of wild-type and PPAR␣-null mice exposed to the RXR pan-agonist 3,7-dimethyl-6S,7S-methano, 7-[1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphth-7-yl]-2E,4E-heptadienoic acid (AGN194,204) or the PPAR pan-agonist WY-14,643 (WY; pirinixic acid) and compared with the profiles from the livers of wild-type and LXR␣/LXR␤-null mice after exposure to the LXR agonist N- (2,2,2-trifluoroethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethylethyl)phenyl] sulfonamide (T0901317). All 218 WY-regulated genes altered in wild-type mice required PPAR␣. Remarkably, ϳ80% of genes regulated by AGN194,204 required PPAR␣ including cell-cycle genes, consistent with AGN-induced hepatocyte proliferation having both PPAR␣-dependent and -independent components. Overlaps of ϳ31 to 62% in the transcript profiles of WY, AGN194,204, and T0901317 required PPAR␣ and LXR␣/LXR␤ for statistical significance. Ofthe 50 overlapping genes regulated by T0901317 and WY, all but one were regulated in a similar direction. These results 1) identify new transcriptional targets of PPAR␣ and RXR important in regulating lipid metabolism and liver homeostasis, 2) illustrate the importance of PPAR␣ in regulation of gene expression by a prototypical PP and by an RXR agonist, and 3) provide support for an axis of PPAR␣-RXR-LXR in which agonists for each nuclear receptor regulate an overlapping set of genes in the mouse liver.

The Bryan Alzheimer's Disease Research Center Rapid Autopsy Program at Duke University Medical Center obtains postmortem human brain tissue for experimental investigations. We evaluated 19 brains for RNA integrity and mRNA gene expression. Nine were from patients diagnosed with Alzheimer's disease, and ten were from nondemented controls. In all cases, the following variables were recorded: postmortem procurement delay (range, 1 hour and 10 minutes to 14 hours), pH of cerebrospinal fluid, premortem fever or sepsis, provision of supplemental oxygen in the agonal period, and temporal relation to time of death (either sudden death or protracted illness). Total RNA was extracted, quantified, and evaluated by agarose gel electrophoresis and quantitative gene expression analysis of 18S rRNA and edg-1 using TaqMan technology. All samples appeared to yield intact RNA without significant degradation, and expression of the edg-1 gene was detected by the real time reverse transcriptase polymerase chain reaction in all cases. We conclude that intact RNA can be obtained from postmortem human brain tissue, even in patients with severe premortem illnesses and delayed postmortem tissue procurement intervals. However, we caution that the successful expression of certain genes from postmortem brain tissue may require enhanced procurement efforts to maximize RNA integrity.