Adipocyte-specific Gene Expression and Adipogenic Steatosis in the Mouse Liver Due to Peroxisome Proliferator-activated Receptor gamma 1 (PPARgamma 1) Overexpression*

Songtao YuDagger , Kimihiko Matsusue§, Papreddy KashireddyDagger , Wen-Qing CaoDagger , Vaishalee YeldandiDagger , Anjana V. YeldandiDagger , M. Sambasiva RaoDagger , Frank J. Gonzalez§, and Janardan K. ReddyDagger

From the Dagger  Department of Pathology, Northwestern University, the Feinberg School of Medicine, Chicago, Illinois 60611-3008 and § The Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, October 1, 2002, and in revised form, October 18, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator activated-receptor (PPAR) isoforms, alpha  and gamma , function as important coregulators of energy (lipid) homeostasis. PPARalpha regulates fatty acid oxidation primarily in liver and to a lesser extent in adipose tissue, whereas PPARgamma serves as a key regulator of adipocyte differentiation and lipid storage. Of the two PPARgamma isoforms, PPARgamma 1 and PPARgamma 2 generated by alternative splicing, PPARgamma 1 isoform is expressed in liver and other tissues, whereas PPARgamma 2 isoform is expressed exclusively in adipose tissue where it regulates adipogenesis and lipogenesis. Since the function of PPARgamma 1 in liver is not clear, we have, in this study, investigated the biological impact of overexpression of PPARgamma 1 in mouse liver. Adenovirus-PPARgamma 1 injected into the tail vein induced hepatic steatosis in PPARalpha -/- mice. Northern blotting and gene expression profiling results showed that adipocyte-specific genes and lipogenesis-related genes are highly induced in PPARalpha -/- livers with PPARgamma 1 overexpression. These include adipsin, adiponectin, aP2, caveolin-1, fasting-induced adipose factor, fat-specific gene 27 (FSP27), CD36, Delta 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 PPARalpha -/- mice, failed to induce the expression of these PPARgamma -regulated adipogenesis-related genes in steatotic liver. These results suggest that a high level of PPARgamma 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 PPARgamma activity can lead to the development of a novel type of adipogenic hepatic steatosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator-activated receptor gamma  (PPARgamma ),1 a member of the nuclear receptor superfamily, is a key regulator of adipogenesis (1-4). The PPAR subfamily consists of three isotypes, namely PPARalpha , PPARgamma , and PPARbeta /delta , and like all other nuclear receptors, PPARs possess a highly conserved DNA-binding domain that recognizes peroxisome proliferator response elements (PPREs) in the promoter regions of target genes (5-9). After ligand binding PPARs heterodimerize with retinoid-X-receptors (RXR) and PPAR-RXR heterodimers bind to PPRE to initiate the transcriptional regulation of target genes, in particular those involved in lipid homeostasis (5-10). The three PPAR isotypes are products of separate genes, and they exhibit distinct patterns of tissue distribution (7). PPARalpha is expressed at a relatively high concentration in liver, plays a central role in regulating enzymes involved in the oxidation of fatty acids, and is essential for the pleiotropic responses induced in liver by structurally diverse chemicals known as peroxisome proliferators (8-10). PPARgamma is present in two isoforms, PPARgamma 1 and PPPARgamma 2, resulting from alternate promoter usage (1, 8, 9). PPARgamma 2 contains an additional 30 amino acids at the N-terminal end relative to PPARgamma 1. PPARgamma 2 expression is limited exclusively to adipose tissue where it play a key role in adipogenesis (1, 2). On the other hand, PPAR gamma 1 is expressed at relatively low levels in many tissues including liver, but the function of this isoform in non-adipose tissue locations is not well delineated (1, 9, 13). Forced expression of PPARgamma 2 or PPARgamma 1 can initiate the differentiation of fibroblasts to adipocytes, and in the process the fibroblasts express adipocyte-specific genes and accumulate lipid (2, 14). Although there is some suggestion that PPARgamma ligands may induce adipocyte-specific gene expression in certain tumor cells, it is uncertain as to whether PPARgamma 1 or PPARgamma 2 expression in vivo, in non-fibroblast mesenchymal cells, or in epithelial tissues can lead to adipocyte-specific gene expression and adipogenesis (15).

It is well known that CCAAT/enhancer binding family of transcription factors C/EBPalpha , -beta , and -delta have been shown to play an important role in adipogenesis in that high expression of each member of this family will direct fibroblasts to differentiate into adipocytes, and this conversion is mediated through down-stream regulator PPARgamma and PPAR coactivator PBP/TRAP220/DRIP205 (4, 16-19). C/EBPs and PBP are expressed in liver, but the significance of this expression in terms of adipogenesis and or lipid accumulation in liver cells remains unclear. The inability of C/EBP and PBP to induce adipogenesis in normal hepatocytes may be due to the fact that down-stream regulator PPARgamma (PPARgamma 1 in liver) may be rate-limiting in hepatocytes (19-21). In this study, we used an adenoviral gene delivery system to overexpress PPARgamma 1 in mouse liver to determine whether this would trigger the expression of adipocyte-specific genes and lipid accumulation (steatosis) in liver cells. To avoid the confounding effect of PPARalpha , we used PPARalpha -/- mice (22), and found that overexpression of PPARgamma 1 in these livers leads to adipocyte-specific gene expression and lipid accumulation. We also demonstrate that fatty liver induced by starvation or that developing after feeding a diet deficient in choline is not associated with the induction of genes associated with adipogenesis unlike that accompanying PPARgamma 1 overexpression in liver. These results strongly suggest that the low level of PPARgamma 1 appears to prevent liver cells from becoming adipocytes despite the prominence of C/EBPalpha gene expression in these cells and that overexpression of PPARgamma 1 leads to adipogenic hepatic steatosis.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice and Treatment-- Wild type (C57BL/6J) mice and PPARalpha -/- mice (22), 3 to 4 months of age and weighing 25-35 g, were used in this study. PPARalpha -/- mice were maintained on powdered chow with or without troglitazone (0.1% w/w) for 5 days prior to adenovirus injection and killed on day 2, 3, 4, 5, or 6 after injections while still on the same diet. For dose response of Ad/PPARgamma 1, mice were maintained on powdered chow, injected with different concentrations of virus, and killed 6 days after injection. For the induction of fatty liver, PPARalpha -/- mice were either fasted for 96 h (23) or fed a choline-deficient diet (Dyets, Bethlehem, PA) for 15 days (24, 25). All animal procedures used in this study were reviewed and preapproved by the Institutional Review Boards for Animal Research of the Northwestern University.

Primers for Endogenous Mouse PPARgamma -- To assess the level of expression of endogenous PPARgamma 1 under forced expression of Ad/PPARgamma 1, the following primers were used to distinguish the ectopic expression from endogenous. They are endogenous PPARgamma 1 sense: 5'-cggagggacgcggaagaagag-3'; endogenous PPARgamma 2:sense: 5'-tgacccagagcatggtgccttc-3'; adenoviral PPARgamma 1:sense: 5'-cggggatcctctagagtcga-3'. All three PPARgamma RT-PCRs were performed using the same antisense primer: 5'-tgtggcatccgcccaaacc-3'. Primers for the internal control beta -actin were sense: 5'-ggttccgatgccctgaggctc-3', antisense: 5'-tgctccaaccaactgctgtcgc-3'. PCR reactions were denatured at 94 °C for 2 min, cycled at 94 °C for 10 s, 60 °C for 30 s, 72 °C for 45 s, 30 cycles for PPARgamma and 25 cycles for beta -actin by using the GeneAmp PCR system 9700 (PE Applied Biosystems).

Adenoviral Gene Transfer-- Construction of recombinant adenovirus containing the mouse PPARgamma 1 cDNA (Ad/mPPARgamma 1) was as follows. Mouse PPARgamma 1 cDNA (8) was cloned into pShuttle-CMV expression vector at SalI site (Quantum Biotechnologies, Inc.). The linearized shuttle vector and AdEasy vector (Quantum Biotechnologies, Inc.) were then co-transformed into Escherichia coli strain BJ5183. Positive recombinant plasmid Ad/mPPARgamma 1 was selected. The Ad/mPPARgamma 1 virus was then generated as described previously (26). Adenoviral construct of Ad/LacZ was the generous gift of Dr W. El-Deiry (University of Pennsylvania, Philadelphia, PA) and has been described (27). Mice were intravenously injected (tail vein) in a volume of 200 µl with 1 ×× 1011 virus particles of Ad/LacZ or Ad/mPPARgamma 1 and killed 6 days later. Mice injected with PBS served as controls in some cases.

Morphology-- Tissue fixed in 10% neutral buffered formalin was embedded in paraffin by using standard procedures. Sections (4-µm thick) were cut and stained with hematoxylin and eosin. For visualizing beta -galactosidase activity, sections of liver were incubated in PBS containing 5 mM potassium ferricyanate, 2 mM MgCl2, and 1/20 volume of 20 mg/ml 5-bromo-4-chloro-3-indolyl beta -D-galactoside in dimethylformamide at 37 °C (26, 27). Immunohistochemical localization of PPARgamma was performed as described previously using polyclonal anti-PPARgamma antibodies (Santa Cruz, CA). Frozen sections of formalin-fixed liver (5-µm thick) were stained with Oil Red O and counterstained with Giemsa. Histological analysis and image processing were carried out using Leica DMRE microscope equipped with Spot digital camera as described (24, 26-30).

Northern and Immunoblot Procedures-- Total RNA isolated from liver using Trizol reagent (Invitrogen) was glyoxylated, electrophoresed on 0.8% agarose gel, and then transferred to nylon membrane. These nylon membranes were then hybridized at 42 °C in 50% formamide hybridization solution using 32P-labeled cDNA probes. Equal loading was verified by the intensity of methylene blue-stained 18 S and 28 S RNA or by hybridizing the filters for glyceraldehyde-3-phosphate dehydrogenase. For immunoblotting, liver extracts were subjected to 7.5% or 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted as previously described (26, 28, 29). The aP2 antibody was a generous gift from Dr. G. S. Hotamisligil (Harvard School of Public Health, Boston, MA).

Microarray Approach-- Total RNA was isolated from the livers of PPARalpha -/- mice injected with Ad/LacZ or Ad/mPPARgamma 1 and killed 6 days after injection (26). Reverse transcription, second-strand synthesis, and probe labeling were all performed using 10 µg of total liver RNA as a template for cDNA synthesis. Biotin-labeled cRNA was produced using the above cDNA as a template, purified, fragmented, and hybridized to U74Av2 arrays (Affymetrix, Santa Clara, CA). After hybridization, bound cRNA was fluorescently labeled using R-phycoerythrin-streptavidin (Molecular Probes), and the fluorescence was intensified by the antibody-amplification method. Data were collected and analyzed using the Affymetrix Microarray suite 4.01 software.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fatty Liver Resulting from PPARgamma 1 Overexpression-- We have investigated the morphological changes in the liver of PPARalpha -/- mice injected with 1 × 1011 adenoviral-PPARgamma 1 particles intravenously. PPARgamma 1 overexpression in the liver of PPARalpha -/- mice caused extensive lipid accumulation in hepatocytes located in the periportal and midzonal regions of liver lobules at 6 days (Fig. 1). The accumulation of fat in liver was grossly evident between 3 and 6 days after Ad/PPARgamma 1 injection. The degree and zonality of lipid accumulation in liver in mice overexpressing PPARgamma 1 appeared essentially similar whether or not they were on troglitazone, the PPARgamma 1 ligand (Fig. 1, B and C). Mice given troglitazone and injected with either PBS or with Ad/LacZ failed to reveal lipid accumulation in hepatocytes (Fig. 1, A and D). Oil red O staining of liver sections obtained from PPARalpha -/- mice infected with Ad/mPPARgamma 1 confirmed hepatic lipid accumulation (Fig. 1E), whereas no appreciable lipid accumulation was evident in the liver of mice treated with Ad/LacZ (Fig. 1F). Immunohistochemical analysis revealed PPARgamma nuclear staining in ~70% of hepatocytes with microvesicular steatosis between 4 and 6 days following Ad/mPPARgamma 1 injection into the PPARalpha -/- mice (Fig. 1G). No detectable immunostaining for PPARgamma was noted in the livers of Ad/LacZ-injected mice (Fig. 1H). As expected, ~60-70% of hepatocytes stained positively for beta -galactosidase after Ad/LacZ injection (Fig. 1, I). Wild type (PPARalpha +/+) mice dosed with Ad/mPPARgamma 1 also exhibited hepatic lipid accumulation but not as severe as that seen in PPARalpha -/- livers with PPARgamma 1 overexpression (not illustrated), suggesting that the presence of PPARalpha in the liver facilitates the up-regulation of fatty acid oxidation systems and reduces lipid accumulation (11, 23).


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Fig. 1.   Adenovirus-PPARgamma 1 expression and hepatic steatosis in PPARalpha -/- mouse. PPARalpha -/- mice were treated with troglitazone for 5 days and then injected with PBS (A), Ad/PPARgamma 1 (B), or Ad/LacZ (D) and then maintained on troglitazone for another 6 days. (C) The PPARalpha -/- mouse not treated with troglitazone was injected with Ad/PPARgamma 1. Livers harvested 6 days after tail vein injection of Ad/PPARgamma 1, with or without the ligand, show severe periportal steatosis (B, C). Oil Red O stain demonstrates fatty change in the Ad/PPARgamma 1-injected mouse that was not on troglitazone (E) in contrast to the mouse given Ad/LacZ (F). Liver of the PPARalpha -/- mouse injected with Ad/PPARgamma 1 (G) or with Ad/LacZ (H, I) was immunostained for PPARgamma localization 6 days after injection (G, H). I, beta -galactosidase staining of 6-day Ad/LacZ PPARalpha -/- liver.

PPARgamma 1-induced Adipogenic Gene Expression in PPARalpha -/- Mouse Liver-- PPARgamma exists as two isoforms, PPARgamma 1 and PPARgamma 2, generated by alternate promoter usage (1, 9). The expression of PPARgamma 2 is restricted mainly to adipocytes and is the key regulator of adipogenesis, although forced expression of PPARgamma 1 isoform can also induce adipogenesis in fibroblasts (2, 14). Since we noted a striking degree of lipid accumulation in hepatocytes following PPARgamma 1 overexpression, it appeared necessary to investigate the adipogenic action of overexpressed PPARgamma 1 in liver (Fig. 2). Northern analysis of RNA isolated from PPARalpha -/- mouse liver revealed dramatic induction mRNAs for fat differentiation markers aP2, adipsin and adiponectin (adipoQ/acrp30) (see Refs. 31, 32) in PPARgamma 1 overexpressing livers but not after Ad/LacZ infection (Fig. 2, A and B). Since these mice are PPARalpha -/-, it is reasonable to conclude that PPARalpha plays no role in the observed induction of these adipogenic genes. The mRNA of the adipsin gene reached peak level at 3 days postinjection, whereas aP2 mRNA level was maximally expressed at 6 days after Ad/PPARgamma 1 injection (Fig. 2A). The induction of adiponectin appeared somewhat delayed when compared with aP2 and adipsin. Glucose-6-phophatase (Glc-6-P) mRNA level in liver increased 2 days after Ad/PPARgamma 1 injection (Fig. 2A). We also determined the hepatic mRNA levels of several genes 6 days after Ad/PPARgamma 1 injection to assess the adipogenic and lipogenic profile. Noticeable increases in CD36, glucokinase, malic enzyme, low density lipoprotein receptor, microsomal triglyceride transfer protein, and Delta 9d mRNA levels were detected at 6 days after Ad/PPARgamma injection (Fig. 2B). We did not observe perceptible increases in the levels of C/EBPalpha , SREBP, glucokinase, phospho(enol)pyruvate carboxykinase, and Glut-2 mRNAs in liver (Fig. 2B). We also assessed the mRNA levels of PPARalpha -regulated peroxisomal fatty acid beta -oxidation system genes and found modest increases in straight chain fatty acyl-CoA oxidase, L-PBE, and peroxisomal 3-ketoacyl-CoA thiolase mRNAs (Fig. 2A), suggesting that PPARgamma 1 at very high levels can transcriptionally activate PPARalpha target gene expression and regulate fatty acid beta -oxidation in liver in the absence of PPARalpha .


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Fig. 2.   Inducibility of PPARgamma and PPARalpha target gene expression in PPARalpha -/- mouse liver. A, Northern blot showing the expression levels of PPARgamma , aP2, adipsin, adiponectin, Glc-6-P, straight chain fatty acyl-CoA oxidase, L-PBE, and peroxisomal 3-ketoacyl-CoA thiolase genes after viral injection. PPARalpha -/- mice maintained on troglitazone were infected with 1 × 1011 Ad/PPARgamma 1 viral particles and killed at 2, 3, 4, 5, and 6 days after infection. PPARalpha -/- mice injected with Ad/LacZ were killed 6 days after the injection. This blot includes samples from 2, 3, and 6-day post-treatment groups (2 animals). RNAs, 28 S and 18 S, are shown as a measure of loading control. B, Northern blot of C/EBPalpha , SREBP1, glucokinase, phosphoenolpyruvate carboxykinase, Glut-2, low density lipoprotein receptor, aP2, microsomal triglyceride transfer protein, CD36, Delta 9d and other genes after viral injection. PPARalpha -/- mice (three in each group) maintained on normal diet were infected with 1 × 1011 Ad/PPARgamma 1 or Ad/LacZ viral particles and killed 6 days after infection. Total RNAs from liver samples were hybridized with aforementioned probes. Glyceraldehyde-3-phosphate dehydrogenase is used for loading control. C, immunoblot of PPARgamma , aP2, and L-PBE genes. PPARalpha -/- mice (two in each group) maintained on troglitazone were infected with 1 × 1011 Ad/PPARgamma 1 viral particles and killed 2, 3, 4, 5, and 6 days after infection. PPARalpha -/- mice injected with Ad/LacZ were killed 6 days after injection. Liver samples were immunoblotted for PPARgamma , aP2, L-PBE, and catalase. The nonresponsive gene catalase was shown as loading control.

Immunoblotting confirmed the induction of white adipose tissue marker protein aP2 in PPARgamma 1-overexpressing liver beginning at day 3 (Fig. 2C). This protein was not detected in normal liver or Ad/LacZ-injected mouse livers (Fig. 3C). Increase in PPARgamma 1 and in L-PBE protein levels were also seen in Ad/PPARgamma 1-expressing livers (Fig. 2C). As expected, no change in the amount of catalase protein, the peroxisomal marker enzyme, was discerned.


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Fig. 3.   Expression of aP2 in PPARgamma -induced adipogenic hepatic steatosis but not in other forms of fatty liver. A, immunoblot analysis of aP2 expression in PPARalpha -/- PPARalpha +/+ wild type mouse liver. Mice were injected with PBS, Ad/LacZ, or Ad/PPARgamma 1 through tail vein with or without treatment with troglitazone and killed 6 days later. Liver samples from each group were immunoblotted for PPARgamma and aP2. The non-responsive gene catalase is used as loading control. B, expression of PPARgamma and aP2 genes in fatty livers. Steatosis that developed in PPARalpha -/- mouse liver after infection with Ad/PPARgamma 1 for 6 days (lanes 1 and 2, following fasting for 4 days (lanes 3 and 4), or fed a choline-deficient diet for 15 days (lanes 5 and 6). Representative liver samples were immunoblotted (two mice in each group) for PPARgamma , aP2 (PPARgamma -responsive gene), and catalase (nonresponsive gene). C, Northern blot analysis of RNA obtained from fatty livers that developed in PPARalpha -/- mice after infection with Ad/PPARgamma 1 (6 days), starvation for 4 days, or after 2 weeks of feeding a choline-deficient diet. Expression of adipogenesis-associated genes is confined to PPARgamma -induced adipogenic hepatic steatosis and not with other forms of fatty liver.

aP2 Protein Gene Expression in Liver is PPARgamma -dependent-- We observed that the gene expression of aP2 in liver was in a PPARgamma dose-dependent manner (Fig. 2C). We also established that the induction of aP2 expression in the liver of PPARalpha -/- and wild type (PPARalpha +/+) mice is dependent upon PPARgamma 1 overexpression whether or not these mice were on troglitazone, the synthetic PPARgamma ligand (Fig. 3A). Since PPARgamma 1 overexpression induced the adipogenic aP2 protein as well as hepatic steatosis in PPARalpha -/- mice, it appeared necessary to study the relationship if any of PPARgamma 1 and aP2 gene expression with hepatic steatosis and to distinguish hepatic adipogenesis (hepatic adiposis) from the typical non-alcoholic hepatic steatosis (30). We induced fatty liver in PPARalpha -/- mice (Fig. 4) by either fasting these mice for 96 h (23) or feeding them a diet deficient in choline for 15 days (24). Immunoblotting failed to reveal PPARgamma and aP2 protein in fatty livers induced by starvation or by a choline-deficient diet, but these two proteins appeared prominent in the fatty liver induced by PPARgamma 1 overexpression (Fig. 3B). These observations clearly demonstrate that hepatic fatty change itself is not enough to induce either PPARgamma or its target gene aP2, adipsin, and adiponectin and that PPARgamma 1 overexpression leads to a novel type of adipogenic hepatic steatosis designated hepatic adiposis to distinguish it from the common hepatic steatosis.


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Fig. 4.   Histological evidence of hepatic steatosis in PPARalpha -/- mice. A, fatty liver following 4 days of starvation. B, fatty liver after 2 weeks of feeding a diet deficient in choline. C, hepatic steatosis in Ad/PPARgamma 1-injected mouse. Insets show high magnification of boxed areas. Note the presence of steatohepatitis in choline-deficient liver.

Gene Expression Profiling-- To investigate whether PPARgamma 1 overexpression in liver indeed leads to adipogenic transdifferentiation of hepatocytes, we performed global transcriptional profiling using RNA isolated from liver 6 days after injection of Ad/PPARgamma 1 into PPARalpha -/- mice. Biotin-labeled RNA probes from either Ad/PPARgamma liver or Ad/LacZ liver were hybridized to Affymatrix microarray chips containing 12,000 genes. When a 4-fold change is used as the cut off for either up- or down-regulation, we found that 184 genes were up-regulated and 87 were down-regulated in Ad/PPARgamma 1-overexpressing livers. Table I lists transcripts displaying a 6-fold or greater increase in liver as a result of overexpression of PPARgamma 1. A majority of these genes participate in adipogenic differentiation or lipid metabolism indicating that the observed lipid accumulation in hepatocytes reflects transformation/transdifferentiation of hepatocytes toward adipocytes. Adipocyte marker genes adipsin (33), aP2 (34, 35), adiponectin (36, 37), and caveolin-1 (38, 39) were increased to ~137-, 66-, 48-, and 28-fold, respectively. E-FABP (40), FSP27 (41), CYP4A10 and CYP4A14 (25), hormone-sensitive lipase (42), monoglyceride lipase (43), and others involved in lipid metabolism were induced >10-fold in PPARgamma 1-overexpressing livers. Three apoptosis-associated genes, namely cell death-inducing DNA fragmentation factor (CIDE) (44), cyclophilin C (45), and nur77 (46, 47) were increased to ~101-, 33-, and 15-fold, respectively, suggesting that induction of the adipogenic differentiation program in liver augments hepatocyte apoptosis. Northern analysis confirmed marked increases in the levels of cavelolin-1, CIDE-A, and nur77 mRNAs in liver beginning about 3 days after Ad/PPARgamma 1 injection (Fig. 5). Previously, we found up-regulation of CIDE in the liver following treatment with Wy-14,643, a PPARalpha ligand (43). However, since CIDE was not up-regulated in mice lacking straight chain fatty acyl-CoA oxidase, which exhibit spontaneous PPARalpha activation, we concluded that induction of CIDE may not be dependent upon PPARalpha (43). Further studies are needed to ascertain the mechanisms and implications of CIDE, cyclophilin C, and nur77 up-regulation following overexpression of PPARgamma 1 in liver. Among the genes up-regulated between 5- and 10-fold are farnesyl diphosphate synthetase (~9-fold), diacylglycerol acyltransferase ~8-fold), pyruvate carboxylase (~7-fold), long chain fatty acyl elongase (~7-fold), CD36 (~7-fold), adiponutrin-like protein (~7-fold), acetyl-CoA acyltransferase (~6-fold), dicarboxylate transporter (~6-fold), and S3-12 gene (~6-fold), among others (Table I). A majority of these genes participate in lipid metabolism.

                              
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Table I
Genes up-regulated (6-fold or greater) in liver with PPARgamma 1 overexpression


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Fig. 5.   Northern blot analysis for caveolin-1, CIDE-A, and nur77. Total RNA isolated from liver of PPARalpha -/- mice at 2, 3, 4, 5, and 6 days after Ad/mPPARgamma 1 injection or 6 days after Ad/LacZ injection. Note marked increases in caveolin-1 and CIDE-A mRNAs at 5 and 6 days after Ad/mPPARgamma 1 injection. Increases in nur77 mRNA are also evident.

Table II lists the genes that were down-regulated 6-fold or higher in the liver with PPARgamma 1 overexpression. These include genes encoding for interferon-induced protein with tetratricopeptide repeats 1 and 2, interferon-induced 15-kDa protein, interferon-gamma -induced 47-kDa protein, and asialoglycoprotein receptor among others.

                              
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Table II
Genes down-regulated (6-fold or greater) in liver with PPARgamma 1 overexpression

Endogenous PPARgamma Expression-- We have ascertained that forced expression of PPARgamma 1 does not up-regulate endogenous PPARgamma (Fig. 6). PPARgamma 2 sense primer was designed to include the N-terminal region of PPARgamma 2 to distinguish it from endogenous PPARgamma 1. For ectopic PPARgamma 1 the sense primer was from the cytomegalovirus promoter. The RT-PCR data show no increase in endogenous PPARgamma 1 or PPARgamma 2 mRNA in the liver 6 days after Ad/PPARgamma 1 injection.


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Fig. 6.   RT-PCR to determine the endogenous and ectopic PPARgamma mRNA level in liver. RNA from liver of mice 6 days after injecting Ad/LacZ or Ad/PPARgamma 1 was analyzed for endogenous PPARgamma 1 (Endo-PPARgamma 1), and endogenous PPARgamma 2 (Endo-PPARgamma 2) and for ectopic PPARgamma 1 (Viral PPARgamma ). beta -Actin served as internal control. A slight increase in endogenous PPARgamma in Ad/LacZ livers is attributed to subtle inflammatory response.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The nuclear receptor PPARgamma is essential for adipogenesis and lipid storage (1-4). PPARgamma is present in two isoforms, PPARgamma 1and PPARgamma 2, generated by alternate promoter usage and splicing (9). PPARgamma 2 isoform, which is expressed exclusively in adipocytes, plays a pivotal role in adipocyte differentiation and adipocyte-specific gene expression, but there is some controversy as to whether PPARgamma 1 isoform can initiate and sustain adipogenic gene expression (2, 14, 48). Tontonoz et al. (2) reported that both PPARgamma 1and PPARgamma 2 could stimulate adipogenesis when introduced into fibroblasts, whereas Ren et al. (48), using engineered zinc finger repressors to inhibit the expression of PPARgamma isoforms, have concluded that PPARgamma 1 overexpression exerted no adipogenic effect. Recently, Mueller et al. (14) using PPARgamma null fibroblasts demonstrated convincingly that both PPARgamma 1and PPARgamma 2 isoforms have intrinsic ability to induce robust adipogenesis. While most of the work on PPARgamma -induced adipogenesis involved the use of fibroblast cell lines, the key question as to whether PPARgamma 1 isoform, which is the only isoform expressed in liver and many other tissues, can stimulate fat cell differentiation or transformation of hepatocytes into adipocytes. In this study, we have shown that overexpression of PPARgamma 1 in mouse liver induced adipocyte-specific gene expression as well as microvesicular steatosis in this organ. Furthermore, we have demonstrated that hepatic steatosis resulting from PPARgamma 1 overexpression is associated with adipogenic gene expression, whereas hepatic steatosis induced by starvation or developing after feeding a choline-deficient diet failed to stimulate adipogenic gene expression. Accordingly, we propose that PPARgamma overexpression leads to the development of a novel form of hepatic steatosis, which is designated adipogenic hepatic steatosis or simply "hepatic adiposis" to distinguish this entity from the common forms of hepatic steatosis.

Our results showed the expression of adipocyte-specific genes in liver upon overexpression of PPARgamma 1 whether or not these mice were treated with troglitazone. This may be due to the availability of putative endogenous ligands in liver generated as part of the normal lipid metabolism (49, 50). Because PPARgamma 1 is endogenously expressed at a low level in mouse liver it is unlikely that this receptor exerts any adipogenic effect under normal physiological conditions. Furthermore, the relative abundance of PPARalpha in normal liver serves as a key regulator of fatty acid catabolism thereby minimizing the need for adipogenesis in liver to store lipids (11, 30). In that sense functionally active PPARalpha and fatty acid oxidation systems keep the PPARgamma 1 in check (30). Our results clearly establish that the overexpression of PPARgamma 1 isoform in liver in PPARalpha null background triggers the expression of adipogenesis-related genes and fatty change in liver. This process essentially represents conversion of hepatocytes into cells with active adipogenic gene expression profile, and in that regard this represents transformation or transdifferentiation of hepatocyte into adipocytes, i.e. the conversion of fat-burning hepatocytes into fat storage cells (31, 32, 51). It is important to note that the expression of adipocyte-specific genes and microvesicular steatosis observed in liver is not associated with overexpression of the endogenous PPARgamma gene. RT-PCR analysis showed no induction of endogenous PPARgamma 1 or PPARgamma 2 isoforms (Fig. 6). Thus all the changes observed in gene expression patterns and morphological changes are the result of the exogenous expression of adenovirally driven PPARgamma 1 overexpression. These results suggest that maintenance of low levels of PPARgamma in liver is crucial for preventing hepatocytes from encountering an adipogenesis fate. The maintenance of marginal PPARgamma 1 gene expression in liver may be achieved by unknown mechanisms designed to prevent positive feedback between C/EBPalpha and PPARgamma because the downstream adipogenic events could manifest successfully in the presence of abundant PPARgamma protein (4).

We performed Northern analysis and global transcriptional profiling to define the pattern of genes expressed in liver as a result of PPARgamma 1 overexpression. Our studies indicate that overexpression of PPARgamma 1 isoform in liver results in the up-regulation of many genes known to be up-regulated during adipocyte differentiation of 3T3-L1 fibroblasts (31, 32, 52-54). Adipogenesis genes up-regulated in liver include PPARgamma target genes adipsin, aP2, adiponectin, caveolin, fat-specific protein 27, and others (Table I). Caveolae and caveolin-1 protein expression are most abundant in adipocytes (39, 55). The overexpression of caveolin-1 mRNA in liver expressing PPARgamma is further indication of adipogenic transformation of hepatocytes in that caveolin-1 appears to participate in facilitating the conversion of triglycerides in lipoprotein form to triglycerides in lipid droplet storage from (39). Caveolin-1 appears functionally necessary to maintain lipid droplet integrity, and the absence of this protein in caveolin-1-deficient mice leads to abnormalities in adipocyte function (39). Increase in caveolin-1 gene expression in PPARgamma 1-overexpressing livers is further indication of attaining an adipocyte phenotype. In addition, numerous adipocyte-enriched genes involved in lipogenesis and lipid metabolism, in particular E-FABP (keratinocyte lipid binding protein) (40), cyp4a10, cyp4A14 (25), fasting-induced adipose factor (angiopoietin-like 4) (56, 57), CD36 (58), glycerophosphate dehydrogenase (52), and hormone-sensitive lipase (42), were markedly up-regulated in liver following PPARgamma 1 overexpression. Thus, our in vivo observations clearly establish the adipogenic conversion of liver when PPARgamma 1 is overexpressed in this organ.

In this study, we noted marked up-regulation of three genes that have recently been shown to participate in apoptosis. These include CIDE-A (44), cyclophilin C (45), and nur77 (46, 47). CIDEs belong to a novel family of recently identified cell death-inducing proteins that induce apoptosis when overexpressed (59). CIDE is not normally expressed in liver, but increased levels of CIDE mRNA have been noted in livers of mice that were treated with Wy-14,643, a PPARalpha ligand (43). However, the CIDE expression was not dependent upon PPARalpha (43). The functional implications of up-regulation of three apoptotic genes in liver with adipogenic hepatic steatosis suggests that the acquisition of adipogenic phenotype places hepatocytes at greater risk for apoptosis. Additional studies are needed to ascertain if the CIDE, cyclophilin C, and nur77 genes are PPARgamma targets for regulation. Studies are also needed to ascertain if DNA synthesis is a prerequisite for the adipogenic conversion of hepatocytes.

It is of interest that this new form of hepatic adiposis or adipogenic hepatic steatosis resulting from PPARgamma 1 overexpression may have potential clinical implications. Individuals with relatively low hepatic levels of PPARalpha and hyperactive PPARgamma 1 resulting from single nucleotide polymorphism or by some other mechanism could develop adipogenic hepatic steatosis similar to that described in this report. It is also worth noting that ob/ob mice exhibit increased levels of PPARgamma mRNA in their livers (60). Increased expression levels of aP2 and CD36 mRNA were seen in these livers when these mice were given PPARgamma ligand troglitazone (61). Thus, it is important to consider PPARgamma 1 overexpression or hyperactivity as a possible molecular mechanisms responsible for a special type of non-alcoholic hepatic steatosis, designated as hepatic adiposis or adipogenic hepatic steatosis.

Finally, we have identified a number of novel genes as modestly up-regulated in PPARgamma 1-overexpressing liver, which remain to be characterized (Table I). This includes a gene we designated as promethin (AY167031), which is induced in liver by PPARgamma overexpression. Additional studies are needed to further characterize these and other genes to gain appreciation of the role of PPARgamma in liver in relation to glucose homeostasis, energy utilization, and insulin resistance (61).

    ACKNOWLEDGEMENT

We thank Dr. Pradip Raychaudhuri of the University of Illinois School of Medicine for the nur77 cDNA.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM23750 (to J. K. R.), CA84472 (to M. S. R.), and the Joseph L. Mayberry, Sr., Endowment Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology, Northwestern University, the Feinberg School of Medicine, 303 E. Chicago Ave., Chicago, IL 60611-3008. Tel.: 312-503-8144; Fax: 312-503-8249; E-mail: jkreddy@northwestern.edu.

Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M210062200

    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element(s); L-PBE, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein; Glc-6-P, glucose-6-phosphatase; C/EBPalpha , CCAAT enhancer-binding protein alpha ; SREBP1, sterol regulatory element-binding protein 1; aP2, adipose fatty acid-binding protein; RT, reverse transcriptase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Tontonoz, P., Hu, E., Graves, R. A., and Spiegelman, B. M. (1994) Genes Dev. 8, 1224-1234[Abstract]
2. Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994) Cell 79, 1147-1156[Medline] [Order article via Infotrieve]
3. Rosen, E. D., Walkey, C. J., Puigserver, P., and Spiegelman, B. M. (2000) Genes Dev. 14, 1293-1307[Free Full Text]
4. Lazar, M. A. (2002) Genes Dev. 16, 1-5[Free Full Text]
5. Kliewer, S. A., Umesono, K., Noonan, D. J., Heyman, R. A., and Evans, R. M. (1992) Nature 358, 771-774[CrossRef][Medline] [Order article via Infotrieve]
6. McKenna, N. J., Lanz, R. B., and O'Malley, B. W. (1999) Endocr. Rev. 20, 321-344[Abstract/Free Full Text]
7. Desvegne, B., and Wahli, W. (1999) Endocr. Rev. 20, 649-650[Abstract/Free Full Text]
8. Zhu, Y., Alvares, K., Huang, Q., Rao, M. S., and Reddy, J. K. (1993) J. Biol. Chem. 268, 26817-26820[Abstract/Free Full Text]
9. Zhu, Y., Qi, C., Korenberg, J. R., Chen, X. N., Noya, D., Rao, M. S, and Reddy, J. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7921-7925[Abstract]
10. Surapureddi, S., Yu, S., Hashimoto, T., Yeldandi, A. V., Kashireddy, P., Cherkaoui-Malki, M., Qi, C., Zhu, Y.-J., Rao, M. S., and Reddy, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11836-11841[Abstract/Free Full Text]
11. Reddy, J. K., and Hashimoto, T. (2001) Annu. Rev. Nutr. 21, 193-230[CrossRef][Medline] [Order article via Infotrieve]
12. Reddy, J. K., and Krishnakantha, T. P. (1975) Science 190, 787-789[Medline] [Order article via Infotrieve]
13. Braissant, O, Foufelle, F., Scotto, C., Duaca, M., and Wahli, W. (1996) Endocrinology 137, 354-366[Abstract]
14. Mueller, E., Drori, S., Aiyer, A., Yie, J., Sarraf, P., Chen, H., Hauser, S., Rosen, E.D., Ge, K., Roeder, R.G., Spiegelman, B.M. (2002) J. Biol. Chem., in press
15. Agarwal, V. R., Bischoff, E. D., Hermann, T., and Lamph, W. W. (2000) Cancer Res. 60, 6033-6038[Abstract/Free Full Text]
16. Lin, F. T., and Lane, M. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8757-8761[Abstract]
17. Hamm, J. K., Park, B. H., and Farmer, S. R. (2001) J. Biol. Chem. 276, 18464-18471[Abstract/Free Full Text]
18. Zhu, Y., Qi, C., Jain, S., Rao, M. S., and Reddy, J. K. (1997) J. Biol. Chem. 272, 25500-25506[Abstract/Free Full Text]
19. Ge, K., Guermah, M., Yuan, C. X., Ito, M., Wallberg, A. E., Spiegelman, B. M., and Roeder, R. G. (2002) Nature 417, 563-567[CrossRef][Medline] [Order article via Infotrieve]
20. Darlington, G. J., Wang, N., and Hanson, R. W. (1995) Curr. Opin. Genet. Dev. 5, 565-570[CrossRef][Medline] [Order article via Infotrieve]
21. Spiegelman, B. M., and Flier, J. S. (1996) Cell. 87, 377-389[Medline] [Order article via Infotrieve]
22. Lee, S. S., Pineau, T., Drago, J., Lee, E. J., Owens, J. W., Kroetz, D. L., Fernandez-Salguero, P. M., Westphal, H., and Gonzalez, F. J. (1995) Mol. Cell. Biol. 15, 3012-3022[Abstract]
23. Hashimoto, T., Cook, W. S., Qi, C., Yeldandi, A. V., Reddy, J. K., and Rao, M. S. (2000) J. Biol. Chem. 275, 28918-28928[Abstract/Free Full Text]
24. Rao, M. S., Papreddy, K., Musunuri, S., and Okonkwo, A. (2002) In Vitro 16, 145-152
25. Leclercq, I. A., Farrell, G. C., Field, J., Bell, D. R., Gonzalez, F. J., and Robertson, G. R. (2000) J. Clin. Invest. 105, 1067-1075[Abstract/Free Full Text]
26. Yu, S., Cao, W,-Q., Kashireddy, P., Meyer, K., Jia, Y., Hughes, D. E., Tan, Y., Feng, J., Yeldandi, A. V., Rao, M. S., et al.. (2001) J. Biol. Chem. 276, 42485-42491[Abstract/Free Full Text]
27. Mitchell, K. O., and El-Deiry, W. S. (1999) Cell Growth Differ. 10, 223-230[Abstract/Free Full Text]
28. Qi, C., Zhu, Y., Pan, J., Yeldandi, A. V., Rao, M. S., Maeda, N., Subbarao, V., Pulikuri, S., Hashimoto, T., and Reddy, J. K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1585-1590[Abstract/Free Full Text]
29. Hashimoto, T., Fujita, T., Usuda, N., Cook, W., Qi, C., Peters, J. M., Gonzalez, F. J., Yeldandi, A. V., Rao, M. S., and Reddy, J. K. (1999) J. Biol. Chem. 274, 19228-19236[Abstract/Free Full Text]
30. Reddy, J. K. (2001) Am. J. Physiol. Gastrointest. Liver Physiol. 281, G1333-G1339[Abstract/Free Full Text]
31. Li, Y., and Lazar, M. A. (2002) Mol. Endocrinol. 16, 1040-104819[Abstract/Free Full Text]
32. Walczak, R., and Tontonoz, P. (2002) J. Lipid Res. 43, 177-186[Abstract/Free Full Text]
33. Cook, K. S., Min, H. Y., Johnson, D., Chaplinsky, R. J., Flier, J. S., Hunt, C. R., and Spiegelman, B. M. (1987) Science 237, 402-405[Medline] [Order article via Infotrieve]
34. Bernholer, D. A., Angus, C. W., Lane, M. D., Bolanowsky, M. A., and Kelly, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 5468-5472[Abstract]
35. Hunt, C. R., Ro, J. H.-S., Dobson, D. E., Min, H. Y., and Spiegelman, B. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3786-3790[Abstract]
36. Hu, E., Liang, P., and Spiegelman, B. M. (1996) J. Biol. Chem. 271, 10697-10703[Abstract/Free Full Text]
37. Scherer, P. E., Williams, S., Fogliano, M., Baldini, G., and Lodish, H. (1995) J. Biol. Chem. 270, 26746-26749[Abstract/Free Full Text]
38. Bernholer, D. A., Coe, N. R., and LiCata, V. J. (1999) Semin. Cell Dev. Biol. 10, 43-49[CrossRef][Medline] [Order article via Infotrieve]
39. Razani, B., Combs, T. P., Wang, X. B., Frank, P. G., Park, D. S., Russell, R. G., Li, M., Tang, B., Jelicks, L. A., Scherer, P. E., and Lisanti, M. P. (2002) J. Biol. Chem. 277, 8635-8647[Abstract/Free Full Text]
40. Bleck, B., Hohoff, C., Bina, B., Rustow, B., Dixkens, C., Hameister, H., Borchers, T., and Spencer, F. (1998) Gene 215, 123-130[CrossRef][Medline] [Order article via Infotrieve]
41. Danesch, U., Hoeck, W., and Ringold, G. M. (1992) J. Biol. Chem. 267, 7185-7193[Abstract/Free Full Text]
42. Kawamura, M., Jensen, D. F., Wancewiz, E. W., Joy, L. L, Khoo, J. C., and Steinberg, D. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 732-736[Abstract]
43. Cherkaoui-Malki, M., Meyer, K., Cao, W.-Q., Latruffe, N., Yeldandi, A. V., Rao, M. S., Bradfield, C. A., and Reddy, J. K. (2001) Gene Expr. 9, 291-304[Medline] [Order article via Infotrieve]
44. Lugovskoy, A. A., Zhou, P., Chou, J. J., McCarty, J. S., Li, P., and Wagner, G. (1999) Cell 99, 747-755[Medline] [Order article via Infotrieve]
45. Montague, J. W., Hughes, F. M., Jr., and Cidlowsky, J. A. (1997) J. Biol. Chem. 272, 6677-6684[Abstract/Free Full Text]
46. Hazel, T. G., Nathans, D., and Lau, L. F. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8444-8449[Abstract]
47. Li, H., Kolluri, K. S., Gu, J., Dawson, M. I., Cao, X., Hobbs, P. D., Lin, B., Chen, G., Lu, J.-S., Lin, F., Xie, Z., Fontana, J. A., Reed, J. C., and Zhang, X.-K. (2000) Science 289, 1159-1164[Abstract/Free Full Text]
48. Ren, D., Collingwood, T. N., Rebar, E. J., Wolffe, A. P., and Camp, H. S. (2002) Genes Dev. 16, 27-32[Abstract/Free Full Text]
49. Forman, B. M., Chen, J., and Evans, R. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4312-4317[Abstract/Free Full Text]
50. Hu, E., Tontonoz, P., and Spiegelman, B. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9856-9860[Abstract]
51. Lee, Y., Yu, X., Gonzalez, F., Mangelsdorf, D. J., Wang, M.-Y., Richardson, C., Witters, L. A., and Unger, R. H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11848-11853[Abstract/Free Full Text]
52. Soukas, A., Socci, N. D., Saatkamp, B. D., Novelli, S., and Friedman, J. M. (2001) J. Biol. Chem. 276, 34167-34174[Abstract/Free Full Text]
53. Ross, S. E., Erickson, R. L., Gerin, I., DeRose, P. M., Bajnok, L., Longo, K. A., Misek, D. E., Kuick, R., Hanash, L., Atkins, K. B., Andersen, S. M., Nebb, H. I., Madsen, L., Kristiansen, K., and MacDougald, O. A. (2002) Mol. Cell. Biol. 22, 5989-5999[Abstract/Free Full Text]
54. Gerhold, D. L., Liu, F., Jiang, G., Li, Z., Xu, J., Lu, M., Sachs, J. R., Bagchi, A., Fridman, A., Holder, D. J., Doebber, T. W., Berger, J., Elbrecht, A., Moller, D. E., and Zhang, B. B. (2002) Endocrinology 143, 2106-2118[Abstract/Free Full Text]
55. Fan, J. Y., Carpenter, J.-L., van Obberghen, E., Grunfeld, C., Gorden, P., and Orci, L. (1983) J. Cell Sci. 61, 219-230[Abstract]
56. Kersten, S., Mandard, S., Tan, N. S., Escher, P., Metzger, D., Chambon, P., Gonzalez, F. J., Desvergne, B., and Wahli, W. (2000) J. Biol. Chem. 275, 28488-28493[Abstract/Free Full Text]
57. Yoon, J. C., Chickering, T. W., Rosen, E. D., Dussault, B., Qin, Y., Soukas, A., Friedman, J. M., Holmes, W. E., and Spiegelman, B. M. (2000) Mol. Cell. Biol. 20, 5343-5349[Abstract/Free Full Text]
58. Chinetti, G., Lestavel, S., Bocher, V., Remaley, A. T., Neve, B., Torra, I. P., Brewer, H. B., Fruchart, J. C., Clavey, V., and Staels, B. (2001) Nat. Med. 7, 53-57[CrossRef][Medline] [Order article via Infotrieve]
59. Inohara, N., Koseki, T., Chen, S., Wu, X., and Nunez, G. (1998) EMBO J. 17, 2526-2533[Abstract/Free Full Text]
60. Memon, R. A., Tecott, L. H., Nonogaki, K., Beigneux, A., Moser, A. H., Grunfeld, C., and Feingold, K. R. (2000) Endocrinology 141, 4021-4031[Abstract/Free Full Text]
61. Olefsky, J. M. (2000) J. Clin. Invest. 106, 467-472[Free Full Text]


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