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A Dominant Negative Peroxisome Proliferator-activated Receptor-γ Knock-in Mouse Exhibits Features of the Metabolic Syndrome

2005; Elsevier BV; Volume: 280; Issue: 17 Linguagem: Inglês

10.1074/jbc.m407539200

1083-351X

Bethany D. Freedman, Eun Jig Lee, Youngkyu Park, J. Larry Jameson,

Metabolism, Diabetes, and Cancer

Peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPARγ mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPARγ L466A mutation, shown previously to inhibit wild-type PPARγ action in vitro. Homozygous PPARγ L466A knock-in mice die in utero. Heterozygous PPARγ L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPARγ function and the metabolic syndrome. Peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPARγ mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPARγ L466A mutation, shown previously to inhibit wild-type PPARγ action in vitro. Homozygous PPARγ L466A knock-in mice die in utero. Heterozygous PPARγ L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPARγ function and the metabolic syndrome. The metabolic syndrome, or syndrome X, is characterized by a constellation of insulin resistance, dyslipidemia, obesity, and hypertension (1Reaven G.M. Diabetes. 1988; 37: 1595-1607Crossref PubMed Google Scholar). The prevalence of the metabolic syndrome is increasing rapidly and is estimated to affect 24% of the United States population (2Ford E.S. Giles W.H. Dietz W.H. J. Am. Med. Assoc. 2002; 287: 356-359Crossref PubMed Scopus (5800) Google Scholar). Thiazolidinediones, a class of antidiabetic compounds that activate the peroxisome proliferator-activated receptor-γ (PPARγ), 1The abbreviations used are: PPARγ, peroxisome proliferator-activated receptor-γ; FFA, free fatty acid; TG, triglyceride; PPARKI, PPARγ L466A knock-in; eWAT, epididymal white adipose tissue; WT, wild type; SREBP, sterol regulatory element-binding protein; HF, high fat; KI, knock-in; 2-DG, deoxyglucose; FKOγ, fat-specific PPARγ knock-out; TNFα, tumor necrosis factor α; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labeling; RT, reverse transcription. stimulate adipocyte differentiation, lower free fatty acids (FFAs), and enhance insulin sensitivity, thereby correcting several features of the metabolic syndrome (3Mukherjee R. Davies P.J. Crombie D.L. Bischoff E.D. Cesario R.M. Jow L. Hamann L.G. Boehm M.F. Mondon C.E. Nadzan A.M. Paterniti Jr., J.R. Heyman R.A. Nature. 1997; 386: 407-410Crossref PubMed Scopus (576) Google Scholar). The insulin-sensitizing action of the thiazolidinediones suggests that PPARγ function may be central to the development and treatment of the metabolic syndrome. PPARγ is a member of the nuclear hormone receptor superfamily and plays a pivotal role in adipogenesis (4Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar, 5Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar, 6Auwerx J. Diabetologia. 1999; 42: 1033-1049Crossref PubMed Scopus (583) Google Scholar). Although PPARγ is most highly expressed in adipose tissue, it is also present in colon, monocytes/macrophages, and at lower levels in many other tissues including skeletal muscle and liver (7Vidal-Puig A.J. Considine R.V. Jimenez-Linan M. Werman A. Pories W.J. Caro J.F. Flier J.S. J. Clin. 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Spiegelman B.M. Mol. Cell. Biol. 2003; 23: 7222-7229Crossref PubMed Scopus (129) Google Scholar). Muscle-specific PPARγ knock-out mice show progressive insulin resistance combined with increased adipose tissue mass (14Hevener A.L. He W. Barak Y. Le J. Bandyopadhyay G. Olson P. Wilkes J. Evans R.M. Olefsky J. Nat. Med. 2003; 9: 1491-1497Crossref PubMed Scopus (420) Google Scholar, 16Norris A.W. Chen L. Fisher S.J. Szanto I. Ristow M. Jozsi A.C. Hirshman M.F. Rosen E.D. Goodyear L.J. Gonzalez F.J. Spiegelman B.M. Kahn C.R. J. Clin. Investig. 2003; 112: 608-618Crossref PubMed Scopus (367) Google Scholar). Fat-specific PPARγ knock-out mice have lipodystrophy (hypocellularity and hypertrophy), elevated plasma FFAs and triglycerides (TGs) and decreased plasma leptin and adiponectin. These mice have insulin resistance in fat and liver but not in muscle (13He W. Barak Y. Hevener A. Olson P. Liao D. Le J. Nelson M. Ong E. Olefsky J.M. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15712-15717Crossref PubMed Scopus (798) Google Scholar). There are several reports of heterozygous PPARγ mutations in humans (18Agarwal A.K. Garg A. J. Clin. Endocrinol. Metab. 2002; 87: 408-411Crossref PubMed Scopus (256) Google Scholar, 19Hegele R.A. Cao H. Frankowski C. Mathews S.T. Leff T. Diabetes. 2002; 51: 3586-3590Crossref PubMed Scopus (230) Google Scholar, 20Barroso I. Gurnell M. Crowly V.E.F. Agosttini M. Schwabw J.W. Soos M.A. Malsen G. Williams T.D. Lewis H. Schafer A.J. Chatterjee V.K.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1166) Google Scholar, 21Savage D.B. Tan G.D. Acerini C.L. Jebb S.A. Agostini M. Gurnell M. Williams R.L. Umpleby A.M. Thomas E.L. Bell J.D. Dixon A.K. Dunne F. Boiani R. Cinti S. Vidal-Puig A. Karpe F. Chatterjee V.K. O'Rahilly S. Diabetes. 2003; 52: 910-917Crossref PubMed Scopus (363) Google Scholar). These patients exhibit partial lipodystrophy, severe insulin resistance, steatohepatitis, and hypertension (20Barroso I. Gurnell M. Crowly V.E.F. Agosttini M. Schwabw J.W. Soos M.A. Malsen G. Williams T.D. Lewis H. Schafer A.J. Chatterjee V.K.K. O'Rahilly S. Nature. 1999; 402: 880-883Crossref PubMed Scopus (1166) Google Scholar, 21Savage D.B. Tan G.D. Acerini C.L. Jebb S.A. Agostini M. Gurnell M. Williams R.L. Umpleby A.M. Thomas E.L. Bell J.D. Dixon A.K. Dunne F. Boiani R. Cinti S. Vidal-Puig A. Karpe F. Chatterjee V.K. O'Rahilly S. Diabetes. 2003; 52: 910-917Crossref PubMed Scopus (363) Google Scholar). These clinical manifestations include many features of the metabolic syndrome and are thought to result from a combination of loss of function and the dominant negative effects of the mutant PPARγ receptors. In this study, we created a knock-in mouse model containing a PPARγ L466A mutation, shown previously to inhibit wild-type PPARγ action in vitro (22Park Y. Freedman B.D. Lee E.J. Park S. Jameson J.L. Diabetologia. 2003; 46: 365-377Crossref PubMed Scopus (264) Google Scholar). The potent dominant negative activity of the L466A mutation, located in the transactivation domain of PPARγ, is because of preserved DNA binding activity in combination with loss of coactivator binding but preserved corepressor recruitment (22Park Y. Freedman B.D. Lee E.J. Park S. Jameson J.L. Diabetologia. 2003; 46: 365-377Crossref PubMed Scopus (264) Google Scholar). Homozygous PPARγ L466A knock-in mice die in utero, but heterozygous PPARγ L466A knock-in (PPARKI) mice are viable and manifest many of the features seen in patients with dominant negative PPARγ mutations. Generation of Mice—An exon 6 fragment of PPARγ was used to screen a 129Sv/J mouse genomic library, resulting in the isolation of a 16-kb fragment of the PPARγ gene. Site-directed mutagenesis was used to mutate the leucine residue at codon 466 to alanine (L466A). Three nucleotides were modified (CCT to AGC) to generate a novel Eco47III enzyme restriction site. A targeting vector was constructed that contained most of the 16-kb clone, modified to include the L466A mutation, along with loxP-flanked neomycin and thymidine kinase cassettes in reverse orientation (Fig. 1A). The vector was linearized by KpnI digestion, electroporated into 129Sv/J R1 ES cells (23Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (1992) Google Scholar), and subjected to selection with G418 and gancyclovir. PCR and Southern blot analysis were used to identify homologous recombination at the PPARγ locus. Two clones identified as carrying the L466A mutation were injected into murine blastocysts and implanted into pseudo-pregnant mice. Male chimeric mice were mated with female 129Sv/J mice, and germ line transmission was confirmed by PCR screening and direct sequencing. Mice were bred to CMV-Cre transgenic mice (129Sv/J background) to excise the neomycin cassette and were maintained on a 129Sv/J background. The 3-bp difference between the L466A mutated allele (CCT) and the wild-type PPARγ allele (AGC) was used to confirm genotype (Fig. 1B). Phenotypic Evaluation of Mice—Mice were housed in a pathogen-free barrier facility, maintained on a 12-h light/dark cycle, and had ad libitum access to chow (Harlan Teklad Laboratory, Madison, WI) that contained 8 kcal % from fat unless otherwise specified. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Northwestern University Feinberg School of Medicine or Vanderbilt Medical School in accordance with National Institutes of Health guidelines. In some experiments, mice were fed a high fat diet that contained 30 kcal % from fat (Harlan Teklad) or 45 kcal % from fat (Research Diets Inc., New Brunswick, New Jersey). In these experiments, other mice were fed standard control diets, 10 kcal % from fat, provided by the same suppliers. Animals were weighed on a weekly basis. When the animals were fasted for glucose and insulin tolerance tests, the fast lasted 6–8 h and was typically performed between 8 a.m. and 2 p.m. Blood Collection and Serum Measurements—Blood samples were obtained by either the tail-cut method for small samples (<50 μl) or by retro-orbital bleeding under light anesthesia (halothane). Serum was stored at –80 °C. Fasting (6–8 h) and fed blood glucose levels were measured using a glucometer (FreeStyle™, TheraSense, Alameda, CA). Serum FFA measurements were performed using a diagnostic kit (Roche Diagnostics). Enzyme-linked immunosorbent assay kits were used to measure serum insulin (Linco Research, St. Charles, MO), adiponectin (Linco), and TNFα (Chemicon International, Inc., Temecula, CA). Tissue Histology, Morphology, Immunohistochemistry, and Cell Counting—Tissues were fixed in either Bouin's buffer (white adipose tissue) or 10% neutral buffered formalin (brown adipose tissue, liver) and paraffin-embedded. 5-μm sections were stained by hematoxylin/eosin. Immunohistochemical staining for adipophilin (guinea pig polyclonal, Research Diagnostics, Inc.), immunofluorescent staining for proliferating cellular nuclear antigen (PCNA; mouse monoclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL, Roche Applied Science) were performed. Alternatively, tissues were directly embedded in Tissue-Tek® O.C.T. Compound (Sakura Finetek, Inc., Torrance, CA), and 8-μm frozen sections were stained by Oil red O. Digital images were obtained using a Zeiss Axioskop optical microscope (Carl Zeiss International) and an Optronics Magnafire SP digital camera (Goleta, CA). The measurements feature of the Image-Pro Express software program (Media Cybernetics, Inc., Silver Spring, MD) was used to count the number of cells and to determine cell surface area in the digital images. Lipid Extraction and Triglyceride Measurement—Mouse liver was homogenized in 1 m NaCl. Liver tissue homogenate (100 mg/500 μl) was extracted with 3 ml of chloroform/methanol (2:1) and 0.5 ml of 1 m NaCl. The organic phase was collected, dried, and resuspended in 0.5 ml of Triton X-100/methanol (2:1). Muscle triglycerides were extracted as described previously (24Frayn K.N. Maycock P.F. J. Lipid Res. 1980; 21: 139-144Abstract Full Text PDF PubMed Google Scholar). Triglycerides were measured using the GPOTrinder kit (Sigma). RNA Isolation and Real-time RT-PCR—RNA from epididymal white adipose tissue (eWAT) was isolated using TRIzol (Invitrogen). The isolated RNA was subjected to DNase treatment and reverse-transcribed, and 100 ng of cDNA was used as template in a 25-μl real-time PCR reaction using an ICycler iQ System (Bio-Rad). Primers and 5′-FAM labeled probes for real-time detection were designed using the Primer Express software (PerkinElmer Life Sciences), purchased from Integrated DNA Technologies (Coralville, IA). Primer/probe sets (Table I) were designed for aP2, Acrp30, CD36, FAS, GLUT4, LPL, PPARγ, RPL19, SREBP1c, and TNFα (GenBank™ accession numbers K02109, AF304466, L23108, AF127033, AB008453, BC003305, U10374, M62952, AF374266, and M38296, respectively). Amplification of the ribosomal housekeeping gene RPL19 was used as a control, and cycle thresholds were corrected relative to RPL19 expression.Table IPrimer and probe sequences used for real-time RT-PCRGenePrimersProbeaP25′-CACCATCCGGTCAGAGAGTACTTT-3′5′-CACCGAGATTTCCTTCAAACTGGGCG-3′5′-GCCATCTAGGGTTATGATGCTCTT-3′Acrp305′-GCAAGCTCTCCTGTTCCTCTTAAT-3′5′-CTCCTGCTTTGGTCCCTCCACCCA-3′5′-CCATCCAACCTGCACAAGTTC-3′CD365′-GCCAAGCTATTGCGACATGAT-3′5′-CACAGACGCAGCCTCCTTTCCACCT-3′5′-TCAGATCCGAACACAGCGTAGAT-3′FAS5′-AGGTATCCATTCTGGGTTCTAGCC-3′5′-CCTACCCGTGTGACCGCCATCTATATCG-3′5′-GCTCGTTGTCACATCAGCCA-3′GLUT45′-CTGCTTCTGGCTCTCACAGTACTC-3′5′-ATTCTGCTGCCCTTCTGTCCTGAGAGC-3′5′-AGGTTCCGGATGATGTAGAGGTAT-3′LPL5′-GATGCCCTACAAAGTGTTCCATTA-3′5′-CAAGCAACACAACCAGGCCTTCGAA-3′5′-CCACTGTGCCGTACAGAGAAAT-3′PPARγ5′-CTGCTCAAGTATGGTGTCCATGAG-3′5′-CATCTACACGATGCTGGCCTCCCTGA-3′5′-GAGGAACTCCCTGGTCATGAATC-3′RPL195′-CAACTCCCGTCAGCAGATCAG-3′5′-TGACTGTCCATTCCCGGGCTCG-3′5′-TACCCTTCCTCTTCCCTATGCC-3′SREBP1c5′-AAGGCCATCGACTACATCCG-3′5′-CAGCACAGCAACCAGAAGCTCAAGCA-3′5′-GCTTTTGTGTGCACTTCGTAGG-3′TNFα5′-AGCCGATGGGTTGTACCTTGT-3′5′-CGTCAGCCGATTTGCTATCTCATACCAGG-3′5′-CGGCAGAGAGGAGGTTGACTT-3′ Open table in a new tab Protein Isolation and Western Blotting—Whole cell extracts were prepared from adipose tissue that had been removed from mice and immediately frozen in liquid N2. The adipose tissue was pulverized and homogenized in lysis buffer, and the protein concentration was determined by the DC protein assay (Bio-Rad). The extracts were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham Biosciences). Immunodetection was carried out using the following antibodies in 5% milk/Tris-buffered saline with 0.05% Tween 20: rabbit SREBP-1 polyclonal (1:200) (Santa Cruz Biotechnology), rabbit PPARγ polyclonal (1:2000) (Abcam, Inc., Cambridge, MA), and goat anti-rabbit horseradish peroxidase-conjugated IgG (1:10,000) (Cell Signaling, Beverly, MA). Proteins were visualized with an ECL Plus detection kit (Amersham Biosciences) according to the manufacturer's instructions. Indirect Calorimetry—Oxygen consumption (VO2) and the respiratory exchange ratio were measured by an Oxymax indirect calorimeter (Columbus Instruments, Columbus, OH) with an air flow of 0.75 liters/min as described previously (25Ma L.J. Mao S.L. Taylor K.L. Kanjanabuch T. Guan Y. Zhang Y. Brown N.J. Swift L.L. McGuinness O.P. Wasserman D.H. Vaughan D.E. Fogo A.B. Diabetes. 2004; 53: 336-346Crossref PubMed Scopus (353) Google Scholar). VO2 is expressed as the volume of O2 consumed per kilogram of body weight per h. After 1 h, to allow for adaptation to the metabolic chamber, VO2 was measured, starting at 10:00 in individual mice for 1 min at 15-min intervals for a total of 22 h under a consistent environmental temperature (22 °C). The respiratory exchange ratio is the ratio of the volume of CO2 produced to the volume of O2 consumed. Tail Cuff Blood Pressure Measurements—Systolic blood pressure was determined with the use of the Visitech Systems BP-2000 blood pressure monitoring system, an automated device for measuring systolic blood pressure by tail cuff, as described previously (26Krege J.H. Hodgin J.B. Hagaman J.R. Smithies O. Hypertension. 1995; 25: 1111-1115Crossref PubMed Scopus (489) Google Scholar). Eleven-month-old male and female mice were subjected to tail cuff blood pressure measurements prior to catheterization for clamp studies. Mice were trained for 3 consecutive days; base line systolic pressure was measured and averaged over 5 days. Hyperinsulinemic-Euglycemic Clamp and 2-Deoxyglucose Uptake Measurements—Clamp studies were performed in chronically catheterized conscious mice in the Mouse Metabolic Phenotyping Center at Vanderbilt University Medical Center. Male mice, 10–11 months of age, had two catheters surgically implanted into the right jugular vein and left carotid artery as described previously (27Halseth A.E. Bracy D.P. Wasserman D.H. Am. J. Physiol. 1999; 276: E70-E77PubMed Google Scholar). Five days post-surgery, animals were fasted for 6 h, and hyperinsulinemic-euglycemic clamp experiments with [3H]deoxyglucose (2-DG) tracer were performed as described previously (27Halseth A.E. Bracy D.P. Wasserman D.H. Am. J. Physiol. 1999; 276: E70-E77PubMed Google Scholar). After basal sampling at time 0, insulin was continuously infused at 4 milliunits kg–1 min–1 for the duration of the 2-h study. Blood glucose levels were measured from 8 μl of blood every 5–10 min using a HemoCue glucose analyzer (Lake Forest, CA). Glucose was infused at a variable rate to maintain clamped blood glucose levels at 120–130 mg/dl. Tracer was added at t = 120 min and continued until t = 145 min, at which time the animals were anesthetized with an intravenous infusion of pentobarbital sodium. Tissues (soleus, gastrocnemius, superficial vastus lateralis, adipose tissue, liver, diaphragm, and brain) were excised and rapidly frozen in liquid nitrogen for subsequent glucose uptake analysis. 2-[3H]DG removal from the blood and uptake by various tissues was determined as described previously (27Halseth A.E. Bracy D.P. Wasserman D.H. Am. J. Physiol. 1999; 276: E70-E77PubMed Google Scholar). Statistical Analysis—Data are presented as means ± S.E. Comparisons between two experimental groups were performed using the twotailed, unpaired Student's t test; analysis of variance was used for multivariate comparisons. Heterozygous PPARKI mice were viable and fertile. RT-PCR of mRNA from adipose tissue was used to verify equivalent expression of both PPARγ alleles. Routine breeding of heterozygotes failed to produce pups homozygous for the L466A allele. Timed matings of heterozygotes demonstrated that PPARγ L466A homozygotes die in utero by embryonic day 10.5 (Fig. 1C). The PPARγ L466A mutant has no inherent transcriptional activity (22Park Y. Freedman B.D. Lee E.J. Park S. Jameson J.L. Diabetologia. 2003; 46: 365-377Crossref PubMed Scopus (264) Google Scholar). The lack of homozygote viability is consistent with the embryonic death seen in homozygous PPARγ null animals (9Barak Y. Nelson M.C. Ong E.S. Jones Y.Z. Ruiz-Lozano P. Chien K.R. Koder A. Evans R.M. Mol. Cell. 1999; 4: 585-595Abstract Full Text Full Text PDF PubMed Scopus (1659) Google Scholar). Histological analysis of PPARKI eWAT and brown adipose tissue revealed altered adipocyte morphology (Fig. 2A). PPARKI eWAT contains a heterogeneous population of adipocytes with a predominance of small and medium sized cells, whereas eWAT in wild-type (WT) mice is composed of a nearly homogenous population of larger adipocytes. PPARKI brown adipocytes contain large multiloculated fat vacuoles that were not present in WT mice (Fig. 2A). The number of PPARKI eWAT adipocytes per high powered field (×400) is ∼2-fold greater than that of WT mice (Fig. 2B), suggesting that the decreased size of the adipocytes is the cause of an overall reduction in fat mass. To investigate whether there is altered cellular proliferation or apoptosis in PPARKI eWAT, immunofluorescent staining for PCNA and TUNEL was performed. PCNA positivity (5.3 ± 4.8 and 4.0 ± 1.8) and TUNEL staining (1.5 ± 1.2 and 2.8 ± 1.6; number of nuclei per 100 4′,6-diamidino-2-phenylindole-labeled nuclei) were not different between PPARKI and WT eWAT, respectively. Real-time RT-PCR analysis was performed to investigate whether there is altered expression of adipocyte-specific genes in eWAT. The expression of adiponectin (Acrp30), aP2, CD36, GLUT4, and SREBP-1c mRNAs in PPARKI mice was 25–50% of that measured in WT mice (Fig. 2C). Western blot analysis showed that the amount of 125-kDa nuclear envelope-bound SREBP-1 was greater in PPARKI eWAT relative to WT eWAT (Fig. 2D). In contrast, the transcriptionally active 68-kDa protein was the predominant form of SREBP-1 expressed in WT eWAT (Fig. 2D). This finding suggests decreased activation of SREBP-1 in PPARKI adipose tissue. The levels of PPARγ protein expression did not differ between PPARKI and WT. We also performed Western blot analysis of insulin-signaling proteins in adipose tissue 10 min after an intraperitoneal insulin injection. No difference in the protein levels of IRβ, IRS-1, IRS-2, Akt, or phospho-Akt was found between PPARKI and WT mice (data not shown). PPARKI mice had normal birth weights. Weight gain was similar for PPARKI and WT littermates for the first 3 months. However, from 3 months of age, PPARKI mice gained less weight on either a standard (10 kcal % from fat) or high fat (HF) diet (45 kcal % from fat) than WT mice (Fig. 3A). These mice had reduced eWAT depots, directly correlating with total body weight. To investigate the cause of abrogated weight gain in PPARKI mice, food intake and oxygen consumption were evaluated. No significant changes in food intake (Fig. 3B) or oxygen consumption were detected (Fig. 3C). In the fed state, PPARKI mice had higher serum FFA levels on a standard diet than WT mice. Serum FFAs were elevated in WT mice on a HF diet, but no further elevation was observed in PPARKI mice (Fig. 3D). An intraperitoneal injection of insulin (0.75 units/kg) lowered serum FFAs in fasting male mice (7–8 months old) by ∼30% in both WT (preinjection: 0.77 ± 0.12 mm, postinjection: 0.54 ± 0.21 mm) and PPARKI (preinjection: 0.96 ± 0.16 mm, postinjection: 0.64 ± 0.28 mm) mice. Serum adiponectin levels in PPARKI mice were significantly lower (p < 0.001) than those in WT mice on either a standard or HF diet (Fig. 3E). No difference in serum TNFα was detected (Fig. 3F), although mRNA expression for TNFα was decreased in PPARKI eWAT. Fasting (for 6 h) and fed blood glucose levels were not significantly different between PPARKI (96 ± 18 and 102 ± 12 mg/dl) and WT (98 ± 22 and 94 ± 9 mg/dl) mice (7–8-month-old males) on a standard diet. Histological examination of the livers of 5-month-old male PPARKI mice showed macrovesicular fatty changes in mid-zonal and centrilobular locations, indicating hepatic steatosis. Hepatic steatosis becomes apparent as early as 4 months of age. With a HF diet (for 3 months), macrovesicular lipid accumulation was observed in both genotypes. Additional microvesicular fatty changes were seen around the central vein in PPARKI livers (Fig. 4A). The lipid accumulation was confirmed by Oil red O staining and adipophilin immunohistochemistry (data not shown). Inflammatory infiltrates were also detected in some PPARKI livers (not shown). The hepatic TG content (22.1 ± 5.2 mg/g liver) of PPARKI mice was 1.8-fold greater than that (12.3 ± 4.9) of WT mice (Fig. 4B). Although the hepatic steatosis appeared to be exacerbated by a HF diet in PPARKI livers, the hepatic TG content (24.9 ± 6.8) increased by only 13% when compared with a standard diet. WT mice exhibited a 39% increase in hepatic TG content (17.1 ± 4.6) on a HF diet. Muscle TG content was also measured, but there was no difference between PPARKI and WT mice (data not shown). Hyperinsulinemic-euglycemic clamp studies and 2-[3H]DG tracer studies were used to further investigate insulin action in PPARKI mice. Four groups of male mice were studied at 11 months of age, having been subjected to a standard diet (10 kcal % from fat) or a high fat diet (30 kcal % from fat) for 8 months. Basal glucose and insulin levels were comparable in PPARKI and WT mice on a standard diet (Fig. 5A). PPARKI mice on a standard diet exhibited no apparent impairment in 2-DG uptake and no insulin resistance compared with WT (Fig. 5). HF diet-fed PPARKI mice exhibited basal hyperinsulinemia (Fig. 5A). In clamp studies, glucose infusion rates were significantly lower (p < 0.05) in PPARKI mice on a HF diet, indicating insulin resistance (Fig. 5B). 2-DG uptake into skeletal muscle was also severely impaired (p < 0.05) (Fig. 5C). These results indicate that PPARKI mice have insulin resistance when exposed to a HF diet. Tail cuff blood pressure (B.P.) measurements on 11-month-old mice demonstrate that female PPARKI mice are overtly hypertensive on either a standard or HF diet (Fig. 6A). However, male PPARKI did not have hypertension (Fig. 6B). In this study, we created a knock-in mouse model (PPARKI) containing a dominant negative PPARγ L466A mutation, shown previously to inhibit wild-type PPARγ action in vitro. The compromised PPARγ function in PPARKI mice leads to a lipodystrophy with decreased expression of adipogenic genes, resulting in high circulating FFAs, low circulating adiponectin, hepatic steatosis, and HF diet-induced insulin resistance. The PPARKI mouse model confirms the importance of PPARγ in adipose tissue maintenance and insulin-resistant states (28Kahn B.B. Flier J.S. J. Clin. Investig. 2000; 106: 473-481Crossref PubMed Scopus (2488) Google Scholar). The heterogeneity and predominance of smaller sized adipocytes may reflect a combination of loss of function and the dominant negative activity of the PPARγ L466A allele. Smaller adipocytes were also described in the heterozygous PPARγ-deficient mouse model, and these smaller adipocytes were associated with enhanced insulin sensitivity (10Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999;