PPARγ Transcription Deficiency Exacerbates High-Fat Diet-Induced Adipocyte Hypertrophy and Insulin Resistance in Mice

Figure 1. R134/135/138A mutations abolish the PPRE-binding ability and the transcriptional activity of PPARγ. (A) The structure of PPRE and PPARγ interaction shows that Arg134, Arg135 and Arg138 locate into the major groove of PPRE. PPRE is colored in light orange, and PPARγ DBD is in sky blue. (B) Binding model of PPARγ DBD on PPRE. Zinc finger domains of PPARγ DBD and Arg134, Arg135 and Arg138 are shown in the sequence of PPARγ DBD. (C, D) The binding of wild type (WT) PPARγ with PPRE (C) and the predicted binding model of R134/135/138A (3RA) mutant PPARγ with PPRE (D). Hydrogen bonds are indicated by dotted yellow lines. PPRE is colored in light orange, and the helix 1 of PPARγ DBD is in blue. The different groups of arginine and alanine are shown. The structure images in Figures 1A, C, D were generated by open source software PyMOL 099rc6 (www.pymol.org) and the chemical structures were drawn by chemdraw2014. (E) Dose response curve of biotin-PPRE with 100 nM of WT or 3RA mutant PPARγ CDE by AlphaScreen assay. (F) Dose response curve of WT or 3RA mutant PPARγ CDE with 1 nM of biotin-PPRE by AlphaScreen assay. Rosiglitazone (1 μM) does not affect the binding affinity of both WT and 3RA…

Figure 2. Generation of PPARγ 3RA mutant mouse model. (A) Schematics of the targeting strategy. Top: WT allele. Middle: recombinant allele. Bottom: constitutive knock-in allele after Cre recombination. (B) Genotyping. Genomic DNA extracted from mouse tail was used for PCR with primer pair F1/R1. The PCR product with one band of 348 bp represents the WT PPARγ, and PCR product with two bands (348 bp and 461 bp) represents the heterozygotes. (C) Sequencing analyses of PCR product using primer pair F2/R2 verified the mutations in PPARγ gene. Heterozygotes show two peaks in the codes of Arg134, Arg135 and Arg138, where one peak indicates the allele of wild-type (CGAAGA and CGA), the other is 3RA mutant (GCAGCA and GCA). The mutated nucleotides were indicated by arrows. (D) Homozygous PPARγ 3RA mutant (PPARγ3RA/3RA) mice are embryonic death. Heterozygous pregnant (mate with a heterozygous male) was dissected, about a quarter of the fetal mice in the womb were absorbed by the mother, leaving only the placenta. The ratio of newborn cubs (homozygote/heterozygote) is about 1/2 when we calculated total 74 cubs (n=74). (E) The protein level of PPARγ in the fat tissues of WT and PPARγ3RA/+ mice. (F) In…

Figure 3. PPARγ 3RA mutations in mice exacerbate HFD-induced obesity and adipocyte hypertrophy. (A) Body weight of PPARγ3RA/+ and WT littermates during a 15 weeks HFD feeding or normal chow diet. Data from the final time point were compared. Samples were collected from 15-week HFD-fed mice for assays. (B) Weights of white adipose tissue (inguinal white fat, iWAT; gonadal white fat, gWAT), brown fat (BAT) and (C) liver mass from PPARγ3RA/+ and WT littermates. (D) Representative histological analysis of iWAT, gWAT and BAT from PPARγ3RA/+ and WT littermates under a HFD by H&E staining. Inflammatory infiltration was labeled by red arrows. Original magnification, 200×. (E) Quantitative statistics of adipocyte size in (D). (F) Representative images of H&E stained liver sections of PPARγ3RA/+ and WT littermates. Original magnification: top, 100×; bottom, 200×. (G) Hepatic triglyceride level of PPARγ3RA/+ and WT littermates. (H, I) Fasting serum levels of total cholesterol (TCHO) (H), triglycerides (TG) (I), free fatty acids (FFA) (J), LDL-C (K) and HDL-C (L) of PPARγ3RA/+ and WT littermates. Values are means ± SEM, n=12 per group, *p < 0.05, **p < 0.01 and ***p < 0.001 by Student’s t test. For…

Figure 4. PPARγ 3RA mutations exacerbate HFD-induced insulin resistance in mice. Oral glucose tolerance test (OGTT) (A) and intraperitoneal insulin tolerance test (IPITT) (B) of 8-week old PPARγ3RA/+ and WT littermates with chow diet. From 8 weeks age, mice were fed with HFD for 15 weeks. Fasting blood glucose (C) and insulin (D) levels of mice fed with HFD. OGTT (E) and IPITT (F) of mice fed with HFD for 15 weeks. For (C, D), values are means ± SEM, n=6 per group, *p < 0.05, **p < 0.01 by Student’s t test.

Figure 5. Metabolic disorders in HFD-fed PPARγ3RA/+ mice were improved by rosiglitazone treatment. 8-week old male PPARγ3RA/+ and WT littermates were fed with HFD for 15 weeks, and then i.p. injected with 3 mg/kg of rosiglitazone once daily for 6 days. (A–F) Fasting serum levels of TCHO (A), TG (B) FFA (C), LDL-C (D) HDL-C (E) and glucose (F). (G) Representative images of H&E stained BAT and iWAT (original magnification, 200×). (H) Quantitative statistics of adipose cell size in (G). (I) Representative images of H&E stained liver sections (original magnification: top, 100×; bottom, 200×). (J) mRNA levels of PPARγ direct target genes involved in glucose and lipid metabolism in liver tissues of mice. For (A–F, J), measurement were repeated three times with similar results. Data show a representative experiment. Values are means ± SEM, n=6 per group, *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA followed by the Dunn’s test.