PMC

P465L PPARγ mutation remodels the global lipidome of adipose tissue. Shown are fractional contributions to the total TG/PC pool. Significantly changed TG/PC values in P465L versus wt adipocytes are indicated.

(A to C) prkaca, d-akap1, and lipid droplet protein mRNA expression in adipose tissue from lean versus morbidly obese low-insulin-resistance and morbidly obese high-insulin-resistance subjects. (B and C) Adipocytes from obese subjects after acute exposure to rosiglitazone. (A) d-akap1 and prkaca mRNA expression is decreased in human obesity. (B)Rosiglitazone (Rosi) increases expression levels of d-akap1 and prkaca. *, differentiated (Diff) adipocytes. (C) Obesity severely affects the lipid droplet protein pool in human subjects. The data represent means ± SE. Differences in expression between groups were assessed by ANOVA and the Student t test. ★, P < 0.05 versus Diff; ★★, P < 0.005 versus Diff.

Obesity and P465L PPARγ mutation modify the architecture of the lipid droplet protein pool. Shown is lipid droplet protein expression analysis of P465L mutant adipocytes from lean and obese mice versus mature adipocytes isolated from 12- to 16-week-old wt counterparts. The data represent means ± SE for 8 to 12 animals per group. Fold changes were established using the wt as 100. Differences in expression between groups were assessed by ANOVA (G, effect of P465L mutation; OB, effect of obesity; G, OB, interactive effect) and the Student t test. *, P465L versus wt or P465L/ob/ob versus ob/ob; °, wt versus ob/ob or P465L versus P465L/ob/ob. The level of probability was set at a P value of <0.05.

(A) atgl mRNA levels are decreased in P465L mutant carriers. Shown is lipase expression analysis of P465L mutant adipocytes from lean and obese mice versus mature adipocytes isolated from 12- to 16-week-old wt counterparts. The data represent means ± SE for 8 to 12 animals per group. Fold changes were established using the wt as 100. Differences in expression between groups were assessed by ANOVA and the Student t test. *, P465L versus wt or P465L/ob/ob versus ob/ob; °, wt versus ob/ob or P465L versus P465L/ob/ob. The level of probability was set at a P value of <0.05. (B) HSL and ATGL protein levels are decreased in P465L carriers. Shown are the results of Western blotting of ser660-HSL (Tot HSL), ser565-HSL HSL, and ATGL. Differences in expression between groups were assessed by ANOVA (G, OB, and G × OB) and the Student t test. *, P465L versus wt or P465L/ob/ob versus ob/ob. The level of probability was set at a P value of <0.05.

PKAcatα and D-AKAP1 mRNA levels are decreased in P465L mutant carriers. (A) PKA subunit expression analysis of P465L mutant adipocytes from lean and obese mice versus mature adipocytes isolated from 12- to 16-week-old wt counterparts. (B) Protein levels of PKA subunits of P465L mutant adipocytes from lean and obese mice versus mature adipocytes isolated from wt counterparts. (C) akap-1 expression analysis of P465L mutant adipocytes from lean and obese mice versus mature adipocytes isolated from 12- to 16-week-old wt counterparts. The data represent means ± standard errors (SE) for 6 to 8 animals per group. Fold changes were established using the wt as 100. Differences in expression between groups were assessed by analysis of variance (ANOVA) (G, effect of P465L mutation; OB, effect of obesity; G × OB, interactive effect) and the Student t test. *, P465L versus wt or P465L/ob/ob versus ob/ob; °, wt versus ob/ob or P465L versus P465L/ob/ob. The level of probability was set at a P value of <0.05.

(A) P465L carriers are more sensitive to the antilipolytic action of insulin. Dose-response curves show the inhibitory effect of insulin on the induced glycerol release in isolated mature white adipocytes from wt and P465L adipocytes in lean and obese mice treated with NA. (B) Increased levels of AKT in P465L carriers. Shown are the effects of the selective phosphodiesterase inhibitors cilostamide (PDE3) and rolipram (PDE4) on the lipolytic inhibition elicited by insulin in the presence of NA in lean mice. (C) PDE proteins are not responsible for the increased sensitivity to the antilipolytic action of insulin in P465L carriers. Shown are the results of Western blotting of Ser473-AKT and total AKT. Differences in expression between groups were assessed by the Student t test. The level of probability was set at a P value of <0.05.

Transactivation of the PKAcat subunit α promoter-luciferase reporter construct (pka-luc) by PPARγ and the PPARγ P465L mutant in HEK293T cells. (A) PPARγ transcriptionally activates prkaca. The pka-luc reporter plasmid was transiently cotransfected with a plasmid expressing RXRα together with PPARγ, the P465L mutant, or a combination of both into HEK293T cells. At 24 h posttransfection, the cells were incubated with 1 μM rosiglitazone (open bars) or vehicle (solid bars) and further incubated for 24 h before being harvested and assayed for luciferase activity. Here, we show a representative experiment out of 3 independent experiments (n = 12 for each experimental condition). The luciferase activity measured in cells transfected with pka-luc and RXRα with pcDNA 3.1 as a control for PPARγ and the P465L mutant (basal condition) in the absence of rosiglitazone was set at 1. **, P < 0.0001, and *, P < 0.001 compared to the basal condition. &, P < 0.001 compared to PPARγ transfection (− rosiglitazone). §§, P < 0.0001, and §, P < 0.001 compared to the basal condition (+ rosiglitazone). #, P < 0.0001 compared to PPARγ transfection (+ rosiglitazone). (B and C) PPARγ binds a new PPRE identified in prkaca and d-akap1 promoters. Shown are electrophoretic mobility shift and supershift assays on putative PPRE of the murine PKA (mPKA) catalytic subunit α promoter and the mAKAP1 promoter. The 3 biotin-labeled double-stranded oligonucleotide probes corresponding to the mouse PKAcat subunit α PPRE (prkaca-ppre) (B) and d-akap-PPRE (C) were incubated with 5 μg of nuclear extract (NE) of HEK293T cells overexpressing PPARγ and RXRα in the absence or presence of PPAR antibody. The gel was transferred to a nylon membrane, and the shifted bands were detected by incubating the membrane with streptavidin-horseradish peroxidase, followed by chemiluminescence detection. Lanes 1, biotin-labeled double-stranded aP2-ppre probe incubated with nuclear extract; lanes 2, biotin-labeled aP2-ppre probe incubated with nuclear extract in the presence of PPAR antibody; lanes 3, PKAcat α-ppre or D-AKAP1 biotinylated probes alone; lanes 4, PKAcat α-ppre or d-akap1 biotinated probes with nuclear extract; lane 5, PKAcat α-ppre or d-akap1 biotinated probes incubated with nuclear extract in the presence of PPAR antibody; lanes 6 and 7, for both PKAcatα and D-AKAP1, 20× and 40× excesses of unlabeled double-stranded wt oligonucleotides were included as competitors with nuclear extract and labeled wt probe; lanes 8 and 9, for both PKAcatα and D-AKAP1, 20× and 40× excesses of unlabeled double-stranded mutated oligonucleotides were included as competitors with nuclear extract and labeled wt probes. (D) PPARγ binds a new PPRE identified in the prkaca promoter in vivo. Shown is a chromatin immunoprecipitation assay of the putative PPRE of the mPKAcat subunit α promoter and mAKAP1 promoter. Isolated mature adipocytes from gonadal pads of wt mice were collected, and ChIP assays were performed, using an anti-PPARγ antibody to immunoprecipitate PPARγ-linked DNA. Quantification of PPRE sequences for both prkaca and d-akap1 promoters was performed by real-time PCR, considering the fold change with respect to a control sequence in the same gene and normalized to the input DNA as described in Materials and Methods. The data are expressed as means and standard errors of the mean (SEM) from six independent experiments. gWAT, gonadal WAT.