Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARγ activation to decrease diet-induced obesity

Abstract

De novo lipogenesis in adipocytes, especially with high fat feeding, is poorly understood. We demonstrate that an adipocyte lipogenic pathway encompassing fatty acid synthase (FAS) and PexRAP (peroxisomal reductase activating PPARγ) modulates endogenous PPARγ activation and adiposity. Mice lacking FAS in adult adipose tissue manifested increased energy expenditure, increased brown fat-like adipocytes in subcutaneous adipose tissue, and resistance to diet-induced obesity. FAS knockdown in embryonic fibroblasts decreased PPARγ transcriptional activity and adipogenesis. FAS-dependent alkyl ether phosphatidylcholine species were associated with PPARγ and treatment of 3T3-L1 cells with one such ether lipid increased PPARγ transcriptional activity. PexRAP, a protein required for alkyl ether lipid synthesis, was associated with peroxisomes and induced during adipogenesis. PexRAP knockdown in cells decreased PPARγ transcriptional activity and adipogenesis. PexRAP knockdown in mice decreased expression of PPARγ-dependent genes and reduced diet-induced adiposity. These findings suggest that inhibiting PexRAP or related lipogenic enzymes could treat obesity and diabetes.

Figure 1. Targeted Deletion of Adipose Tissue FAS Decreases Adiposity

(A) FAS protein by Western blot in brown (BAT) and white (WAT) adipose tissue of Lox/Lox control (without Cre), adiponectin-Cre control (without floxed alleles), and FASKOF mice. (B) Tissue distribution of FAS protein by Western blot. An apparent increased expression of hepatic FAS protein in FASKOF mice was not consistently observed. (C) FAS enzyme activity assay. *P=0.031. N=4/genotype. (D) Body weight of HFD-fed control and FASKOF male mice. Similar results were also obtained in two additional feeding experiments with different cohorts of male mice. *P=0.03. **P=0.0068 at 16 weeks, 0.0028 at 20 weeks. N=6–8/genotype. Additional data including females are provided in Table S1. (E) MRI analysis of body composition in HFD-fed mice. **P<0.0001. N=6/genotype. (F) Tissue weights of HFD-fed control and FASKOF mice. **P=0.005. N=6/genotype. (G) Histologic appearance of WAT harvested from chow-fed or HFD-fed mice. (H) Adipocyte size distribution determined with the NIH Image J program. Error bars in panels C–F represent SEM.

Figure 2. Altered Metabolism in Mice with Adipose-specific Knockout of FAS

(A) Oxygen consumption (VO₂) by indirect calorimetry in HFD-fed mice. Indicated P value by ANOVA. N=8–10/genotype. (B) Glucose tolerance testing in HFD-fed mice. P=0.0477 at 0 min, 0.0415 at 60 min. N=6–8/genotype. Serum insulin values at 30 min point shown in the inset. (C) Insulin tolerance testing in the mice of panel B. *P=0.039. (D) RT-PCR analysis of gene expression in inguinal WAT of HFD-fed control and FASKOF male mice. Gene expression analysis in inguinal WAT of ZFD-fed mice is presented in Supplementary Figure 1H. **P=<0.0001 for UCP1, 0.0017 for Cidea, 0.0001 for PRDM16, 0.0008 for PGC1α, 0.0012 for PPARa, and 0.0001 for CPT1; *P=0.042 for ACO. (E) Measurement of fatty acid oxidation in epididymal (eWAT) and inguinal (iWAT) fat of control and FASKOF mice fed HFD. *P=0.0355 for HFD iWAT. N=3 animals/genotype for each diet. (F) Western blot analysis in inguinal WAT of ZFD-fed control and FASKOF male mice. Each lane represents a separate mouse. (G) Immunocytochemical analysis of UCP1 expression in inguinal WAT of ZFD-fed control and FASKOF mice. Images are from two separate mice per genotype. (H) Rectal temperature of ZFD-fed control and FASKOF mice at room temperature (23°C) and after 1 hr exposure to 4°C. N=6–8 animals/genotype. *P=0.011. Error bars in panels A–E and H represent SEM.

Figure 3. FAS is Required for Adipogenesis and PPARγ Activation

(A) Western blot analysis of FAS knockdown in primary MEFs from FAS^lox/lox mice treated with an adenovirus expressing GFP or Cre at the indicated multiplicity of infection (MOI). (B) Oil red O staining of FAS^lox/lox MEFs treated with Ad-GFP or Ad-Cre and differentiated to adipocytes in the presence or absence of rosiglitazone. (C) HEK 293 cells treated with control or FAS siRNA were transfected with plasmids encoding PPRE-luciferase, Renilla luciferase and wild type PPARγ in the presence or absence of rosiglitazone. **P<0.0001 vs. control, #P<0.0001 vs. FAS siRNA basal. N=3/condition. (D) HEK 293 cells treated with control or FAS siRNA were transfected with plasmids encoding PPRE-luciferase, Renilla luciferase and wild type PPARγ or VP16-PPARγ DBD (an N-terminal fragment of PPARγ encompassing the DNA binding domain fused to the VP16 transactivation domain). **P<0.0001 vs. control, #P<0.0001 vs. FAS siRNA/WT PPARγ. N=3/condition. (E) RT-PCR analysis of gene expression in FAS-deficient (Ad-Cre-treated) or control (Ad-GFP-treated) MEFs subjected to the adipogenesis protocol. **vs. Ad-GFP, P=0.0060 for aP2, 0.0010 for CD36, 0.0051 for MyoD, 0.0007 for Myogenin. #vs. Ad-Cre, P=0.0015 for aP2, 0.0013 for CD36, 0.0099 for MyoD, 0.0019 for Myogenin. (F) FAS-deficient (Ad-Cre-treated) or control (Ad-GFP-treated) MEFs cultured to promote myogenesis and stained with a skeletal muscle myosin heavy chain antibody. (G) Detection of proteins induced by PPARγ in 3T3-L1 fibroblasts and adipocytes treated with control or FAS shRNA. (H) Restoration of PPARγ target gene expression with human FAS using 3T3-L1 adipocytes with endogenous knockdown of FAS. 3T3-L1 cells stably expressing retrovirally encoded human FAS were infected with a lentivirus expressing scrambled control (SC) or mouse FAS shRNA. The cells were induced to differentiate into adipocytes. The upper panel shows real-time PCR analysis of aP2 expression and the bottom panel shows a Western blot with antibodies against FAS, HA and actin. *P=0.0224 (vs. SC shRNA, empty vector). #P<0.0001 (vs. FAS shRNA, empty vector). (I) RT-PCR analysis of gene expression in control and FASKOF gonadal WAT. **P=0.007. *P=0.0493 for C/EBPα, 0.010 for LPL, 0.039 for CD36. N=4/genotype. (J) Western blot analysis in gonadal WAT of ZFD-fed control and FASKOF female mice. Each lane represents a separate mouse. Error bars in panels C–E and H–I represent SEM.

Figure 4. Isolation of FAS-dependent Diacyl and 1-O-alkyl Ether Phosphatidylcholine Species Associated with PPARγ

(A) Strategy for detection of PPARγ-associated lipids. (B) Detection of FLAG-PPARγ protein immunoprecipitated from adipocytes treated with control or FAS shRNA. (C) Mass spectrometric analyses of [M+Li]⁺ ions of glycerophosphocholine (GPC) lipids bound to FLAG-PPARγ or control protein (GFP) immunoprecipitated from control and FAS knockdown adipocytes. Ions of m/z 752 and 780 represent 1-O-alkyl GPC species. (D) CV-1 cells were transfected with plasmids encoding UAS-luciferase, Renilla luciferase and Gal4-PPARγ LBD (a C-terminal fragment of PPARγ encompassing the ligand binding domain fused to the Gal4 DNA binding domain) or Gal4 alone. The cells were treated with 18:1e/16:0-GPC (corresponding to m/z 752 in panel C), rosiglitazone or DMSO. After 48 hrs, UAS-luciferase reporter activity was measured and normalized to Renilla luciferase reporter activity. **P=0.0001. *P=0.018 (10 μM), 0.024 (20 μM), 0.019 (80 μM). (E) 3T3-L1 cells were induced to differentiate in DMEM+10% FBS with supplemental dexamethasone, insulin and IBMX in the presence of 20 μM 18:1e/16:0-GPC or DMSO. After 3 days, the cells were re-treated with the GPC in media containing supplemental insulin alone. The next day, the cells were harvested for RNA extraction and real-time PCR analysis. The data are representative of 3 separate experiments. *P=0.0147 (aP2), 0.0006 (LPL), 0.0102 (CD36). Error bars in panels D and E represent SEM.

Figure 5. Cloning and Characterization of the Terminal Component in the Mammalian Peroxisomal Ether Lipid Synthetic Pathway

(A) The peroxisomal acyl-DHAP pathway of lipid synthesis. FAS, fatty acid synthase; ACS, acyl CoA synthase; G3PDH, glycerol 3-phosphate dehydrogenase; DHAP, dihydroxyacetone phosphate; DHAPAT, DHAP acyltransferase; FAR1, fatty acyl CoA reductase 1; ADHAPS, alkyl DHAP synthase; ADHAP Reductase, acyl/alkyl DHAP reductase activity; LPA, lysophosphatidic acid; AGP, 1-O-alkyl glycerol 3-phosphate. (B) Mouse DHRS7b is homologous to yeast acyl DHAP reductase, Ayr1p. TMD, transmembrane domain; Adh_short, short chain dehydrogenase/reductase domain. (C) PexRAP (Peroxisomal Reductase Activating PPARγ, detected using anti-DHRS7b antibody) is enriched in peroxisomal fractions isolated from 3T3-L1 adipocytes. S, supernatant; P, pellet after sedimentation. (D) Pex19 co-immunoprecipitates with Myc-tagged PexRAP. WCL, whole cell lysates. (E) Pex19 interacts with PexRAP in GST pulldown experiments using 3T3-L1 adipocytes. (F) RT-PCR analysis of PexRAP expression with PexRAP knockdown in MEFs. **P=0.0084. (G) Mass spectrometric analyses of [M+H]⁺ ions of GPC lipids in MEFs after PexRAP knockdown. Quantification of the 1-O-alkyl ether GPC lipid peak at m/z 746 [M+H]⁺ (identical to the lithium adduct at m/z 752 in Figure 4C) is shown in the inset. **P=0.0009. (H) Mouse tissue distribution of PexRAP protein by Western blotting. (I) Protein abundances of PexRAP and FAS increase prior to increases in C/EBPα and aP2 during differentiation of 3T3-L1 adipocytes. Error bars in panels F and G (inset) represent SEM.

Figure 6. PexRAP is Required for Adipogenesis and PPARγ Activation

(A) Nile red staining of 3T3-L1 adipocytes treated with control or PexRAP shRNA in the presence or absence of rosiglitazone. (B) Triglyceride content for the cells of panel A. **P=0.0066 vs. control, #P=0.0071 vs. PexRAP shRNA vehicle. N=3/condition. (C) RT-PCR analysis of gene expression following PexRAP or DHAPAT knockdown. P vs. control: DHAPAT, *0.0278, **0.007; PexRAP, *0.040; aP2, **0.0060 for PexRAP shRNA and 0.0058 for DHAPAT shRNA; C/EBPα, *0.0160 for PexRAP shRNA and 0.0165 for DHAPAT shRNA; LPL, **0.0014, *0.0450; CD36, *0.0113 for PexRAP shRNA and 0.0132 for DHAPAT shRNA. N=3–5/condition. (D) Rosiglitazone treatment rescues the effect of PexRAP or DHAPAT knockdown on PPARγ target gene expression. 3T3-L1 cells infected with lentivirus expressing control, PexRAP, or DHAPAT shRNA were induced to differentiate into adipocytes and then treated with 2.5 μM rosiglitazone. Expression of PPARγ target genes was analyzed by quantitative RT-PCR. For aP2, exact P values from left to right= 0.0038, 0.0119, 0.0024, 0.0032. For CD36, P values= 0.0022, 0.0015, 0.0110, <0.0001. Error bars in panels B–D represent SEM.

Figure 7. Knockdown of PexRAP in Mice Alters Body Composition and Metabolism

(A) PexRAP knockdown using antisense oligonucleotides (ASOs) in Hepa1-6 cells. (B) Western blot analysis using epididymal WAT of C57BL/6J mice treated intraperitoneally with the indicated doses of control or PexRAP ASO twice a week for 3 weeks. (C) RT-PCR analysis of epididymal WAT expression following ASO treatment. P vs. control: PexRAP **0.0078; PPARγ **0.0051; CD36 *0.0420; LPL **0.0030. N=4/condition. (D) Body composition by MRI following 4 weeks of HFD feeding (baseline) and after 3.5 weeks of ASO treatment while still eating a HFD. **P=0.0098 for fat, 0.0071 for lean. N=4/condition. (E) Glucose tolerance testing in HFD-fed mice following ASO treatment. *P=0.0220 at 15 min. and 0.0434 at 120 min. **p=0.0019. (F) Insulin levels at the 30 min point from panel E. *P=0.0363. (G) Models of PPARγ and PPARα gene expression in WT and FAS-deficient adipocytes. Error bars in panels C–F represent SEM.