Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes - PubMed (original) (raw)

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Tsuyoshi Goto et al. J Lipid Res. 2011 May.

Abstract

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.

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Figures

Fig. 1.

Fig. 1.

Effects of PPARα activator on adiposity and insulin resistance. A, B: White adipose tissue (WAT) weight from male mildly obese KK mice (A) and wild-type (WT) or PPARα-deficient (PPARα−/−) mice (B) fed high-fat diet control (Cont) or high-fat diet containing 0.2% bezafibrate (Beza) for four and six weeks. In each experiment, the mice were housed under pair-fed conditions. C, D: Plasma glucose levels during oral glucose tolerance test (GTT) (C) and insulin tolerance test (ITT) (D). Both GTT and ITT were performed on KK mice fed high-fat diet (Cont) or high-fat diet containing 0.2% bezafibrate (Beza) for five weeks under pair-fed condition. Plasma glucose level was measured by enzymatic colorimetric assay. Data are means ± SEM of 4-6 animals per group. *P < 0.05, **P < 0.01, compared with control diet group.

Fig. 2.

Fig. 2.

mRNA expression levels of PPARα in cultured cells and WAT. A, B: mRNA expression levels of murine PPARα and adiponectin or human PPARα during adipocyte differentiation. 3T3-L1 cells and human multipotent adipose tissue-derived stem cells were harvested at the indicated times after differentiation induction. Semiquantitative PCR conditions are described in “Materials and Methods.” C, D: mRNA expression levels of PPARα in WAT in obese model mice and lean control mice. PPARα mRNA expression levels in C57BL/6 mice fed high-fat diet (HFD) or normal diet (control) for 12 months (C) and in ob/ob mice or lean control mice (D) were quantified by a real-time PCR as described in “Materials and Methods.” E: The mRNA expression levels of PPARα in WAT in C57BL/6 under fed (control), fasted (24 h), and fasted/refed (24 h after refeeding) conditions were quantified by real-time PCR. Data are means ± SEM of 4-6 animals per group. *P < 0.05, **P < 0.01.

Fig. 3.

Fig. 3.

PPARα activation increased mRNA expression levels of marker factors for adipocyte differentiation but not TG content. A: PPARα expressions in WAT and cultured cells. PPARα mRNA levels in several WATs [subcutaneous (Sub), mesenteric (Mes), perirenal (Rea), and epididymal (Epi) WAT from C57BL/6 mice], stromal-vascular (SV) cells from epididymal WAT in C57BL/6 mice, and cultured cells [3T3-L1 cells overexpressing PPARα (L1-PPARα), mock control (L1-Mock), RAW264.7 macrophages, and LPS-stimulated RAW264.7 macrophages] were visualized by semiquantitative PCR (left). PPARα protein levels in L1-Mock and L1-PPARα were visualized by Western blotting (right). B-F: L1-Mock and L1-PPARα cells were induced to differentiate and cultured with or without GW7647 (30 or 100 nM) for 6-10 days. B-E: The mRNA expression levels of adipogenic marker genes (aP2 and PPARγ) (on day 10) (B), glycerol-3-phosphate dehydrogenase (GPDH) activity (on day 6) (C), the capacity of insulin-stimulated 2-DG transport (on day 10) (D), and intracellular triglyceride (TG) amounts (E) in L1-Mock and L1-PPARα were determined as described in “Materials and Methods.” F: Microscopy views of representative L1-Mock and L1-PPARα cells treated with or without 100 nM GW7647, fixed with formalin, and stained with Oil Red O. The original magnification is 100×. Data are means ± SEM; n = 4-6. *P < 0.05, **P < 0.01 compared with DMSO-treated L1-Mock cells. #P < 0.05, ##P < 0.01 compared with L1-Mock cells treated with the same compounds. G, H: 3T3-L1 cells were induced to differentiate and cultured with or without GW7647 (300 nM) and GW6471 (100 nM) for 10 days. The mRNA expression levels of aP2 and PPARγ (G) and microscopy views of representative cells stained with Oil Red O (H). The original magnification is 100×. Data are means ± SEM; n = 4. *P < 0.05, **P < 0.01.

Fig. 4.

Fig. 4.

Effects of PPARα activation on FA oxidation in cultured adipocytes. A-D: 3T3-L1 cells overexpressing PPARα (L1-PPARα) and mock control (L1-Mock) cells were induced to differentiate and cultured with or without GW7647 (30 or 100 nM) for 10 days. The mRNA expression levels of genes involved in FA oxidation (ACO, CPT1b, and UCP3) (A), oxidation of [14C]palmitic acid to CO2 (B), and acid-soluble FA metabolites (ASM) (C) for 16 h, and oxygen consumption rate (OCR) (D) in L1-Mock and L1-PPARα was determined as described in “Materials and Methods.” Data are means ± SEM; n = 3-6. *P < 0.05, **P < 0.01 compared with DMSO-treated L1-Mock. #P < 0.05, ##P < 0.01 compared with L1-Mock treated with the same compounds. E: 3T3-L1 cells were induced to differentiate and cultured with or without GW7647 (300 nM) and GW6471 (100 nM) for 10 days. The mRNA expression levels of ACO and CPT1b was measured by real-time PCR. Data are means ± SEM; n = 4. **P < 0.01.

Fig. 5.

Fig. 5.

Effects of PPARα activator on adipocyte differentiation and FA oxidation in WAT. KK mice (A, B, E-G) and wild-type (WT) or PPARα-deficient (PPARα−/−) mice (C, D) fed HFD (Cont) or HFD containing 0.2% bezafibrate (Beza) were housed under the pair-fed condition for 4-6 weeks. A-D: The mRNA expression levels of adipogenic marker genes (aP2, PPARγ, and adiponectin) (A, C) and genes involved in FA oxidation (ACO, CPT1b, and UCP3) (B, D) in WAT were determined by real-time PCR. E-G: Oxidation of [14C]palmitic acid to CO2 in isolated adipocytes (E), microscopy views of representative histological sections of epididymal WAT (F), and adipocyte size distribution in epididymal WAT (G) from mice fed each experimental diet for 6 weeks were examined. Data are means ± SEM; n = 3-8. *P < 0.05, **P < 0.01 compared with mice fed control diet.

Fig. 6.

Fig. 6.

ChIP assays of aP2 and CPT1b promoter in 3T3-L1 adipocytes treated with or without GW7647. ChIP assays were performed as described in “Materials and Methods.” Soluble chromatin from 3T3-L1 cells, whose differentiation was induced and maintained for 14 days with or without of 30 nM GW7647, was immunoprecipitated with control mouse IgG (lanes 2 and 4) or antibodies against PPARα (lanes 3 and 5). Immunoprecipitates were analyzed by PCR using specific primers for the mouse aP2 or CPT1b promoter region containing potential PPRE. PCR was performed with total chromatin input (lane 1).

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