Growth hormone controls lipolysis by regulation of FSP27 expression

in Journal of Endocrinology
Correspondence should be addressed to V Puri or K Y Lee: puri@ohio.edu or leek2@ohio.edu

*(R Sharma, Q Luong and V M Sharma contributed equally to this work)

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Growth hormone (GH) has long been known to stimulate lipolysis and insulin resistance; however, the molecular mechanisms underlying these effects are unknown. In the present study, we demonstrate that GH acutely induces lipolysis in cultured adipocytes. This effect is secondary to the reduced expression of a negative regulator of lipolysis, fat-specific protein 27 (FSP27; aka Cidec) at both the mRNA and protein levels. These effects are mimicked in vivo as transgenic overexpression of GH leads to a reduction of FSP27 expression. Mechanistically, we show GH modulation of FSP27 expression is mediated through activation of both MEK/ERK- and STAT5-dependent intracellular signaling. These two molecular pathways interact to differentially manipulate peroxisome proliferator-activated receptor gamma activity (PPARγ) on the FSP27 promoter. Furthermore, overexpression of FSP27 is sufficient to fully suppress GH-induced lipolysis and insulin resistance in cultured adipocytes. Taken together, these data decipher a molecular mechanism by which GH acutely regulates lipolysis and insulin resistance in adipocytes.

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    GH acutely induces lipolysis and reduces FSP27 expression in a time and dose-dependent manner. (A) qPCR analysis of mRNA levels of regulators of lipolysis: ATGL, HSL, Perilipin, Cidea, Comparative gene identification-58 (CGI-58), Tail-interacting protein of 47 kD (TIP47), G0/G1 switch gene 2 (G0S2s), and FSP27 mRNA was compared in RNA isolated from perigonadal fat of 4 month old male bGH mice. Data are shown as mean ± s.e.m. of 8–10 Samples. *P < 0.05; **P < 0.01; ***P < 0.01. (B) qPCR analysis of Atgl, Hsl, Perilipin, Cidea, CGI-58, TIP47, and FSP27 mRNA in RNA isolated from 3T3-L1 adipocytes treated with 500 ng/mL recombinant bovine GH (bGH) for 2 h. Data are shown as mean ± s.e.m. of three independent experiments. (C) qPCR and Western Blot analysis of FSP27 mRNA and protein isolated from 3T3-L1 adipocytes treated with 500 ng/mL recombinant bovine GH (bGH). Data are shown as mean ± s.e.m. of three independent experiments. (D) Lipolysis as measured by glycerol release from 3T3-L1 adipocytes treated with bGH for two hours. Data are shown as mean ± s.e.m. of three independent experiments. (E) qPCR and Western Blot analysis of FSP27 mRNA and protein isolated from 3T3-L1 adipocytes treated with bGH for 2 h. Data are shown as mean ± s.e.m. of three independent experiments.

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    GH regulates lipolysis in a MEK and Pparγ dependent manner. (A) Lipolysis as measured by glycerol release from 3T3-L1 adipocytes treated with 500 ng/mL bGH for 2 h after 2 hours of pre-treatment with 10 µM U0126 or 1 µM Rosiglitazone. Data are shown as mean ± s.e.m. of three independent experiments. Asterisks indicate a significant differences in all panels *P < 0.05; **P < 0.01. (B) Representative Western blot analysis of pERK T202/Y204, total ERK, pSTAT5Y694, total STAT5, pHSL S563, and total HSL in 3T3-L1 adipocytes treated with 500 ng/mL bGH for either 20 min or 2 h after 2 hours of pre-treatment with 10 µM U0126 or 1 µM Rosiglitazone. Actin is used as a loading control. (C and D) qPCR and Western blot analysis of FSP27 mRNA and protein isolated from cells treated with 500 ng/mL bGH for 2 h after 2 hours of pre-treatment with 10 µM U0126 or 1 µM Rosiglitazone. Actin is used as a loading control. Quantitation of Western blot data are shown as mean ± s.e.m. of four independent experiments.

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    GH treatment results in rapid PPARγ translocation. (A) Representative Western blot analysis of pPPARγ S112 and total pPPARγ in 3T3-L1 adipocytes treated with 500 ng/mL bGH for 20 min after 2 hours of pre-treatment with 10 µM U0126 or 1 µM Rosiglitazone. (B) Representative Western blot analysis for PPARγ in 3T3-L1 adipocytes treated with 500 ng/mL bGH for 1 h after 2 h of pre-treatment with MEK1 inhibitor, 10 µM U0126, or 1 µM Rosiglitazone following nuclear fractionation. The positive control is whole cell lysate of 3T3-L1 adipocytes. Gapdh and PCNA are loading controls for the cytoplasmic and nuclear fractions, respectively. (C) Immunofluorescence for PPARγ (green) with nuclei counterstained with DAPI (blue) of in 3T3-L1 adipocytes treated with 500 ng/mL bGH for 1 h after 2 h pre-treatment with 10 µM U0126 or 1 µM Rosiglitazone.

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    STAT5 and PPARγ regulate FSP27 expression. (A) qPCR analysis of FSP27 in RNA isolated from 3T3-L1 adipocytes treated with vehicle or 500 ng/mL bGH for 2 h after 2 hours of pre-treatment with 10 µM U0126, 1 µM Rosiglitazone, or 200 µM STAT5 inhibitor. Data are shown as mean ± s.e.m. of three independent experiments. Asterisks indicate a significant differences in all panels *P < 0.05; **P < 0.01; ***P < 0.001. (B) Lipolysis as measured by glycerol release from 3T3-L1 adipocytes treated with vehicle or 500 ng/mL bGH for 2 h after 2 hours of pre-treatment with 200 µM STAT5 inhibitor. Data are shown as mean ± s.e.m. of two independent experiments, each with three replicates/group. (C) Expression level of FSP27 mRNA was compared using quantitative real-time PCR (qPCR) of RNA isolated from subcutaneous (SC) and perigonadal (PG) fat of 4 month old male Stat5ΔN/ΔN mice. Data are shown as mean ± s.e.m. of six samples. (D, E and F) Luciferase activity of 293T cells transfected with a 0.9-kb WT FSP27 luciferase reporter, with the PPARγ response element mutated, or with a presumptive STAT5 response element mutated. The reporter vector was co-transfected either a vector control or 25 ng of PPARγ expression vector and 25 ng of its obligate heterodimer RXRα. The cells were also co-transfected with either a vector control or 25 ng of a STAT5 expression vector. Data are shown as mean ± s.e.m. of three independent experiments, each with three replicates.

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    FSP27 over-expression prevents GH-induced lipolysis and insulin resistance. (A) Representative Western blot analysis of FSP27 in WT 3T3-L1 adipocytes and FSP27 over-expressing adipocytes treated with 500 ng/mL bGH for 2 h. (B) Lipolysis as measured by glycerol release from WT 3T3-L1 adipocytes and FSP27 over-expressing adipocytes treated with 250 ng/mL bGH for two hours. Data are shown as mean ± s.e.m. of three independent experiments. Asterisks indicate a significant differences: *P < 0.05, **P < 0.01, ***P < 0.001. (C) Immunofluorescence of Nile Red Stained WT and FSP27 over-expressing 3T3-L1 adipocytes treated with vehicle or 500 ng/mL bGH for 24 h. (D) Representative Western blot analysis of pAKT S473 and total AKT in WT 3T3-L1 adipocytes and FSP27 over-expressing adipocytes treated with 250 ng/mL bGH for 2 h and 10 nM insulin for 15 min. (E) Proposed mechanism of GH-induced lipolysis.

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