Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes - PubMed (original) (raw)

Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes

Srijana Ranjit et al. J Lipid Res. 2011 Feb.

Abstract

The lipid droplet-associated fat specific protein 27 (FSP27) suppresses lipolysis and thereby enhances triglyceride accumulation in adipocytes. We and others have recently found FSP27 to be a remarkably short-lived protein (half-life, 15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, we tested the hypothesis that lipolytic agents such as tumor necrosis factor-α (TNF-α) and isoproterenol modulate FSP27 levels to regulate FFA release. Consistent with this concept, we showed that the lipolytic actions of TNF-α, interleukin-1β (IL-1β), and IFN-γ are accompanied by marked decreases in FSP27 expression and lipid droplet size in mouse adipocytes. Similar depletion of FSP27 using short interfering RNA (siRNA) mimicked the lipolysis-enhancing effect of TNF-α, while maintaining stable FSP27 levels using expression of hemagglutinin epitope-tagged FSP27 blocked TNF-α-mediated lipolysis. In contrast, we show the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 levels and a delayed degradation rate corresponding to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism for restraining excessive lipolysis by catecholamines, is mimicked by forskolin or 8-bromo-cAMP treatment and is prevented by the protein kinase A (PKA) inhibitor KT5720 or by PKA depletion using siRNA. Taken together, these data identify the regulation of FSP27 as an important intermediate in the mechanism of lipolysis in adipocytes in response to TNF-α and isoproterenol.

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Figures

Fig. 1.

FSP27 has a short half-life and is degraded by the proteasome after ubiquitination. A: Representative Western blots of adipocyte lysates following 15, 30, and 60 min treatments with 5 µg/ml cycloheximide (CHX) compared with untreated (0) cells show rapid degradation of FSP27. B: Densitometric measurement of FSP27 protein levels after cycloheximide treatment (as shown in panel A). C: Representative Western blots of FSP27 show protection of FSP27 from degradation in lysates from cells treated with the proteasome inhibitor MG132 but not by peptidase inhibitor leupeptin (Leu) or aprotinin (Aprot) or lysosomal inhibitor NH4Cl. D: Densitometric measurements showing protection of FSP27 by MG132. E: FSP27 immunoblot (IB) of total lysates of 3T3-L1 adipocytes with or without MG132. F: Lysates from control (−) or MG132-treated (+) cells were immunoprecipitated (IP) with anti-FSP27 antibody (FSP27) or nonimmune rabbit IgG (IgG), and immunoprecipitates were probed with anti-ubiquitin (Ub) monoclonal antibody. G: Immunoprecipitates (as shown in panel F) probed with anti-FSP27 antibody. Data shown are representative of four or more separate experiments.

Fig. 2.

TNF-α as well as IL-1β and IFN-γ but not IL-6 decrease FSP27 levels and increase glycerol release in 3T3-L1 adipocytes. A–C: Cells treated with 10 ng/ml TNF-α for 16 h. A: Quantitative RT-PCR of Fsp27 and perilipin mRNA levels; B) representative immunoblot of FSP27, perilipin, and actin (loading control); C: densitometric quantitation of immunoblots of FSP27 and perilipin protein levels. A–C, Data are expressed as means ± SEM for six different experiments; P values were calculated using Student's _t_-test; *, P < 0.05; **, P < 0.0005. A.U., arbitrary units. D: 3T3-L1 adipocytes were treated with IL-6 at the indicated concentrations and times. D, left panel, glycerol release; D, right panel, quantitative RT-PCR of FSP27 mRNA levels. E, F: Cells treated with IL1-β or IFN-γ, respectively, at the indicated concentrations. and times are shown. Upper panels, glycerol release; lower panels, quantitative RT-PCR of FSP27 mRNA levels. D–F, Data are expressed as means ± SEM from three to five experiments performed in duplicate. P values were calculated using Student's _t_-test relative to the time-matched untreated condition; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

TNF-α also decreases FSP27 levels and increases glycerol release in adipocytes isolated from mouse adipose tissue. A: Primary adipocytes were isolated from subcutaneous adipose tissue of C57Bl/6J mice and treated with 10 ng/ml TNF-α for 20 or 40 h. A, left panel: glycerol released into medium; right panel: quantitative RT-PCR of FSP27 mRNA level (shown as a percentage of the time-matched untreated sample). Data are expressed as means ± SEM from two different experiments performed in duplicate. A.U., arbitrary units. B: Primary preadipocytes from the SVF were induced to differentiate as described in Materials and Methods. Mature adipocytes were then treated with 10–20 ng/ml TNF-α for 24 or 48 h. B, left panel: glycerol release; B, right panel: quantitative RT-PCR. Data are expressed as means ± SEM from one experiment performed three to five times. P values were calculated using Student's _t_-test relative to the time-matched untreated condition; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 4.

TNF-α treatment of 3T3-L1 adipocytes increases lipolysis that is enhanced by further depletion of FSP27 using siRNA. A: Glycerol released in medium was measured in 3T3-L1 adipocytes electroporated with scrambled (Scr) or FSP27 siRNA and treated with 10 ng/ml TNF-α for 16 h. A.U., arbitrary units. B: Western blot analysis using FSP27, perilipin (PLIN), and actin antibodies showing FSP27 and PLIN protein levels in control and under TNF-α-treated conditions. MW, molecular weight. C: Densitometric analysis of FSP27 and PLIN protein levels in siRNA or TNF-α-treated cells. Data are expressed as means ± SEM for six different experiments. KD, knock-down. P values were calculated using ANOVA; *, P < 0.05; **, P < 0.0005.

Fig. 5.

TNF-α-mediated decrease in FSP27 is associated with reduced lipid droplet size and increased glycerol release. Mature 3T3-L1 adipocytes were treated with TNF-α for 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h. A: Confocal microscope images showing lipid droplets stained with Oil Red O (red) and nuclei stained with DAPI (blue). Images are representative of 90 random fields imaged from three different experiments. B: Quantification of lipid droplet volume using MetaMorph imaging software. Data are means ± SEM of 10 cells for each condition from different experiments. Con, control. C: Quantification of numbers of lipid droplets per cell for each time point. D: Glycerol released into medium after 10 ng/ml TNF-α treatment for 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h. E: Western blot analysis showing FSP27 levels after 10 ng/ml TNF-α treatment for 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h. F: Densitometry showing decrease of FSP27 protein levels after TNF-α treatment. Data are expressed as means ± SEM from three different experiments. P values were calculated using ANOVA;*, P < 0.05; **, P < 0.005.

Fig. 6.

Adenoviral expression of FSP27 protects against TNF-α-mediated lipolysis and lipid droplet size diminution. 3T3-L1 adipocytes were infected with control virus (Ad-GFP) or HA-FSP27-expressing virus (Ad-HA-FSP27) on the fourth day of differentiation. On the fifth day, cells were serum starved overnight, treated with 10 ng/ml TNF-α for 24 h, and analyzed for protein, glycerol release, and lipid droplet morphology. A: Western blot analysis using FSP27, HA, GFP, perilipin (PLIN), actin, and Vti1a antibodies show maintenance of HA-tagged FSP27 protein levels even after treatment of TNF-α. Actin is the loading control for HA, GFP, and PLIN, and Vti1a is the loading control for FSP27. B: Glycerol released into the medium in 24 h with or without TNF-α treatment. A. U., arbitrary units. C: Confocal microscopy image showing lipid droplets stained with Oil Red O (red), nuclei stained with DAPI (blue), and GFP expression (green) in 3T3-L1 adipocytes expressing Ad-GFP or Ad-HA-FSP27 in the presence or absence of TNF-α. Data are expressed as means ± SEM for five individual experiments. P values were calculated using ANOVA; *, P < 0.05; **, P < 0.0005.

Fig. 7.

Isoproterenol delays degradation of FSP27 to increase protein level in 3T3-L1 adipocytes. A: Lysates from adipocytes treated with 10 µM isoproterenol (Iso) for 15, 30, 60, 120, or 180 min were immunoblotted for FSP27, perilipin (PLIN), or Vti1a (loading control). B: Densitometry of immunoblot shows increased FSP27 protein level after 180 min of 10 µM isoproterenol treatment of mature adipocytes. A.U., arbitrary units; Con, control. C: Mature adipocytes treated with 10 µM isoproterenol, pulsed with 5 µg/ml cycloheximide (CHX), and chased for 15, 30, and 60 min show delayed degradation of FSP27 in the presence of isoproterenol compared with that of control (0). D: Densitometry of FSP27 level in the presence or absence of 10 µM isoproterenol shows delayed FSP27 degradation in the presence of isoproterenol. E: Adipocytes transfected with either Scr or PKA siRNA and treated with or without 10 µM isoproterenol. The effect of isoproterenol on FSP27 is abrogated when PKA is knocked down (KD). The absence of perilipin phosphorylation despite isoproterenol treatment in adipocytes treated with PKA siRNA is used as the positive control. F: Mature adipocytes were treated with 10 µM isoproterenol, 5 µM forskolin (For), 1 mM 8-bromo-cAMP (B-cAMP) plus IBMX, or 80 µM KT5720 plus 10 µM isoproterenol. The effect of isoproterenol on FSP27 is mimicked by forskolin and 8-bromo-cAMP and inhibited by KT5720. G: Mature adipocytes treated with 10 µM isoproterenol were harvested for RNA and analyzed for FSP27 mRNA. It shows no change in FSP27 transcript with isoproterenol treatment. Data are expressed as means ± SEM for six individual experiments.

Fig. 8.

Expression of adenoviral FSP27 protects against isoproterenol-mediated lipid droplet size diminution but not lipolysis. The 3T3-L1 adipocytes were infected with control virus (Ad-GFP) or HA-FSP27-expressing virus (Ad-HA-FSP27) on the fourth day of differentiation. On the fifth day, cells were serum starved overnight, treated with 10 µM isoproterenol (Iso) for 3 h, and analyzed for protein, glycerol release, and lipid droplet morphology. A: Western blot analysis using FSP27, HA, GFP, PLIN, Actin, and Vti1a antibodies. Actin is the loading control for HA, GFP, and PLIN, and Vti1a is the loading control for FSP27. B: Glycerol released into the medium in 3 h with or without isoproterenol. C: Difference in the rate of glycerol released [calculated using the equation (FSP27 KD + Iso) – (Scr + iso), where KD is knockdown] in response to isoproterenol from 3T3-L1 adipocytes treated with Scr or FSP27 siRNA shows increased rate of lipolysis at 3 h compared with 1 h. D: Confocal microscopy image shows lipid droplets stained with Oil Red O (red), nuclei stained with DAPI (blue), and GFP (green) expression in 3T3-L1 adipocytes expressing Ad-GFP or Ad-HA-FSP27 in the presence or absence of isoproterenol. Data are expressed as means ± SEM for three individual experiments. P values were calculated using ANOVA for data shown in panel B and Student's _t_-test for data shown in panel C; *, P < 0.05.

Fig. 9.

Isoproterenol (Iso) alters ubiquitination of FSP27 to modulate FSP27 in 3T3-L1 adipocytes. Mature adipocytes were treated with MG132 alone or with TNF-α or isoproterenol, FSP27- immunoprecipitated using FSP27 antibody, and probed with ubiquitin and FSP27 antibody. A: Western blot showing total lysate of adipocytes treated with MG132 alone or with TNF-α or isoproterenol. B: FSP27 immunoprecipitated (IP) with FSP27 antibody and probed with ubiquitin (Ub) antibody. C: FSP27 immunoprecipitated with FSP27 antibody and probed with FSP27 antibody. Data are representative of four separate experiments. Lanes G, IgG; lanes F, FSP27.

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Regulation of fat specific protein 27 by isoproterenol and TNF-α to control lipolysis in murine adipocytes - PubMed (original) (raw)