Fat-specific protein 27 (FSP27) interacts with adipose triglyceride lipase (ATGL) to regulate lipolysis and insulin sensitivity in human adipocytes

Abstract

In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120-220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120-220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.

Keywords: Adipocyte; Adipose Tissue Metabolism; Adipose Triglyceride Lipase; Diabetes; Diacylglycerol; Fatty Acid; Fatty Acid Metabolism; Insulin Resistance; Obesity.

FIGURE 1.

FSP27 depletion increased both basal and stimulated lipolysis in human adipocytes. Nonspecific scrambled (Scr) siRNA was used as a control in all experiments. A, relative mRNA levels in siRNA-transfected human adipocytes. B, immunoblot analysis and quantification of protein expression levels of FSP27 and β-tubulin (loading control) of siRNA-transfected human adipocytes. C, biochemical quantification of basal and stimulated lipolysis on the basis of measurement of glycerol release after 2 h. Data are mean ± S.E. *, p < 0.05; **, p < 0.001, n = 3 (unpaired Student's t test).

FIGURE 2.

FSP27 coimmunoprecipitated with ATGL. A, in human adipocytes expressing FSP27-FLAG-HA, FSP27 was immunoprecipitated with anti-FLAG antibodies and immunoblotted with ATGL (top panel) or HA (bottom panel) antibody. B, in human adipocytes, ATGL antibodies were used as a bait to pull down endogenous ATGL, followed by immunoblotting with ATGL (top panel) or FSP27 antibody (bottom panel). C, FSP27-HA and ATGL-CFP were expressed in 293T cells using adenovirus. HA antibodies were used to immunoprecipitate FSP27-HA, followed by immunoblotting with ATGL (top panel) or FSP27 (bottom panel) antibodies. Input represents total cell lysates (15 μg), and control represents lysates from human adipocytes infected with control virus and pulled down with FLAG or HA antibody.

FIGURE 3.

FSP27 expression decreased ATGL-mediated lipolysis. Glycerol released in cell culture media from human adipocytes expressing EGFP, FSP27-HA and/or ATGL (A) and COS-7 cells, measured per well containing an equal number of cells. A and B, control cells were infected with EGFP-containing empty virus. C, human adipocytes after ATGL depletion and/or FSP27-HA expression. A and C, glycerol released in 2.5 h was measured and normalized to total protein. Data are mean ± S.E. *, p < 0.001; **, p < 0.05; n = 3 (unpaired Student's t test).

FIGURE 4.

FSP27-mediated TG accumulation was ATGL-dependent. Biochemical quantification of total triglycerides in human adipocytes treated with scrambled siRNA (Scr, control) (A) or siRNA against ATGL in the presence or absence of adenoviral expression of FSP27-HA or EGFP (as a negative control) and adipocytes differentiated from WT or ATGL-KO MEFs (B). FSP27-HA or EGFP (control) were expressed using adenovirus (adV). Total triglyceride concentration was normalized to total protein. Data are mean ± S.E. (paired Student's t test). *p < 0.001, and **p < 0.05, n = 3.

FIGURE 5.

Identification of the TG accumulatory domain of FSP27. A, deletion mutants of FSP27 fused to EGFP were expressed in COS-7 cells to study their role in TG accumulation. *, p < 0.001 (unpaired Student's t test, n = 5). B, FSP27 (120–220) is sufficient to accumulate TGs in human adipocytes. FSP27 and FSP27 (120–220) were expressed in human adipocytes using lentivirus. EGFP containing lentivirus was used as a control. *, p < 0.005; n = 3 (unpaired Student's t test). C, schematic of FSP27 indicating its functional domains.

FIGURE 6.

FSP27 (120–220) coimmunoprecipitated ATGL and reduced lipolysis. A, in human adipocytes, adenovirally expressed FSP27(120–220)-HA was immunoprecipitated using anti-HA antibody and immunoblotted with ATGL (top panel) or HA (bottom panel) antibody. B, human adipocytes were infected with EGFP (control), FSP27(120–220)-HA, or ATGL using lentivirus or adenovirus, respectively, and glycerol release in the media was measured. *, p < 0.005; n = 3 (unpaired Student's t test).

FIGURE 7.

FSP27 protected human adipocytes against FFA-induced insulin resistance. A, insulin-stimulated AKT phosphorylation in human adipocytes after siRNA-mediated FSP27 knockdown. Scr, scrambled; P-AKT, phospho-AKT. B, insulin-stimulated AKT phosphorylation in human adipocytes after overnight treatment with 100 μm PA/BSA in the presence or absence of FSP27-CFP or EGFP (control). C, FSP27-HA expression protects adipocytes differentiated from WT MEFs against 100 μm PA/BSA-mediated inhibition of insulin-stimulated AKT phosphorylation. D, 100 μm PA/BSA or FSP27-HA expression had no effect on insulin-stimulated AKT activation in adipocytes differentiated from ATGL KO MEFs.

FIGURE 8.

FSP27 (120–220) protected against FFA-induced insulin resistance in human adipocytes. Shown is insulin-stimulated AKT phosphorylation in human adipocytes. The core FSP27domain associated with TG accumulation, aa 120–220, was expressed using lentivirus, with EGFP as a control. The cells were treated overnight with 100 μm PA/BSA. The blots show AKT phosphorylation in basal and insulin-stimulated conditions. FSP27 (120–220) protected human adipocytes from inhibition of AKT phosphorylation by exogenous PA.

FIGURE 9.

A hypothetical model of FSP27 regulation of lipolysis that is supported by the results of this study. The hypothesis is that PLIN1 scaffolds FSP27 at the lipid droplet surface where FSP27 interacts with ATGL and decreases lipolysis. A, under basal conditions, FSP27 decreases the access of ATGL to its coactivator CGI-58 (CGI), thereby diminishing lipolysis (dashed downward arrow). HSL, hormone-sensitive lipase. B, when FSP27 is absent in basal conditions, ATGL is free to interact with CGI-58, leading to increased lipolysis (solid downward arrow). C, upon β-adrenergic stimulation in the presence of FSP27, PKA activation results in phosphorylation of PLIN1 and hormone-sensitive lipase, causing the release of CGI-58, which binds to and stimulates ATGL (15, 18). Unbound ATGL is translocated to lipid droplets, and G0S2 is down-regulated to increase ATGL-mediated lipolysis (downward arrow). D, upon β-adrenergic stimulation in the absence of FSP27, the otherwise FSP27-sequestered ATGL is now available for CGI-58 binding, resulting in even higher levels of lipolysis (downward arrow). TAG, triacyl glycerol; and P, phosphorylation.