FSP27 and PLIN1 interaction promotes the formation of large lipid droplets in human adipocytes

Abstract

Human adipocytes express high levels of two distinct lipid droplet proteins, fat specific protein 27 (FSP27; also called CIDEC), a member of the CIDE family, and perilipin1 (PLIN1), a member of the PAT family. Both proteins play a role in fat metabolism in adipocytes, but how they interact is not known. Our present study demonstrates that FSP27 and PLIN1 co-localize and interact in cultured human primary adipocytes. We also found that the C-terminal domain of FSP27, aa 120-220, interacts with PLIN1. Individual expression of exogenous FSP27 or PLIN1 increased triglyceride content and decreased glycerol release (a measure of lipolysis), but co-expression of both proteins did not further increase triglyceride content or decrease lipolysis in human adipocytes. However, the combination of PLIN1 and FSP27 increased the average size of lipid droplets or caused the formation of unilocular adipocytes. Our data suggest that FSP27 interacts with PLIN1 to regulate lipid droplet size in human adipocytes in a concerted manner.

Figure 1. FSP27 and PLIN1 co-localize and co-immunoprecipitate in human adipocytes

(A) A single LD in a human adipocyte showing the distribution of endogenous FSP27 (green) and PLIN1 (red) at its surface. Nucleus was stained with DAPI (blue). Bar, 10 μm. (B) 4-μm confocal Z-section of a human adipocyte showing localization of endogenous FSP27 (green) and PLIN1 (red). Bar, 10 μm. (C) HA-FSP27 was immunoprecipitated with anti-HA antibodies and immunoblotted with PLIN1 and HA antibodies. (D) Flag-PLIN1 was immunoprecipitated with Flag antibodies and immunoblotted with FSP27 or Flag antibodies. (E) Endogenous PLIN1 was immunoprecipitated with PLIN1 antibodies and immunoblotted with PLIN1 and FSP27 antibodies. (F) HA tagged FSP27(120–220) was immunoprecipitated with HA-antibodies and immunoblotted with PLIN1 or HA antibodies. In panels C, D, E and F, input represents whole cell lysate and control represents the beads conjugated with Flag or HA antibodies which were incubated with the uninfected human adipocytes.

Figure 2. Expressing exogenous FSP27 or PLIN1 increases total TG content while decreasing lipolysis in human adipocytes, but FSP27+PLIN1 has no additional effect on TG content or lipolysis

(A) Adenovirus-mediated expression of HA-FSP27 and Flag-PLIN1 in human adipocytes using FSP27 and PLIN1 antibodies, respectively. GFP-containing adenovirus was used as a control. (B, C) Biochemical quantification of total triglyceride (B) and lipolysis (C) in human adipocytes infected with control, FSP27, PLIN and FSP27+PLIN1 adenoviruses; *, p<0.05 (paired t-test). Control represents GFP-containing empty virus. For lipolysis, glycerol released in 2.5 h was measured and normalized to total triglycerides and total proteins. The data show an average of three independent experiments. Values are means ± standard error.

Figure 3. Transducing FSP27+PLIN1 for 72 hr induces formation of unilocular LDs in cultured human adipocytes

(A) Phase contrast image showing LD morphology after 72 h of transduction of FSP27, PLIN1 or FSP27+PLIN1 adenoviruses in cultured human adipocytes. Control represents GFP-containing empty virus. Bar, 10 μm. (B) Range of the LD size. (C) Average number of LDs per cell. LD size and number was measured in more than 15 cells in each condition from three independent experiments. Values are means ± standard deviation. *, p≤0.01; **, p≤0.05 (paired t-test).