Cell death-inducing DNA fragmentation factor A-like effector A and fat-specific protein 27β coordinately control lipid droplet size in brown adipocytes

Abstract

Adipose tissue stores neutral lipids and is a major metabolic organ involved in regulating whole-body energy homeostasis. Triacylglycerol is stored as unilocular large lipid droplets (LDs) in white adipocytes and as multilocular small LDs in brown adipocytes. Proteins of the cell death-inducing DNA fragmentation factor A-like effector (Cide) family include CideA, CideB, and fat-specific protein of 27 (FSP27). Of these, FSP27 has been shown to play a crucial role in the formation of unilocular large LDs in white adipocytes. However, the mechanisms by which brown adipocytes store small and multilocular LDs remain unclear. An FSP27 isoform, FSP27β, was recently identified. We herein report that CideA and FSP27β are mainly expressed in brown adipose tissue and that FSP27β overexpression inhibits CideA-induced LD enlargements in a dose-dependent manner in COS cells. Furthermore, RNAi-mediated FSP27β depletion resulted in enlarged LDs in HB2 adipocytes, which possess the characteristics of brown adipocytes. Brown adipocytes in FSP27-knock-out mice that express CideA, but not FSP27β, had larger and fewer LDs. Moreover, we confirmed that FSP27β and CideA form a complex in brown adipose tissue. Our results suggest that FSP27β negatively regulates CideA-promoted enlargement of LD size in brown adipocytes. FSP27β appears to be responsible for the formation of small and multilocular LDs in brown adipose tissue, a morphology facilitating free fatty acid transport to mitochondria adjacent to LDs for oxidation in brown adipocytes.

Keywords: adipocyte; brown adipose tissue (BAT); cell death-inducing DNA fragmentation factor A (DFFA)-like effector A (CideA); fat-specific protein of 27 (FSP27); lipid droplet; lipid metabolism; lipid oxidation; lipolysis; white adipose tissue (WAT).

Figure 1.

Expression of FSP27α, FSP27β, and CideA in white and brown adipose tissues. A, RT-PCR analysis of FSP27α, FSP27β, and CideA in the eWAT and BAT of C57BL/6J mice at 10 weeks of age. n = 3. # shows p < 0.01 versus eWAT. B, immunoblot analysis of FSP27α, FSP27β, and CideA in total cell lysates of the eWAT and BAT of C57BL/6J mice at 14 weeks of age. UCP-1 (BAT marker) and α-tubulin (loading control) were also examined. The arrow and arrowhead indicate FSP27β and FSP27α, respectively.

Figure 2.

Effects of the overexpression of FSP27α, FSP27β, and CideA on LD sizes in COS cells. A, COS cells transfected with pIRES2-DSRed2 encoding FSP27α, FSP27β, or CideA were incubated with 400 μm oleic acid for 2 days and then subjected to immunoblot analysis with antibody against FSP27 and CideA. The black triangle, circle, and square represent molecular mass markers of 33.6 kDa, 27.1 kDa, and 47.5 kDa, respectively. β-Actin (loading control) was also examined. B, COS cells transfected with pIRES2-DSRed2 encoding FSP27α, FSP27β, or CideA along with the fluorescence marker DSRed2 were incubated with 400 μm oleic acid for 2 days and then stained with BODIPY 493/503 for triacylglycerol. A merged image of BODIPY 493/503 and DSRed2 fluorescence is shown. Scale bar: 10 μm. C, quantitation of LD sizes in COS cells expressing FSP27α, FSP27β, and CideA. Data are the means ± S.E. of the diameters of the largest LD in 30 cells (left graph). # shows p < 0.01 versus the control. The right graph is a scatter plot of the same results that shows all the data points and means. D, quantitation of intracellular LD numbers in COS cells expressing FSP27α, FSP27β, and CideA. Data are the means ± S.E. of the LD number in 30 cells. # shows p < 0.01 versus the control (left graph). The right graph is a scatter plot of the same results that shows all the data points and means. E, immunofluorescence localization of FSP27α, FSP27β, and CideA in COS cells by confocal laser microscopy. TAG was stained with BODIPY 493/503. The expression of FSP27α and FSP27β was detected with an anti-FSP27 antibody, whereas that of CideA was detected by an anti-CideA antibody. Scale bar: 10 μm.

Figure 3.

Effects of the overexpression of DSRed-Monomer-FSP27α, FSP27β, and CideA fusion proteins in COS cells. A, COS cells transfected with pDSRed-Monomer-C1 vectors encoding FSP27α, FSP27β, or CideA were incubated with 400 μm oleic acid for 2 days and then stained with BODIPY 493/503 for triacylglycerol. A merged image of BODIPY 493/503 and DSRed-Monomer fluorescence is shown. Scale bar: 10 μm. B, quantitation of LD sizes in COS cells expressing FSP27α, FSP27β, and CideA. Data are the means ± S.E. of the diameters of the largest LD in 30 cells. # shows p < 0.01 versus the control (left graph). The right graph is a scatter plot of the same results that shows all the data points and means. C, quantitation of intracellular LD numbers in COS cells expressing FSP27α, FSP27β, and CideA. Data are the means ± S.E. of the LD number in 30 cells. # shows p < 0.01 (left graph). The right graph is a scatter plot of the same results that shows all the data points and means.

Figure 4.

Effects of the overexpression of FSP27β on the CideA-induced enlargement of LD in COS cells. A, COS cells co-transfected with pcDNA encoding CideA and pIRES2-DsRed2 encoding DSRed2 and FSP27β were incubated with 400 μm oleic acid for 2 days and then stained with BODIPY 493/503 for triacylglycerol (green) followed by immunostaining for CideA using an anti-CideA primary antibody and anti-rabbit secondary antibody with Dylight405 and represented in yellow. Scale bar: 10 μm. B, quantitation of LD sizes (left graph) and numbers (right graph) in COS cells expressing FSP27β and CideA. Graphs represent all the data points with means ± S.D. n = 20. # shows p < 0.01 versus CideA. C, dose-dependent inhibition of CideA-induced enlargements in LD by FSP27β. Transfection was performed as in A in addition to changing the amount of the plasmids of pIRES2-DsRed2 encoding FSP27β. n = 20. # shows p < 0.01. D, quantitation of LD sizes in COS cells transfected as in A in addition to changing the amount of the plasmids of pIRES2-DSRed2. n = 20. E, quantitation of LD sizes (left graph) and numbers (right graph) in COS cells expressing CideA, FSP27β, and its mutant (FSP27β-DENQ). Graphs represent all the data points with means ± S.D. n = 20. # shows p < 0.01. F, immunofluorescence localization of CideA and FSP27β expressed by the pcDNA and pDSRed-Monomer-C1 vectors, respectively, in COS cells under confocal laser microscopy. TAG was stained with BODIPY 493/503 (green). The expression of FSP27β was detected with DSRed fluorescence, whereas that of CideA was detected by an anti-CideA antibody (yellow). Scale bar: 10 μm.

Figure 5.

Effects of FSP27β depletion by siRNA on LD sizes in HB2 adipocytes. A, immunoblot analysis of FSP27α, FSP27β, and CideA in HB2 adipocytes in which FSP27α and FSP27β were depleted using siRNA. The black triangle and circle represent molecular mass markers of 33.6 kDa and 27.1 kDa, respectively. B, HB2 adipocytes were stained with BODIPY 493/503 for triacylglycerol for 2 days after the knockdown with siRNA. Scale bar: 10 μm. C, quantitation of LD sizes in HB2 adipocytes in which FSP27α and FSP27β were depleted with siRNA. Data are the means ± S.E. of the diameters of the largest LD in 35 cells. # shows p < 0.01 versus control cells. D, typical LD pattern in HB2 adipocytes stained with BODIPY 493/503 for triacylglycerol for 2 days after the knockdown with siRNA. Scale bar: 10 μm.

Figure 6.

LD size and number in white and brown adipocytes from FSP27 knock-out mice. A, immunoblot analysis of FSP27 and CideA protein in eWAT and BAT from wild-type (WT), FSP27 hetero KO (HE), and FSP27 homo KO (HO) mice. B, sections of eWAT and BAT from WT, FSP27 hetero KO (HE), and FSP27 homo KO (HO) mice were stained by hematoxylin-eosin and examined by light microscopy. Scale bar: 100 μm. C, quantitation of LD sizes in eWAT and BAT from wild-type (WT), FSP27 hetero KO (HE), and FSP27 homo KO (HO) mice. n = 1,593 (WT), 2,201 (HE), and 16,355 (HO) in eWAT and 20,372 (WT), 18,953 (HE), and 8,243 (HO) in BAT. # shows p < 0.01. D, comparison of LD numbers in a unit area of eWAT and BAT from wild-type (WT), FSP27 hetero KO (HE), and FSP27 homo KO (HO) mice. # shows p < 0.01. The LD area in C and LD number in D were measured with BZ-X710, Keyence.

Figure 7.

FSP27β inhibits the homo dimerization of CideA in COS cells and forms a complex with CideA in BAT. A, overexpression of FSP27β inhibits the homodimer formation of CideA in COS cells. Extracts of COS cells transfected with an expression vector pcDNA encoding CideA-MYC alone, CideA-MYC, and CideA-HA with or without a vector pIRES2-DSRed2 encoding FSP27β, as indicated, were subjected to immunoprecipitation (IP) with antibody to HA. Total cells and immunoprecipitates were subsequently subjected to immunoblot analysis with antibody to MYC, FSP27, and HA. Total amounts of plasmids in each cell were adjusted to the same by adding pcDNA and pIRES2-DSRed2 vectors. B, detergent extracts of the BAT and eWAT of 9-week old mice were subjected to immunoprecipitation with the antibody against CideA. Total cell lysates and the resulting immunoprecipitates were analyzed with an immunoblot analysis using antibodies to FSP27 and CideA.

Figure 8.

Proposed mechanisms by which the Cide protein family regulates LD sizes in WAT and BAT. In WAT, FSP27α on neighboring LD induces homo dimerization, resulting in the fusion of LD, subsequent lipid exchange, and formation of larger LD (A). CideA also mediates the formation of large LD in the same manner (B). In BAT, FSP27β inhibits the homo dimerization of CideA and suppresses the formation of large LD (C).