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. 2008 Aug;118(8):2808-21.
doi: 10.1172/JCI34090.

FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets

Naonobu Nishino  1 , Yoshikazu Tamori, Sanshiro Tateya, Takayuki Kawaguchi, Tetsuro Shibakusa, Wataru Mizunoya, Kazuo Inoue, Riko Kitazawa, Sohei Kitazawa, Yasushi Matsuki, Ryuji Hiramatsu, Satoru Masubuchi, Asako Omachi, Kazuhiro Kimura, Masayuki Saito, Taku Amo, Shigeo Ohta, Tomohiro Yamaguchi, Takashi Osumi, Jinglei Cheng, Toyoshi Fujimoto, Harumi Nakao, Kazuki Nakao, Atsu Aiba, Hitoshi Okamura, Tohru Fushiki, Masato Kasuga
Affiliations

FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets

Naonobu Nishino et al. J Clin Invest. 2008 Aug.

Abstract

White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPARgamma in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity.

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Figures

Figure 1
Figure 1. Adipocyte-specific expression of Fsp27.
(A) Northern blot analysis of FSP27 mRNA in 3T3-L1 cells at the indicated times after the onset of induction of adipocyte differentiation. The region of the ethidium bromide–stained gel containing 28S rRNA is also shown. (B) Northern blot analysis of FSP27 mRNA in 3T3-L1 adipocytes, either after incubation for 48 hours with 5 μM BRL49653 or TNF-α (10 ng/ml) or 48 hours after infection with adenoviral vectors encoding wild-type mouse PPARγ (adex-PPARγ-WT) or PPARγ-ΔC (adex-PPARγ-ΔC) or with the corresponding empty vector (adex-empty), at an MOI of 60 PFU per cell. (C) Northern blot analysis of FSP27 mRNA in various organs and tissues of C57BL/6J mice at 4 weeks of age. Sub, subcutaneous; Epi, epididymal. (D) Immunoblot analysis of FSP27 in total lysates of WAT (subcutaneous or epididymal) and BAT isolated from C57BL/6J mice at 4 weeks of age. Both α-tubulin (loading control) and UCP-1 (BAT marker) were also examined. (E) Northern blot analysis of FSP27 mRNA in HB2 and HW cells at the indicated times after the onset of induction of adipocyte differentiation. (F) Immunoblot analysis of FSP27 in HB2 and HW cells at the indicated times after the onset of induction of adipocyte differentiation using the antibodies to a COOH-terminal peptide of FSP27. Both α-tubulin and β-actin (loading controls) as well as COXIV (mitochondrial marker) were also examined.
Figure 2
Figure 2. Glucose and energy metabolism in FSP27-knockout mice.
(A) Body weight of wild-type, FSP27 heterozygous knockout (+/–), and FSP27-KO (–/–) mice maintained on a standard diet (SD) or fed a high-fat diet (HFD). Data are mean ± SEM (n = 7–11). §P < 0.05 versus corresponding values for wild-type or FSP27 heterozygous knockout mice on the high-fat diet; #P < 0.05 versus corresponding value for FSP27 heterozygous knockout mice on the high-fat diet. (B) Plasma glucose concentrations during an intraperitoneal glucose tolerance test (left panel) or an insulin tolerance test (right panel) in 12-week-old wild-type and FSP27-KO (KO) mice, either maintained on a standard diet or fed a high-fat diet from 4 weeks of age. Data are mean ± SEM (n = 11 for standard diet; n = 8 for high-fat diet.). *P < 0.05, **P < 0.01 versus corresponding value for wild-type animals on the standard diet; †P < 0.05, ††P < 0.01 versus corresponding value for wild-type animals on the high-fat diet. (C) Hyperinsulinemic-euglycemic clamp analysis in 10- to 12-week-old wild-type and FSP27-KO mice fed a high-fat diet. GIR, glucose infusion rate; Rd, rate of glucose disappearance; BHGP, basal hepatic glucose production; CHGP, hepatic glucose production during the clamp period. Data are mean ± SEM (n = 6). §§P < 0.05, ##P < 0.01 versus corresponding value for wild type. (D and E) Whole-body oxygen consumption rate (VO2, expressed in milliliters per minute per gram of body weight) during a 12-hour dark/12-hour light cycle in 12-week-old mice fed a standard diet (n = 3 for wild type; n = 5 for FSP27-KO) (D) or a high-fat diet (n = 6) (E) is shown in the left panels. The average values for the 24-hour period are shown in the right panels. Data are mean ± SEM. ‡P < 0.05, ‡‡P < 0.01 versus wild type. (F and G) Average values of respiratory quotient (VCO2/VO2) (F) and energy expenditure (G) for the 24-hour period in the experiments shown in D and E. Data are mean ± SEM. ‡P < 0.05, ‡‡P < 0.01 versus wild type.
Figure 3
Figure 3. Characterization of adipose tissue in FSP27 knockout mice.
(A) Comparison of interscapular brown fat pads (BAT) and epididymal white fat pads (WAT) of 14-week-old wild-type and FSP27-KO mice (left panel). Weights of epididymal WAT, subcutaneous WAT, and BAT isolated from 14-week-old wild-type and FSP27-KO mice (right panels). Data are mean ± SEM (n = 4–8). †P < 0.05 versus the corresponding value for wild type. (B) Sections of subcutaneous WAT and interscapular BAT from 14-week-old wild-type and FSP27-KO mice were stained with hematoxylin-eosin and examined by light microscopy. Scale bar: 20 μm. (C) Transmission electron microscopy of subcutaneous WAT and interscapular BAT from 6-week-old wild-type and FSP27-KO mice. L, lipid droplet; *, mitochondria. Scale bar: 500 nm. (D) Mitochondrial number (left panel) and size (right panel) in subcutaneous WAT, epididymal WAT, and interscapular BAT determined from electron micrographs similar to those in C. Mitochondrial number is expressed per nucleus and was determined for 25 wild-type and 28 FSP27-KO cells for subcutaneous WAT, 33 cells for epididymal WAT, and 56 wild-type and 64 FSP27-KO cells for BAT. Mitochondrial diameter was measured with a scale of 25 nm in 120 cells. Data are mean ± SEM. ††P < 0.01 versus the corresponding value for wild type. (E) Southern blot analysis of total cellular DNA from BAT or WAT of 9-week-old wild-type or FSP27-KO mice with probes specific for the mitochondrial COXI gene and the nuclear 36B4 gene.
Figure 4
Figure 4. Mitochondrion-related gene expression and metabolic rate in WAT of FSP27-knockout mice.
(A and B) Quantitative RT-PCR analysis of the expression of genes related to mitochondrial biogenesis (A) or to FFA oxidation (B) in WAT and BAT of 20-week-old wild-type and FSP27-KO mice. Data were normalized by the amount of 36B4 mRNA and expressed relative to the corresponding value for WAT of wild-type mice; data are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding value for wild-type mice. (C) Northern blot analysis of mRNAs for COXI, COXII, COXIV, and UCP1 in WAT and BAT of 14-week-old wild-type and FSP27-KO mice. (D) Oxygen consumption by adipocytes isolated from inguinal WAT (IWAT; left panel) or interscapular BAT (right panel) of wild-type and FSP27-KO mice. Arrows indicate the addition of the β3-adrenergic agonist CL316,243 or vehicle (Ca2+- and Mg2+-free PBS). Data are mean ± SEM of values from 4 independent experiments. (E and F) Glucose (E) and oleic acid (F) oxidation in adipocytes isolated from epididymal WAT or interscapular BAT of wild-type and FSP27-KO mice. Data are expressed relative to the corresponding value for WAT of wild-type mice and are mean ± SEM of values from 4 WT or 3 KO independent experiments. †P < 0.05, ††P < 0.01 versus the corresponding value for wild-type cells.
Figure 5
Figure 5. Localization of FSP27 to lipid droplets and its role in the pattern of lipid droplet accumulation in HW adipocytes.
(A and B) Immunofluorescence localization of FSP27 (A) and perilipin (B) in HW adipocytes by confocal laser microscopy. TAG was stained with Bodipy 493/503. (C) Immunoblot analysis of FSP27, perilipin, and β-actin (control) in HW adipocytes 2 days after the introduction of FSP27 or perilipin siRNAs as indicated. (D) Pattern of lipid droplet accumulation in HW adipocytes 2 days after siRNA introduction as in C. TAG was stained with Bodipy 493/503. (E) Glycerol release of HW adipocytes during incubation for 4 or 8 hours, 2 days after introduction of siRNAs as in C. Data are mean ± SEM (n = 6) and are expressed relative to the value for control cells incubated for 4 hours. *P < 0.01 versus corresponding value for control siRNA; **P < 0.01 versus corresponding value for FSP27 or perilipin siRNA. (F) Glycerol release of HW adipocytes during incubation for 40 minutes with 10 μM isoproterenol 2 days after introduction of siRNAs as in C. Data are mean ± SEM (n = 5) and are expressed relative to the value for control cells. *P < 0.01 versus corresponding value for control siRNA; †P < 0.01 versus corresponding value for perilipin siRNA. (G) Isolated white adipocytes of wild-type or FSP27-KO mice were incubated for 60 minutes in the absence (basal) or presence of 10 μM isoproterenol, after which concentrations of FFAs (left panel) and glycerol (right panel) in the culture medium were measured. Data are mean ± SEM (n = 5 WT or 4 KO for FFAs; n = 4 for glycerol) and are expressed relative to the corresponding basal value for wild-type cells. §P < 0.05, §§P < 0.01 versus corresponding value for wild type. (H) Serum FFA (left panel) and glycerol (right panel) concentrations in wild-type and FSP27-KO mice measured 20 minutes after intraperitoneal injection of CL316,243 (0.1 mg/kg) or vehicle. Data are mean ± SEM (n = 4 WT or 3 KO) and are expressed relative to the corresponding basal value for wild-type mice. Original magnification, ×630.
Figure 6
Figure 6. Effects of FSP27 depletion for 4 days on gene expression in HW adipocytes.
Quantitative RT-PCR analysis of the expression of genes whose products are associated with lipid synthesis (A), lipolysis (B), lipid droplets (C), mitochondrial biogenesis (D), fatty acid oxidation (E), or oxidative phosphorylation (F) in HW adipocytes 4 days after the introduction of FSP27 siRNA (KD). Data were normalized by the amount of 36B4 mRNA and expressed relative to the corresponding value for control cells; they are mean ± SEM from 4 independent experiments. Cont, control. *P < 0.01 versus control.
Figure 7
Figure 7. Formation of large lipid droplets induced by forced expression of FSP27 in COS cells.
(A) Immunoblot analysis of FSP27 and β-actin (loading control) in COS cells 2 days after transfection with an expression plasmid encoding both FSP27 and DsRed2 (pIRES2-DsRed2-FSP27). (B) COS cells transfected with pIRES2-DsRed2-FSP27 as in A were incubated with 400 μM oleic acid for 24 hours and then stained with Bodipy 493/503 (for TAG). A merged image of Bodipy 493/503 and DsRed2 fluorescence is shown. Arrowheads and arrows indicate cells positive or negative for FSP27 expression, respectively. Original magnification, ×630. (C–E) Quantitation of mean droplet size (C), droplet number (D), and total lipid amount (E) per cell for COS cells treated as in B but also stained with Hoechst 33258 (for nuclei). Total lipid amount was calculated as the product of the area and green fluorescence density of each droplet per cell. Data are mean ± SEM of values from 1,502 and 444 cells negative or positive for DsRed2 fluorescence, respectively, and are expressed relative to the corresponding value for DsRed2-negative cells. *P < 0.001 versus DsRed2-negative cells.
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