Perilipin overexpression in white adipose tissue induces a brown fat-like phenotype

Abstract

Background: Perilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model.

Methodology and principal findings: When compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size.

Conclusions: These findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders.

Figure 1. Oxygen consumption and energy expenditure.

(A) Whole-body oxygen consumption rate during a 12-hour dark/12-hour light cycle for 30-week-old mice fed a high fat diet (n = 7 for WT; n = 7 for Tg). (B) The average value during the 24-hour period. (C) Energy expenditure during 24-hour period in the experiment. FCBW: fat-corrected body weight = body weight−(mass of subcutaneous and perigonadal white adipose tissue). Data are mean ± SEM. *, p<0.05.

Figure 2. DNA microarray analysis.

Metabolic pathways altered in WAT of Tg mice: cell differentiation, lipid synthesis, fatty acid oxidation, and thermogenesis. Fold changes in transcript levels are noted beneath the gene symbols. Atp5g1: ATP synthase, H+ transporting, F1 gamma 1, Bmp: bone morphogenic protein, Cac: carnitine/acylcarnitine translocase, Cox: cytochrome c oxidase, Cpt: carnitine palmitoyl transferase, Dgat: diacylglycerol acyltransferase, Kat: 3-ketoacyl-CoA thiolase, Pgc1a: peroxisome proliferator activated receptor gamma coactivator-1 alpha, Prdm16: PRD1-BF1-RIZ1 homologous domain containing 16, Scd: stearoyl-CoA desaturase.

Figure 3. Quantitative PCR.

Quantitative real-time PCR analysis of the expression of genes in WAT of 30-week-old WT and Tg mice fed a high fat diet, related to (A) fatty acid oxidation and thermogenesis, (B) lipid synthesis, and (C) cell differentiation to white/brown adipocytes. Data were normalized by the amount of 36B4 mRNA and expressed relative to the corresponding value for WAT of WT mice; Data are mean ± SEM (n = 9). *, p<0.05; **, p<0.01. Acc: acetyl-CoA carboxylase, Adrb3: beta-3 adrenergic receptor, Fas: fatty acid synthease, Lpl: lipoprotein lipase, Mcd: malonyl-CoA decarboxylase, Srebp1c: sterol regulatory element binding protein 1c.

Figure 4. Ectopic expression of UCP1.

Ectopic expression of UCP1 in WAT of 30-week-old PeriATg mice fed a chow diet. (A) Quantitative real-time PCR analysis of Ucp1 mRNA expression in WAT of WT and Tg mice. Data are mean ± SEM (n = 7). **, p<0.01. (B) Western blot analysis showing UCP1 or CPT1 protein expression in WT and Tg mice. (C) Innunohistochemistry of PeriA or UCP1 in WAT of WT and Tg mice. Original magnification, ×400.

Figure 5. FSP27 expression in vivo.

Alteration in the expression of lipid droplet proteins in vivo. (A) Quantitative real-time PCR analysis of Fsp27 mRNA expression in WAT of WT and Tg mice. Data are mean ± SEM (n = 7). ***, p<0.001. (B) Western blot analysis for FSP27, RIP140 and PeriA in WAT of WT and Tg mice.

Figure 6. In vitro experiments.

Change of lipid droplet size and protein expression in vitro. (A) Microscopic images of 3T3-L1 adipocytes transfected human PeriA or GFP (control) using adenoviral system plus lipofection methods. (B) Western blot analysis for lipid droplet surface proteins (PeriA and FSP27) and Actin (as an internal control) in 30µg of lysates of day10 3T3-L1 cells treated with adenovirus PeriA (Ad PeriA). PeriA protein content increased dose-dependently with increasing PeriA viral titer in 3T3-L1 cells. Signs in a figure ((−), (+) and (++)) mean the amount of transfected adenovirus (none, single and double quantity). (C) Quantitative real-time PCR analysis of mRNA expression in cultured 3T3-L1 cells (white bar, Ad PeriA(−): black bar, Ad PeriA(++); Data are mean ± SEM (n = 6). *, p<0.05; **, p<0.01; ***, p<0.001). mtTFA: mitochondrial transcription factor A, Nrf1: nuclear respiratory factor 1, Vlcad: very long-chain acyl-CoA dehydrogenase, Lcad: long-chain acyl-CoA dehydrogenase, Mcad: medium-chain acyl-CoA dehydrogenase.