Targeted Deletion of Adipocyte Abca1 (ATP-Binding Cassette Transporter A1) Impairs Diet-Induced Obesity

Abstract

Objective: Adipose tissue cholesterol increases with adipocyte triglyceride content and size during development of obesity. However, how adipocyte cholesterol affects adipocyte function is poorly understood. The aim of this study was to evaluate the role of the cellular cholesterol exporter, Abca1 (ATP-binding cassette transporter A1), on adipose tissue function during diet-induced obesity.

Approach and results: Adiponectin Cre recombinase transgenic mice were crossed with Abca1^flox/flox mice to generate ASKO (adipocyte-specific Abca1 knockout) mice. Control and ASKO mice were then fed a high-fat, high-cholesterol (45% calories as fat and 0.2% cholesterol) diet for 16 weeks. Compared with control mice, ASKO mice had a 2-fold increase in adipocyte plasma membrane cholesterol content and significantly lower body weight, epididymal fat pad weight, and adipocyte size. ASKO versus control adipose tissue had decreased PPARγ (peroxisome proliferator-activated receptor γ) and CCAAT/enhancer-binding protein expression, nuclear SREBP1 (sterol regulatory element-binding protein 1) protein, lipogenesis, and triglyceride accretion but similar Akt activation after acute insulin stimulation. Acute siRNA-mediated Abca1 silencing during 3T3L1 adipocyte differentiation reduced adipocyte Abca1 and PPARγ protein expression and triglyceride content. Systemic stimulated triglyceride lipolysis and glucose homeostasis were similar between control and ASKO mice.

Conclusions: Adipocyte Abca1 is a key regulator of adipocyte lipogenesis and lipid accretion, likely because of increased adipose tissue membrane cholesterol, resulting in decreased activation of lipogenic transcription factors PPARγ and SREBP1.

Keywords: adipose tissue; animals; cholesterol; mice; obesity.

Figure 1

A) Abca1 and GAPDH Western blots of tissues from Cntl, heterozygous ASKO (HT), and ASKO mice. EAT=epididymal adipose tissue, BAT=brown adipose tissue, Peri. Mac.=resident peritoneal macrophages, BMDM=bone marrow-derived macrophages. B) Abca1 gene expression in liver, EAT, SAT and BAT (n=6 per group). *** denotes statistical difference by Student’s t-test, p<0.0001. Plasma concentrations of: C) total cholesterol, D) HDL cholesterol, E) LDL cholesterol, and F) triglyceride (TG) in male Cntl and ASKO mice fed chow for 24 weeks or a HFHC diet for 16 weeks starting at 8 weeks of age. Total plasma cholesterol and TG concentrations were determined from individual plasma samples. LDL and HDL cholesterol concentrations were determined after FPLC fractionation of plasma and enzymatic assay of cholesterol as described in the Methods. Each data point represents an equal volume pooled from 2 mice. Panels C and D, different letters denote statistical difference by ANOVA and Tukey’s multiple comparisons test, p<0.05.

Figure 2

Male Cntl and ASKO mice were fed chow for 24 weeks (n=5–6) or a HFHC diet for 16 weeks starting at 8 weeks of age (n=20). Mice were sacrificed at 24 weeks of age to harvest adipose tissue. A) Body weight gain during dietary feeding. B) Terminal body weights. C) Epididymal adipose tissue (EAT) weight harvested at 24 weeks of age. D) Percentage of body weight as fat was calculated by summing the weight of epididymal, retrorenal, subcutaneous, and brown fat depots and dividing the sum by total body weight X 100% for each mouse. E) Snout to tail length of mice at 24 weeks of age. F) Representative photomicrographs of EAT harvested at 24 weeks of age. G) Histogram of EAT adipocyte cross-sectional area; 300 cell measurements per mouse were taken from n=6 mice per genotype. Adipocyte size was significantly smaller in ASKO vs. Cntl mice by Chi square analysis (p<0.001). Asterisks (panels A and D) and different letters (panels B and C) denote statistical difference by ANOVA and Tukey’s multiple comparisons test or unpaired Student’s t-test, p<0.05.

Figure 2

Male Cntl and ASKO mice were fed chow for 24 weeks (n=5–6) or a HFHC diet for 16 weeks starting at 8 weeks of age (n=20). Mice were sacrificed at 24 weeks of age to harvest adipose tissue. A) Body weight gain during dietary feeding. B) Terminal body weights. C) Epididymal adipose tissue (EAT) weight harvested at 24 weeks of age. D) Percentage of body weight as fat was calculated by summing the weight of epididymal, retrorenal, subcutaneous, and brown fat depots and dividing the sum by total body weight X 100% for each mouse. E) Snout to tail length of mice at 24 weeks of age. F) Representative photomicrographs of EAT harvested at 24 weeks of age. G) Histogram of EAT adipocyte cross-sectional area; 300 cell measurements per mouse were taken from n=6 mice per genotype. Adipocyte size was significantly smaller in ASKO vs. Cntl mice by Chi square analysis (p<0.001). Asterisks (panels A and D) and different letters (panels B and C) denote statistical difference by ANOVA and Tukey’s multiple comparisons test or unpaired Student’s t-test, p<0.05.

Figure 3

A) Male Cntl and ASKO mice were fed chow for 24 weeks or a HFHC diet for 16 weeks starting at 8 weeks of age before EAT was harvested to measure total cholesterol content. B) Expression of genes involved in cholesterol metabolism in EAT from HFHC-fed mice diet for 16 weeks (n= 6 per group). C) Western blot of EAT ABCG1, LDLr and GAPDH from mice fed the HFHC diet diet for 16 weeks. D) Quantification of Western blot data in panel C. E) EAT plasma membrane cholesterol content from mice fed the HFHC diet for 16 weeks. F) Lipid raft staining of EAT from mice fed the HFHC diet for 16 weeks. Lipid rafts detected by fluorescent-labeled beta-cholera toxin, which binds to GM1 gangliosides. Negative control contained no fluorescent-labeled beta-cholera toxin. Different letters (panel A) denote statistical difference by ANOVA and Tukey’s multiple comparisons test. Asterisks (panels B, D, and E) denote statistical differences between genotypes by unpaired Student’s t-test, p<0.05.

Figure 3

A) Male Cntl and ASKO mice were fed chow for 24 weeks or a HFHC diet for 16 weeks starting at 8 weeks of age before EAT was harvested to measure total cholesterol content. B) Expression of genes involved in cholesterol metabolism in EAT from HFHC-fed mice diet for 16 weeks (n= 6 per group). C) Western blot of EAT ABCG1, LDLr and GAPDH from mice fed the HFHC diet diet for 16 weeks. D) Quantification of Western blot data in panel C. E) EAT plasma membrane cholesterol content from mice fed the HFHC diet for 16 weeks. F) Lipid raft staining of EAT from mice fed the HFHC diet for 16 weeks. Lipid rafts detected by fluorescent-labeled beta-cholera toxin, which binds to GM1 gangliosides. Negative control contained no fluorescent-labeled beta-cholera toxin. Different letters (panel A) denote statistical difference by ANOVA and Tukey’s multiple comparisons test. Asterisks (panels B, D, and E) denote statistical differences between genotypes by unpaired Student’s t-test, p<0.05.

Figure 4

A) EAT TG content of male Cntl and ASKO mice fed HFHC diet for 16 weeks starting at 8 weeks of age. B) Plasma NEFA concentrations before and after stimulation with a β-3 specific adrenergic agonist in male mice fed HFHC diet for 8 weeks starting at 8 weeks of age. C) Incorporation of [³H]-oleic acid into EAT explant lipid fractions (n=6 per group). D) Incorporation of [¹⁴C]-acetate into EAT explant lipid fractions (n=4 per group). Different letters (panel B) denote statistical difference by ANOVA and Tukey’s multiple comparisons test. Asterisks (panels A, C, and D) denote statistical difference between genotypes by Student’s t-test, p<0.05.

Figure 5

Male Cntl and ASKO mice were fed chow for 24 weeks or a HFHC diet for 16 weeks starting at 8 weeks of age and then fasted for 12 hours before blood and plasma were collected for glucose (A) and insulin (B) measurements, respectively. C) Male mice fed a HFHC diet for 16 weeks were fasted overnight, anesthetized, and injected with insulin (1U/Kg) in the portal vein. Five minutes later, mice were euthanized and EAT, liver, and skeletal muscle were harvested for Western blot analysis of p-Akt and total Akt expression. D) Western blot quantification of p-Akt/total Akt from blots in C. E) Glucose tolerance tests were performed on mice fed the HFHC diet for 16 weeks after a 12 hour fast. F) Insulin tolerance tests were performed on mice fed the HFHC diet for 16 weeks after a 12 hour fast. Different letters (panel A, B) denote statistical difference by ANOVA and Tukey’s multiple comparisons test, p<0.05.

Figure 5

Male Cntl and ASKO mice were fed chow for 24 weeks or a HFHC diet for 16 weeks starting at 8 weeks of age and then fasted for 12 hours before blood and plasma were collected for glucose (A) and insulin (B) measurements, respectively. C) Male mice fed a HFHC diet for 16 weeks were fasted overnight, anesthetized, and injected with insulin (1U/Kg) in the portal vein. Five minutes later, mice were euthanized and EAT, liver, and skeletal muscle were harvested for Western blot analysis of p-Akt and total Akt expression. D) Western blot quantification of p-Akt/total Akt from blots in C. E) Glucose tolerance tests were performed on mice fed the HFHC diet for 16 weeks after a 12 hour fast. F) Insulin tolerance tests were performed on mice fed the HFHC diet for 16 weeks after a 12 hour fast. Different letters (panel A, B) denote statistical difference by ANOVA and Tukey’s multiple comparisons test, p<0.05.

Figure 6

Male Cntl and ASKO mice were fed a HFHC diet for 16 weeks, starting at 8 weeks of age, before harvesting EAT for gene and protein expression. A) EAT gene expression of lipogenic genes and transcription factors (n=6 per group). B) Western blot for PPARγ, CD36, SCD-1, and GAPDH in EAT tissue. C) Quantification of Western blot results in panel B; asterisk denotes statistical difference by upaired Student’s t-test, p<0.05. D) Western blot of SREBP1 and YY1 in nuclear fraction of EAT isolated adipocytes. E) and F) 3T3-L1 cells were transfected with scrambled control siRNA (C) or with Abca1 siRNA (A) for 6 h and then differentiated for 8 d. Abca1, PPARγ, aP2, and GAPDH expression was examined by Western blot and cellular triglyceride (TG) content was measured by enzymatic TG assay. Two-way ANOVA results showed significant differences for time (0.0001), siRNA treatment (0.03), and interaction (0.01); asterisks denote Bonferroni’s multiple comparisons test results; *** p<0.001.