High-fat diet triggers inflammation-induced cleavage of SIRT1 in adipose tissue to promote metabolic dysfunction

Abstract

Adipose tissue plays an important role in storing excess nutrients and preventing ectopic lipid accumulation in other organs. Obesity leads to excess lipid storage in adipocytes, resulting in the generation of stress signals and the derangement of metabolic functions. SIRT1 is an important regulatory sensor of nutrient availability in many metabolic tissues. Here we report that SIRT1 functions in adipose tissue to protect from inflammation and obesity under normal feeding conditions, and to forestall the progression to metabolic dysfunction under dietary stress and aging. Genetic ablation of SIRT1 in adipose tissue leads to gene expression changes that highly overlap with changes induced by high-fat diet in wild-type mice, suggesting that dietary stress signals inhibit the activity of SIRT1. Indeed, we show that high-fat diet induces the cleavage of SIRT1 protein in adipose tissue by the inflammation-activated caspase-1, providing a link between dietary stress and predisposition to metabolic dysfunction.

Figure 1. SIRT1 Deletion from Adipose Tissue Leads to Obesity

(A) Representative experiment of body weight gain of WT and FKO female mice on chow diet starting at 4 weeks of age (n = 5). (B–D) Gonadal, inguinal and brown fat mass of WT and FKO female mice at 2 months of age (n = 5). (E) Representative H&E staining of gonadal white adipose tissue and quantification of adipocyte size (500 cells/genotype, n = 3). All of the above experiments were repeated at least once, yielding the same conclusions. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed unpaired Student’s t test. See also Figure S1.

Figure 2. SIRT1 Deletion from Adipose Tissue Results in Metabolic Deregulation

(A) Weekly body weight of WT and FKO female mice on low-fat (LFD) and high-fat (HFD) diet. (B) Glucose and insulin tolerance tests (GTT and ITT) of mice after 12 weeks on diet (n = 8). (C and D) Body weight of aged WT and FKO female mice (~1 year old) (C) and glucose and insulin tolerance test (n = 10–12) (D). The above experiments were repeated at least once, yielding the same conclusions. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed unpaired Student’s t test. See also Figures S2 and S3.

Figure 3. Adipose SIRT1 Deletion and High-Fat Diet Induce Similar Changes in Gene Expression

(A) Hierarchical clustering of samples based on changes in gene expression. (B) Representative GSEA plots of gene pathways upregulated or downregulated in both the comparison FKO/LFD versus WT/LFD and WT/HFD versus WT/LFD are shown. Positive and negative enrichment scores indicate correlation and anticorrelation respectively. NES, normalized enrichment score; FDR, false discovery rate. (C) Venn diagram of overlapping genes significantly changed (±1.5-fold) in the comparisons FKO/LFD versus WT/LFD and WT/HFD versus WT/LFD. See also Figure S4 and Table S1.

Figure 4. Obesity and Inflammation Result in Reduced SIRT1 Activity in Adipose Tissue

(A) Relative mRNA levels of SIRT1 in adipose tissue of male mice fed low- or high-fat diet (males, n = 6). (B) Quantitative analysis of SIRT1 protein, using Image J software. Western blots of adipose tissue lysates from the same mice are shown. (C) Western blots in adipose tissue lysates prepared from WT and ob/ob male mice. (D) Western blot in adipose tissue lysates prepared from male mice treated with lipopolysacharides for 1 hr and 6 hr. (E) Western blot in adipose tissue lysates prepared from male mice treated with TNFα, or combination of TNFα and the caspase inhibitor BOC-D-FMK (BOC). In all panels, red arrows indicate the cleaved form of SIRT1 and blue arrows indicate the active form of caspase-1. All of the above experiments were repeated at least once, yielding the same conclusions. Data are expressed as mean ± SEM. **p < 0.01, ***p < 0.001 by two-tailed unpaired Student’s t test. See also Figure S5.

Figure 5. Caspase-1 Cleaves SIRT1

(A) Western blot in whole cell extracts of differentiated 3T3L1 adipocytes treated with TNFα or combination of TNFα and the caspase inhibitor BOC-D-FMK (BOC). (B) Western blot in WT or SIRT1 KO mouse embryonic fibroblasts lysates treated with TNFα or TNFα and BOC. (C) Western blot in WT MEFs cell extracts treated in vitro with the indicated recombinant caspases. (D) Western blot in extracts prepared from KO MEFs expressing WT or the indicated point mutant SIRT1 and treated in vitro with recombinant caspase-1. (E) Western blot in extracts prepared from KO MEFs expressing WT or the indicated mutant SIRT1, treated with TNFα. In all panels, arrows indicate the cleaved SIRT1 protein. All of the above experiments were repeated at least once, yielding the same conclusions. See also Figure S6.

Figure 6. Caspase-1 Cleaved SIRT1 Is Less Stable than Full-Length SIRT1

(A) Western blot in whole-cell extracts of SIRT1 KO MEFs transfected expressing the full-length SIRT1 or the truncated SIRT1 (fragment: D142-end) reveals a defect in p53 acetylation in cells transfected with truncated SIRT1. (B) Western blot of cells treated with cyclohexamide (CHX) to inhibit protein synthesis for the indicated time. The graphs show the relative protein levels of full-length and truncated SIRT1 at time points after inhibition. All of the above experiments were repeated at least once, yielding the same conclusions. Data are expressed as mean ± SEM. *p < 0.05, ***p < 0.001 by two-tailed unpaired Student’s t test.