SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome

Abstract

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.

Fig. 1. Chronic HFD feeding results in global mitochondrial hyperacetylation and reduces hepatic SIRT3

(A) Mitochondria were isolated from livers of wt mice fed a standard or HFD for 1 week or 13 weeks (Jackson Laboratory) and analyzed for mitochondrial protein acetylation by western blot analysis with an antiserum specific anti-acetyllysine; n=3 mice/condition. (B) Mitochondria were isolated from livers of wt mice fed a standard or HFD for 1 week, 5 weeks or 13 weeks (Jackson Laboratory) and analyzed for SIRT3 expression by western blot analysis with an antiserum specific for SIRT3. Integrated density values were calculated for SD and HFD fed wt mice; data represented in arbitrary units (AU) ±SEM, n=3 mice/condition, *p<0.05; (C, D) mRNA transcript levels were quantified by qPCR from wt mice, (from panel B, *p<0.05, n=3/genotype, standard or HFD, ±SEM). (E, F) mRNA transcript levels were quantified by qPCR from wt mice fed a standard (SD) or high-fat (HF) diet overexpressing adenoviral PGC-1α or GFP as a control, (*p<0.05, **p<0.01, n=5/condition, ±SEM).

Fig. 2. Chronic HFD feeding induces LCAD hyperacetylation and reduces enzymatic activity

(A) Mitochondria were isolated from livers of 6-month old wt and SIRT3KO mice fed a standard or HFD and analyzed for mitochondrial protein acetylation with an antiserum specific anti-acetyllysine; average integrated densitometry values (IDV) were calculated relative to wt mice fed a standard diet, ±SEM. (B) Mitochondria were isolated from livers of wt and SIRT3KO mice fed a standard or HFD and analyzed for SIRT3 expression by western blot analysis with an antiserum specific for SIRT3. (C) Liver extracts from wt and SIRT3KO mice fed a standard or HFD were immunoprecipitated with an anti-acetyllysine antiserum and analyzed with anti-LCAD antiserum; average integrated densitometry values (IDV) were calculated relative to one wt mouse fed a standard diet and LCAD input was used as a reference, ±SEM. (D) Liver extracts from wt and SIRT3KO mice fed a standard or HFD were assessed for enzymatic activity ex vivo using 2, 6 dimethylheptanoyl-CoA as a substrate; *p<0.05, all samples from same mice, n=3/condition, representative samples shown, ±SEM.

Fig. 3. SIRT3KO develop diet-induced obesity and insulin resistance

(A) Body weight measurements were recorded from wt and SIRT3KO mice weaned onto and maintained on a HFD (n=20/genotype); (B, C) 3-month old WT and SIRT3KO mice were assessed for the volume of oxygen consumption (VO₂) (B) and carbon dioxide exhalation (VCO₂) (C), in metabolic cages, n=10/genotype, 3 independent trials, data collected over 48 h and normalized to lean body mass, averages were totaled for dark and light cycles, ±SEM. (D, E) 12-month old SIRT3KO and wt mice fed a HFD were tested for glucose (D) and insulin tolerance (E) and measured for blood glucose levels; inset data represent area under the curve (AUC), ±SEM. (F, G) 12-month old SIRT3KO and wt mice fed a standard diet were tested for glucose (F) and insulin tolerance (G) and measured for blood glucose levels; inset data represent AUC, (n=5/genotype, fasted 6 h, standard diet); *p<0.05, **p<0.01, ±SEM.

Fig. 4. SIRT3KO mice fed a HFD develop hepatic steatosis and inflammation

(A) Histological analysis of livers from HFD fed wt and SIRT3KO mice with Oil Red O Stain (fed or fasted 24 h). (B) Livers extracts from wt and SIRT3KO mice fed a HFD were analyzed for total phospholipids, triglycerides and cholesterol esters (n=5/genotype, fasted 24 h, ±SEM). (C) Hepatic lipids were measured in wt mice 1 week after injection with adenovirus expression vectors for GFP or SIRT3. (D) Hepatic sections from 12-month-old wt and SIRT3KO mice fed a HFD were fixed, stained with hematoxylin & eosin (H&E), Masson’s trichrome, or reticulin, and scored for inflammation, steatosis, ballooning, and fibrosis, (n=15/genotype, Wilcoxon rank-sum test, *p<0.05). (E) Cytokine analyses were conducted on serum obtained from 12-month-old wt and SIRT3KO mice maintained on a HFD (n=5/genotype, ±SEM). (F) Serum triglyceride (TG), cholesterol (Chol), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) were measured in 12-month-old wt and SIRT3KO mice fed a HFD ad libitum (fasted 24 h, n=5 mice/genotype, ±SEM).

Fig. 5. SIRT3KO mice have high expression and activity of hepatic SCD1

(A) mRNA transcript levels were quantified by qPCR from wt and SIRT3KO mice, (*p<0.05, n=3/genotype, 3-month old mice, standard diet, ±SEM). (B) Plasma samples from 3-month old SIRT3KO and wt mice were analyzed for desaturation indices in triglyceride (TG) phospholipids (PL) and free fatty acids (FFA) by measuring palmitate (C16), palmitoleate (C16:1), stearate (C18) and oleate (C18:1) (*p<0.05, n=5/genotype, 3-month-old mice, standard diet, ±SEM). (C) Adenoviral SCD1 promoter activity in Huh7 cells after treatment with lipids extracted from plasma or liver tissue in wt or SIRT3KO (*p<0.05, n=3/genotype, 12-week-old mice, standard diet). (D) Hepatic extracts from SIRT3KO and wt mice were analyzed for acyl-CoA lipid species (*p<0.05, n=5/genotype, 12-week-old mice, standard diet). (E) Adenoviral SCD1 promoter activity in Huh7 cells after treatment with palmitate, palmitoleate, stearate, oleate (n=3/condition, ±SEM, *p<0.05) (F) Hepatic triglycerides from wt, SCD1KO, SIRT3KO, and SCD1KO/SIRT3KO (dKO) mice fed a standard (SD) or high-fat (HF) diet were measured (n=5/genotype, fed or fasted 24 h, ±SEM). (G) 2-month old wt, SIRT3KO, SCD1KO, and dKO mice fed a HFD were tested for glucose tolerance and measured for blood glucose levels; inset data represent area under the curve (AUC), ±SEM.

Fig. 6. A functional SNP in the human SIRT3 gene is associated with human metabolic syndrome and encodes a point-mutation

(A) Heat map detailing the pairwise LD among the 7 SNPs spanning the SIRT3 coding region; the pairwise correlations (D’) are rendered within each diamond with greater LD reflected by darker shades of gray; SIRT3 gene structure is depicted above the LD heat map: exons are depicted in gray boxes, introns as connecting black lines, and untranslated regions as smaller boxes shades in pink; approximately 10 kbp of flanking DNA sequence was included in the tagSNP selection procedure; tagSNPs the promoter region is included in the diagram to the right of the transcription start site indicated by the arrow found directly above the SIRT3 gene structure diagram; SNPs rendered in bold are included in the haploblock outlined in the black triangle; the two SNPs that encode for nonsynonymous polymorphisms (rs11246020 and rs28365927; green). (B) Association of SIRT3 rs11246020 with the metabolic syndrome in the NASH-CRN cohort. Abbreviations: P, p-value for the test statistic; OR (95% CI), the odds ratio and 95% confidence interval for the test statistic; VNTR, variable number tandem repeat; UNADJ, unadjusted; ADJ, adjusted for age, sex, and BMI unless otherwise indicated. (C) Association of SIRT3 rs11246020 with the metabolic syndrome in the METSIM cohort, adjusted for age and BMI. ^†Metabolic syndrome criteria based on the IDF guidelines, excluding the presence of type 2 diabetes. *p-value for the overall model. (D) Schematic of SIRT3 protein; mitochondrial targeting sequence (MTS), mitochondrial processing peptidase (MPP) site. (E, F) Steady-state kinetic analyses of SIRT3 activity; rates of activity were measured as a function of [NAD⁺] (E) or [³H-histone H4 peptide] (F), as measured by organic-soluble radioactive signal, n=3 independent measurements/sample, ±SEM.

Fig. 7. Working model

SIRT3 functions to deacetylate mitochondrial proteins, and increase fatty acid oxidation and energy production. In SIRT3KO mice or mice fed a HFD, mitochondrial proteins are hyperacetylated, resulting in reduced energy expenditure and less fatty acid oxidation, which contributes to insulin resistance, obesity, and increased inflammation. Similarly, humans with a unique SNP in the SIRT3 gene have reduced SIRT3 enzymatic efficiency and increased risk to develop the metabolic syndrome.