In this study, we identify SIRT3 and mitochondrial protein acetylation as crucial regulators of metabolic homeostasis and demonstrate that deficiency of SIRT3 leads to accelerated development of the diseases of the metabolic syndrome. While some genes influence several traits associated with the metabolic syndrome, thus far no single gene influences the entire spectrum (Lusis et al., 2008
), and mouse models encompassing a single or several traits of the metabolic syndrome continue to provide a better understanding of the mechanisms underlying the metabolic syndrome in humans. Our observations support the model that SIRT3 deficiency and the associated mitochondrial protein hyperacetylation results in mitochondrial dysfunction, and we identify three distinct conditions associated with decreased SIRT3 expression or activity that all lead to metabolic dysfunction.
First, wt mice fed a HFD develop obesity, hyperlipidemia, type 2 diabetes mellitus, insulin resistance, and non-alcoholic steatohepatitis (Collins et al., 2004
; Petro et al., 2004
; Rossmeisl et al., 2003
; Surwit et al., 1995
), and these deleterious effects of HFD feeding are further exacerbated in mice with a genetic deletion of Sirt3
. We previously reported that SIRT3 deacetylates mitochondrial proteins and that ablation of SIRT3 is associated with LCAD hyperacetylation, reduced LCAD enzymatic activity, and decreased fatty acid oxidation (Hirschey et al., 2010
). Interestingly, LCAD deficiency is also associated with accelerated development of a insulin resistance in mice (Zhang et al., 2007
). Ablation of LCAD in mice results in hepatic steatosis and hepatic insulin resistance, primarily attributed to lipid accumulation from reduced fatty acid oxidation (Kurtz et al., 1998
). Additionally, ablation of malonyl-CoA decarboxylase (MCD), an enzyme that regulates mitochondrial fatty acid oxidation, leads to reduced fatty acid oxidation and insulin resistance (Koves et al., 2008
). We speculate that increased SCD1 expression also contributes to the metabolic syndrome phenotype in SIRT3KO mice. Lipids are implicated in regulating Scd1
gene transcription [for a review, see (Paton and Ntambi, 2009
)], and elevated hepatic lipids in SIRT3KO mice lead to increased transcriptional signals that further activate Scd1
expression. Notably, increased Scd1
expression in SIRT3KO mice precedes the development of the metabolic syndrome phenotype, and ablation of Scd1
in SIRT3KO mice rescues the hepatic steatosis phenotype induced by HFD feeding. Thus, primary lesions in fatty acid oxidation upon ablation of SIRT3, LCAD, or MCD all result in elevated lipids and insulin resistance, and strongly support a role for mitochondrial lipid oxidation in the maintenance of insulin signaling and metabolic homeostasis.
Second, prolonged exposure to HFD feeding in wt mice results in a reduction of hepatic SIRT3 expression and increased mitochondrial protein acetylation, as reported previously (Bao et al., 2010
). Furthermore, HFD feeding results in LCAD hyperacetylation and reduces LCAD activity, demonstrating that HFD feeding partially mimics the phenotype of the SIRT3KO mice. The HFD-induced suppression of Sirt3
occurs at the transcriptional level, and is primarily driven by the HFD-induced suppression of PGC-1α (Crunkhorn et al., 2007
; Li et al., 2007
), which regulates the expression of SIRT3 (Kong et al., 2010
). Overexpression of exogenous PGC-1α was sufficient to rescue the loss of SIRT3 in HFD fed mice. However, overexpression of exogenous PGC-1α did not result in overexpression of SIRT3 in SD fed mice, which displayed basal levels of SIRT3. These data show that SIRT3 levels are tightly regulated at the transcriptional level under SD and HFD feeding. Because fatty acid oxidation is also suppressed by HFD feeding (Ji and Friedman, 2008
; Ji and Friedman, 2007
), we hypothesize that reduced SIRT3 and increased mitochondrial protein acetylation could be a new mechanism to reduce fatty acid oxidation in a HFD fed state.
Third, we find a correlation between a genetic polymorphism in SIRT3
in humans and the development of the metabolic syndrome. Although SNPs in SIRT3
have not been identified in large-scale genome wide association studies in obesity (Heid et al., 2010
; Lindgren et al., 2009
; Speliotes et al., 2010
), diabetes (Dupuis et al., 2010
; Prokopenko et al., 2009
), or cholesterol and lipid metabolism (Musunuru et al., 2010
; Teslovich et al., 2010
), we tested the possibility that SNPs in SIRT3
were associated with metabolic dysfunction in a Caucasian cohort diagnosed with fatty liver disease (The NASH CRN). This cohort was chosen because SIRT3KO mice have clear signs of fatty liver disease, and we hypothesized these patients might be enriched for one of 12 SNPs in SIRT3
. Our unbiased approach identified a single SNP that correlated with increased risk of the metabolic syndrome from over 30 clinical parameters tested.
In a subsequent study focusing specifically on the rs11246020 “A” minor allele, we sought to validate the SNP association in a population of approximately 8000 Finnish men (Stancáková et al., 2009
). We observed an additional correlation between the frequency of this allele and meeting the criteria for diagnosis of the metabolic syndrome. While these data are suggestive and support the findings in the NASH-CRN study, these data are not definitive and further human genetic studies will fully elucidate the association between rs11246020 and the metabolic syndrome. Although, the complexity of different experimental designs and measures introduce heterogeneity in the results that are more difficult to interpret together, the both studies provide evidence of an association between a single SNP in SIRT3
and the metabolic syndrome.
To further define this relationship, we examined the functional impact of the non-synonymous point mutation (V208I) in the SIRT3 protein. Indeed, the V208 lies within the conserved sirtuin catalytic deacetylase domain, and mutation from valine to isoleucine reduces SIRT3 enzyme efficiency, by both increasing the KM for NAD+ and reducing the Vmax. These data are consistent with the model that reduction in SIRT3 enzymatic activity associated with the V208I mutation plays a pathogenic role in humans, as in mice, and increases susceptibility to the metabolic syndrome. Taken together, our observations highlight the importance of using primary cellular and mouse data to direct human genetic studies and the power of integrating these data to glean insights into the relationships between human SNPs and the underlying biology.
We propose that reduction or loss of SIRT3 and the resulting mitochondrial protein hyperacetylation lead to global mitochondrial dysfunction (). Since, every major metabolic pathway in human liver contains acetylated proteins (Zhao et al., 2010
), the function of other critical mitochondrial proteins is likely to be dysregulated in the absence of SIRT3. A number of abnormalities in mitochondria have been identified in prior studies as key pathogenic mechanisms in the development of the metabolic syndrome, including reduced mass (Kelley et al., 2002
), altered morphology (Civitarese et al., 2010
), reduced fatty acid oxidation (Zhang et al., 2007
), lower oxidative phosphorylation (Befroy et al., 2007
; Petersen et al., 2005
), and increased reactive oxygen species (Civitarese et al., 2006
; Patti et al., 2003
; Petersen et al., 2004
; Ukropcova et al., 2007
). SIRT3KO show an overlapping group of mitochondrial abnormalities including reduced fatty acid oxidation (Hallows et al., 2011
; Hirschey et al., 2010
), lower oxidative phosphorylation (Ahn et al., 2008
), and increased reactive oxygen species (Kim et al., 2010
; Qiu et al., 2010
; Someya et al., 2010
; Tao et al., 2010
). Thus, these data support the hypothesis that a primary mitochondrial lesion results in global metabolic dysfunction, and can progress to metabolic disease.
We conclude that mitochondrial protein acetylation is a critical post-translational modification, whose regulation by SIRT3 is necessary to maintain metabolic health in mice and possibly in humans. Future studies will examine the therapeutic potential of manipulating SIRT3 expression or activity in the liver or other tissues to ameliorate manifestations of the metabolic syndrome.