SIRT1 Can Regulate Mitochondria through a PGC-1α/β-Independent Pathway

A central dogma in the sirtuin field is that SIRT1 promotes mitochondrial function in response to fasting and CR by deacetylating PGC-1α (Gerhart-Hines et al., 2007, Ptitsyn et al., 2006). Consistent with this, SIRT1 iKO animals failed to upregulate both nuclear- and mitochondrially encoded OXPHOS genes in response to fasting (Figure S2C). However, our findings in fed animals (see Figure 1) indicated that SIRT1 can regulate mitochondrial genes independently of PGC-1α. To test this, we examined primary myotubes from PGC-1α/β knockout (KO) mice (Zechner et al., 2010) and from PGC-1α muscle-specific null mice (Handschin et al., 2007), and we saw no defect in the ability of SIRT1 and NMNAT1 to induce mitochondrially encoded OXPHOS genes (Figures 2I and 2J). Thus, SIRT1 can induce OXPHOS genes in the absence of PGC-1α/β (Figure S2D).

SIRT1 Regulates Mitochondrially Encoded Genes through HIF-1α

Next, we sought to understand how SIRT1 regulates mitochondria independently of PGC-1α/β. Analysis of SIRT1 iKO animals indicated that genes involved in glycolysis were upregulated, with increased lactate levels (Figures 3A and 3B ) and a switch from slow-twitch oxidative fibers (MyHCIIa) to fast-twitch glycolytic fibers (MyHCIIb) (Figure S1F). These metabolic changes were reminiscent of Warburg remodeling of metabolism in cancer cells, which is known to be mediated, in part, by the stabilization of the transcription factor HIF-1α (Majmundar et al., 2010). The levels of HIF-1α and the expression of HIF-1α target genes were considerably higher in the SIRT1 iKO (Figures 3C and S3A). Despite being cultured under normoxic conditions, primary myoblasts deleted for SIRT1 also had increased HIF-1α protein levels and activity of a HIF-1α reporter (Figures 3C and S3B). Reducing NAD+ levels, either by knocking down NMNAT1 or by treating cells with lactate (which decreases the NAD+/NADH ratio), also caused HIF-1α protein stabilization (Figures 3D, 3E, and S3C).
HIF-1α has been studied extensively in cancer and during hypoxia; however, its role in normal physiology remains largely unknown. To better understand this, HIF-1α was stabilized ectopically in vivo by deleting the EglN1 gene encoding HIF prolyl hydroxylase 2 (PHD2) (Minamishima et al., 2008). Upon EglN1 deletion and HIF-1α stabilization in muscle, there was a specific decline in mtDNA content and decreased levels of mitochondrially encoded, but not nuclear-encoded, OXPHOS mRNA, paralleling the effects of SIRT1 deletion and normal aging (Figures 3F–3H). Pharmacological stabilization of HIF-1α in PGC-1α/β knockout myotubes reduced expression of mitochondrially encoded genes (Figures 3I and S3D), whereas treating PGC-1α/β KO cells with pyruvate (to increase NAD+ levels) upregulated mitochondrially encoded genes, an effect that was prevented by stabilization of HIF-1α (Figure S3E). Stabilization of HIF-1α in primary cells and transgenic mice blocked the ability of SIRT1 to upregulate mitochondrially encoded genes and increase ATP levels, with a specific loss of mitochondrially encoded mRNAs (Figures 3I–3L and S3F–SFI). Overexpression of a stabilized mutant version of the related factor HIF-2α did not have the same effect (Figures 3J–3L and S3I), demonstrating that the inhibition of OXPHOS and mitochondrially encoded genes is HIF-1α specific. In primary myoblasts lacking HIF-1α, deletion of SIRT1 had no effect on mtDNA content, mitochondrially encoded gene expression, or ATP levels (Figures 3M–3P). Together, our results show that HIF-1α, but not HIF-2α, regulates mitochondria in response to SIRT1 activity, which is under the control of nuclear NAD+ levels.

SIRT1 Stabilizes HIF-1α via VHL

HIF-1α can be stabilized by ROS originating from complex III of the ETC as part of retrograde response (Bell et al., 2007). Six hours after inducing SIRT1 deletion in primary myoblasts, HIF-1α levels increased (Figure 5F), and by 12 hr, mitochondrial homeostasis was impaired (Figures 1L, S2A, and S2B). Yet, ROS levels did not increase until the 24 hr time point (Figure S4A). Myoblasts depleted of mitochondrial DNA (rho0), which are unable to produce ROS and signal to the nucleus (Chandel and Schumacker, 1999), were similar to the parental control cells (Figure S4B), indicating that ROS and retrograde signaling are not the cause of HIF-1α stabilization.
HIF-1α stability has been previously reported to be regulated by acetylation of lysine 709 (Geng et al., 2011). To test whether SIRT1-mediated deacetylation was the mechanism, we mutated K709 to glutamine (an acetylation mimetic) or to arginine (nonacetylated mimetic), with K674 serving as a negative control (Lim et al., 2010). Neither of the K709 substitutions stabilized HIF-1α, nor were they affected by SIRT1 deletion (Figure S4C), indicating that SIRT1 does not regulate HIF-1α protein stability by deacetylating K709.
HIFα proteins are regulated by a proteasomal degradation mechanism mediated by the Von Hippel-Lindau (VHL) E3 ubiquitin ligase that recognizes hydroxylated proline residues on HIFα (Kaelin, 2008). Knockout of SIRT1 did not affect HIF-1α hydroxylation (Figure S4D), but in the SIRT1 iKO mouse and transgenic overexpressor the levels of SIRT1 correlated with VHL levels (Figures 4A–4D). VHL promoter activity was not altered by SIRT1 deletion, suggesting posttranscriptional regulation (Figures 4F and 4G). HIF-2α was also stabilized by SIRT1, though HIF-2α target genes were not upregulated (Figures S4E and S4F). The re-establishment of SIRT1 eliminated HIF-1α protein and restored levels of mitochondrial OXPHOS mRNA in SIRT1 iKO myoblasts, but these effects were lost when VHL was knocked down (Figures 4H–4J; also see Figure 5F). Thus, SIRT1 is constantly required to maintain mitochondrial homeostasis by inducing VHL and by ensuring that HIF-1α is degraded efficiently.