Aging and Loss of SIRT1 Leads to a Specific Decline in Mitochondrial-Encoded Genes and Impairment in Mitochondrial Homeostasis in Skeletal Muscle (A) ATP content of 6-, 22-, and 30-month-old mice (n = 5, ∗p < 0.05 versus 6-month-old mice). (B) Cytochrome c oxidase (COX) activity (n = 5, ∗p < 0.05 versus 6-month-old animals). (C and D) Mitochondrial DNA content (C) and DNA integrity (D) (n = 5, ∗p < 0.05 versus 6-month-old animals). (E) Expression of nuclear- and mitochondrially encoded genes (n = 5, ∗p < 0.05 versus 6-month-old animals). (F) Immunoblot for COX2 and COX4 in 6-, 22-, and 30-month-old mice. (G) Expression of nuclear- (NDUFS8, NDUFAS, SDHb, SDHd, Uqcrc1, Uqcrc2, COX5b, Cox6a1, ATP5a1, and ATPc1) and mitochondrially encoded genes (ND1, ND2, ND3, ND4, ND4l, ND5, ND6, Cytb, COX1, COX2, COX3, ATP6, and ATP8) in WT and SIRT1 iKO mice (n = 5, ∗p < 0.05 versus WT). (H and I) (H) Immunoblot for COX2 and COX4 and (I) ATP content in WT and SIRT1 iKO mice (n = 5, ∗p < 0.05 versus WT). (J) Mitochondrial DNA content of WT and SIRT1 iKO mice (n = 5, ∗p < 0.05 versus WT). (K) Electron microscopy of gastrocnemius from WT and SIRT1 iKO mice and mitochondrial area (n = 4). (L) Expression of nuclear- and mitochondrially encoded genes in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle (0 hr) or tamoxifen (OHT) to induce SIRT1 excision for 6, 12, 24, and 48 hr (n = 4, ∗p < 0.05 versus vehicle). (M) Mitochondrial mass by NAO fluorescence in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle (0 hr) or OHT to induce SIRT1 excision for 6, 12, 24, and 48 hr (n = 4, ∗p < 0.05 versus vehicle).

Nuclear- and mitochondrially encoded genes were ND1, Cytb, COX1, ATP6 and NDUFS8, SDHb, Uqcrc1, COX5b, ATP5a1, respectively. Tissue samples are gastrocnemius unless otherwise stated. Values are expressed as mean ± SEM. See also Figure S1.

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).

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.

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.

These results raised the question of how HIF-1α, a nuclear protein, inhibits mitochondrial OXPHOS genes. Analysis of gene expression in SIRT1 iKO mice identified the nuclear-encoded mitochondrial factor TFAM as a candidate (Figures 5A and S5A). Consistent with this, TFAM promoter activity in SIRT1 iKO myoblasts was greatly reduced (Figure 5B), the reintroduction of TFAM into SIRT1 iKO cells restored levels of mitochondrially encoded mRNAs and ATP (Figure 5C-E), and in time course studies, TFAM levels declined 6 hr after VHL and HIF-1α (Figure 5F).

Knockdown of ARNT, a HIF-1α transcriptional binding partner (Wang et al., 1995), had no appreciable effect on mitochondrially encoded OXPHOS genes and ATP levels (Figures S5B–S5D), indicating that HIF-1α acts via a different mechanism. In cancer cells, metabolic reprogramming is mediated by crosstalk between HIF-1α and c-Myc (Gordan et al., 2007), raising the possibility that c-Myc was the missing factor. In fact, c-Myc DNA-binding sites are found at mitochondrial biogenesis genes (Kim et al., 2008, Li et al., 2005). Deletion of SIRT1 in primary myoblasts increased the binding between HIF-1α and c-Myc and reduced c-Myc reporter activity (Figures 5G and S5E). Similarly, knockdown of c-Myc completely blocked the ability of SIRT1 to induce mitochondrially encoded mRNAs and mtDNA (Figures S5F–S5H). Conversely, overexpression of c-Myc in myoblasts treated with a SIRT1 inhibitor, EX-527, prevented loss of mtDNA, mitochondrially encoded mRNA, and cellular ATP levels (Figures S5I–S5L).

We tested whether c-Myc directly controls TFAM promoter in myoblasts and is modulated by SIRT1-HIF-1α. TFAM is known to be regulated by PGC-1α, which interacts with NRF 1/2 bound at positions −311 and −154 in the TFAM promoter (Figure 5H). Knockdown of c-Myc reduced TFAM promoter activity (Figure 5I), consistent with a study in cancer cells (Li et al., 2005). We identified a putative c-Myc consensus sequence, CACGTG, 1,028 bp upstream of the ATG site—the mutation of which decreased promoter activity by about half without affecting PGC-1α-mediated induction (Figures 5J and 5K). Overexpression of SIRT1 also induced the TFAM promoter reporter, and mutation of the c-Myc binding site blocks this effect (Figure 5L). Chromatin IP experiments detected an interaction between c-Myc and the TFAM promoter, which was markedly reduced when SIRT1 was deleted, but not when HIF-1α was also knocked down (Figures 5M–O). We did not detect direct HIF-1α binding to TFAM (Figures 5M and 5N), with LDHA as a positive control (Figures S5M and S5N). Together, these data provide the first direct link between HIF-1α and the regulation of mitochondrially encoded genes in skeletal muscle and identify a mechanism of PGC-1α/β-independent regulation of mitochondrial function.

Next, we determined the mechanisms that determine whether SIRT1 utilizes the PGC-1α-dependent or -independent pathways. Under conditions of low energy, AMPK-mediated phosphorylation of PGC-1α allows it to be deacetylated and activated by SIRT1 (Cantó et al., 2009, Gerhart-Hines et al., 2007, Ptitsyn et al., 2006), whereas under basal conditions, acetylation status is primarily regulated by the acetyltransferase GCN5 (Fernandez-Marcos and Auwerx, 2011). We speculated that the biphasic decline in OXPHOS subunits (in Figure 1L) might be due to AMPK. In time course experiments following SIRT1 deletion, AMPK activation occurred after 48 hr, well after the decline in VHL-TFAM and mitochondrial genes (Figures 1L–1M and 6A ) but coincident with the decline in nuclear-encoded OXPHOS genes and mitochondrial mass (see Figures 1L–1M). An AMPK dominant-negative adenovirus (AMPK-DN) prevented the decline of nuclear OXPHOS mRNAs at 48 hr (Figures 6B and 6C), whereas forced maintenance of TFAM prevented AMPK activation (Figures 6D, 5D, and 5E). Together, these results strongly suggest that AMPK is the switch between the PGC-1α-dependent and -independent pathways. In this model, AMPK activation occurs in the absence of SIRT1 only when ATP levels fall below a threshold. Consistent with this, AMPK was unchanged under fed conditions in the SIRT1 iKO mice and 22-month-old wild-type mice but was markedly increased in fasting animals, when we observe changes in both nuclear- and mitochondrially encoded OXPHOS genes (Figure 6E and 6F).

CR is known to delay numerous diseases of aging in mammals, including cancer and type 2 diabetes. Interestingly, CR (30%–40% instituted at 6 weeks) completely prevented the decline in VHL and the increase in HIF-1α that occurs in ad-libitum (AL)-fed 22-month-old mice (Figure 7A). The observed decreases in NAD+ and ATP levels, COX activity, mtDNA, and mitochondrially encoded OXPHOS components with age were also prevented by CR (Figures 7B–7D, S6A, and S6B). Unlike the accumulation of mutations in mtDNA, the pathway that we describe here should be rapidly reversible. Treatment of 22-month-old mice for 1 week with NMN, a precursor to NAD+ that increases NAD+ levels in vivo (Yoshino et al., 2011), reversed the decline in VHL and accumulation of HIF-1α (Figures 7E and 7F); reduced lactate levels; and increased ATP, COX activity, and mitochondrially encoded OXPHOS transcripts (Figures 7G–7I and S6D). In EglN1 and SIRT1 iKO mice, however, NMN failed to induce mitochondrially encoded genes or to raise ATP levels (Figures 7J–7L). Knockdown of NMNAT1 also prevented NMN from inducing mitochondrially encoded OXPHOS genes (Figure 7M), consistent with nuclear NAD+ being a key regulatory molecule. The SIRT1 iKO and the 22-month-old mice had increased levels of markers of muscle atrophy and inflammation compared to young WT mice, along with impaired insulin signaling and insulin-stimulated glucose uptake (Figures S1G–S1J and S6E–S6H). Strikingly, treatment of old mice with NMN reversed all of these biochemical aspects of aging and switched gastrocnemius muscle to a more oxidative fiber type (Figures S6E–S6H). However, we did not observe an improvement in muscle strength (data not shown), indicating that 1 week of treatment might not be sufficient to reverse whole-organism aging and that longer treatments might be required.

Impairment in mitochondrial homeostasis is one of the hallmarks of aging that may underlie common age-related diseases (Lanza and Nair, 2010, Satoh et al., 2013). Despite its importance, there is still controversy as to why mitochondrial homeostasis is disrupted with age and whether this process can be slowed or even reversed. Here, we present evidence for a PGC-1α/β-independent pathway that ensures OXPHOS function and maintenance of mitochondrial homeostasis (Figure 7N). During aging, however, decline in nuclear energetic state or NAD+ levels reduces the activity of SIRT1 in the nucleus, causing VHL levels to decline and HIF-1α to be stabilized. This program, which likely evolved to modulate mitochondrial metabolism in response to changes in energy supply, becomes chronically activated in old mice, inducing a pseudohypoxic state that disrupts OXPHOS, a phenomenon that is consistent with antagonistic pleiotropy (Williams and Day, 2003).

One of the more surprising findings is the existence of a SIRT1-mediated pathway that regulates mitochondria independently of PGC-1α/β. The data indicate that SIRT1 can regulate these two pathways in response to the energetic state of the cell. Which one predominates depends on AMPK activity and the phosphorylation status of PGC-1α (Cantó et al., 2009).

This study shows that HIF-1α-induced metabolic reprogramming occurs in normal tissue and that it disrupts mitochondrial homeostasis. We consider the metabolic state of the old mice as pseudohypoxic because the downstream effects are similar to hypoxia but occur even when oxygen is abundant, as previously in type 2 diabetes and cancer (Ido and Williamson, 1997, Rodgers et al., 2005, Williams and Day, 2003). An interesting implication is that reprogramming of normal tissue toward a Warburg-like state may increase ROS and establish a milieu for subsequent mutations to initiate carcinogenesis, a possibility that may help explain why cancer risk increases exponentially with age.

All of the main players in the nuclear NAD+-SIRT1-HIF-1α-OXPHOS pathway are present in lower eukaryotes, indicating that the pathway evolved early in life’s history. This pathway may have evolved to coordinate nuclear-mitochondrial synchrony in response to changes in energy supplies and oxygen levels, and its decline may be a conserved cause of aging. In C. elegans, HIF-1α is known to be a key determinant of lifespan, though its precise role is still a matter of debate (Leiser and Kaeberlein, 2010). HIF-1α modulation may have differential effects on lifespan depending on the animal’s diet or whether the mtUPR is activated (Dillin et al., 2002, Durieux et al., 2011, Houtkooper et al., 2013). Though we did not detect mUPR in skeletal muscle, we do not exclude the possibility that mtUPR plays a role in other tissues or under different conditions.

Additional studies will be required to elucidate complex feedback loops that likely regulate the SIRT1-HIF-1α-Myc-TFAM pathway. For example, in cancer cells, SIRT1 directly regulates c-Myc transcriptional activity, either by deacetylation of c-Myc (Menssen et al., 2012) or by binding c-Myc and promoting its association with Max (Mao et al., 2011). Given that SIRT3 and SIRT6 also regulate HIF-1α and compromise respiration (Bell et al., 2011, Finley et al., 2011, Zechner et al., 2010), it will be interesting to test whether a decline in the activity of other sirtuins causes a similar loss of TFAM and mitochondrially encoded OXPHOS components.

How broadly applicable might these findings be? High-fat diet feeding increases levels of HIF-1α in liver (Carabelli et al., 2011) and white adipose tissue, the latter of which correlated with a decline in mitochondrial gene expression (Krishnan et al., 2012). Moreover, insulin-resistant human skeletal muscle has a signature reminiscent of hypoxia (Ptitsyn et al., 2006). In SIRT1 iKO mice, specific dysregulation of mitochondrial OXPHOS genes is also observed in the heart, demonstrating that the pathway is relevant not only to skeletal muscle (Figure S1K–S1N), but not in liver, WAT, or brain. In these tissues, other factors such as SIRT3 or SIRT6 may be responsible for regulation of HIF-1α, or the metabolic status of the tissue at the time of harvest may also be critical. Current dogma is that aging is irreversible. Our data show that 1 week of treatment with a compound that boosts NAD+ levels is sufficient to restore the mitochondrial homeostasis and key biochemical markers of muscle health in a 22-month-old mouse to levels similar to a 6-month-old mouse. Although further work is necessary, this study suggests that increasing NAD+ levels and/or small compounds that prevent HIF-1α stabilization or promote its degradation might be an effective therapy for organismal decline with age. In summary, these findings provide evidence for a new pathway that controls carbon utilization and OXPHOS independently of PGC-1α, a pathway that goes awry over time but is readily reversible, with implications for treating aging and age-related diseases.

The Sinclair lab is supported by the NIH/NIA, the Glenn Foundation for Medical Research, the United Mitochondrial Disease Foundation, the Juvenile Diabetes Research Foundation, and a gift from the Schulak family. A.P.G. was supported by the Portuguese Foundation for Science and Technology (SFRH/BD/44674/ 2008) and B.P.H. by an NSERC PGS-D fellowship. N.T. is supported by an Australian Research Council Future Fellowship. We are grateful to Michael Bonkowski, Carlos Daniel de Magalhaes Filho, Meghan Rego, Nikolina Dioufa, and David Zhang for technical advice and experimental assistance; William Kaelin Jr. for kindly providing the EglN1 KO mice; Daniel Kelly, John Rumsay, and Teresa Leone for unpublished PGC-1α/β KO myoblasts and advice; Bruce Spiegelman for PGC-1α null myoblasts and advice; and Pere Puigserver and Zachary Gerhart-Hines for a SIRT1 adenovirus. D.A.S. is a consultant to Cohbar, OvaScience, HorizonScience, Segterra, MetroBiotech, and GlaxoSmithKline. Cohbar, MetroBiotech, and GlaxoSmithKline work on mitochondrially derived peptides, NAD+, and sirtuin modulation, respectively.

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