EFTA00611136.pdf

Cel Declining NAD÷ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging Ana P. Comes,12.3 Nathan L. Price. Alvin J.Y. Ling, Javid J. Moslehi. • Magdalene K. Montgomery," Luis Rajman,' James P. White/ Joao S. Teodoro.' Christiane D. Wrann. Basil P. Hubbard. . Evi M. Mercken," Carlos M. Palmeira,2•3 Rafael de Cabo,BAnabela P. Rolo, Nigel Turner,' Eric L. Bell,' and David A. Sinclair'-6'* 'Glenn Labs for the Biological Mechanisms of Aging, Department of Genetics. Harvard Medical School. Boston. MA 02115. USA 'Center for Neurosciences and Cell Biology, 3004.517 Coimbra, Portugal *Department of Life Sciences. Faculty of Science and Technology. University of Coimbra, 3004.517 Coimbra, Portugal 'Department of Medical Oncology. Brigham and Women's Hospital and Dana-Farber Cancer Institute. Harvard Medical School, Boston, MA 02115. USA *Division of Cardiovascular Medicine. Department of Medicine, Brigham and Women's Hospital. Harvard Medical School. Boston, MA 02115. USA *Department of Pharmacology. School of Medical Sciences. The University of New South Wales, Sydney NSW 2052, Australia 'Dana-Farber Cancer Institute, Department of Cell Biology. Harvard Medical School. Boston. MA 02115. USA *Laboratory of Experimental Gerontology, National Institute on Aging. National Institutes of Health, Baltimore. MD 21224. USA *Department of Biology. University of Aveiro. 3810.193 Aveiro, Portugal '*Department of Biology. Massachusetts Institute of Technology. Paul F. Glenn Laboratory for the Science of Aging. Cambridge, MA 02139, USA 'Correspondence: david [email protected] httpfidx.doi.org/10.10164.cell.2013.11.0.37 SUMMARY and Nair. 2010: Wallace et al., 2010). There is considerable debate, however, about why mitochondria' homeostasis is Ever since eukaryotes subsumed the bacterial disrupted in the first place. The original idea of Hannan, that ancestor of mitochondria, the nuclear and mitochon- reactive oxygen species (ROS) from mitochondria are a primary drial genomes have had to closely coordinate their cause of aging (Harman. 1972), has been challenged by recent activities, as each encode different subunits of the studies of long-lived species and genetically altered animals (La- oxidative phosphorylation (OXPHOS) system. Mito- pointe and Hekimi, 2010). Though most mitochondrial genes have been transferred chondria] dysfunction is a hallmark of aging, but its to the nuclear genome, 13 subunits of the oxidative phos- causes are debated. We show that, during aging, phorylation (OXPHOS) system remain, demanding functional there is a specific loss of mitochondrial, but not communication between the nucleus and mitochondria to form nuclear, encoded OXPHOS subunits. We trace the stoichiometric OXPHOS complexes. This is mediated in large cause to an alternate PGC-1a/frindependent path- part by the peroxisome proliferator-activated receptor-y coacti- way of nuclear-mitochondrial communication that is vators ct and p (PGC-1x and PGC1-1p), which along with NRF-1 induced by a decline in nuclear HAD' and the accu- and -2, induce nuclear-encoded proteins, such as TFAM (mito- mulation of HIF-1a under normoxic conditions, with chondria] transcription factor A), that carry out the replication, parallels to Warburg reprogramming. Deleting transcription, and translation of mitochondrial DNA (mtDNA) SIRT1 accelerates this process, whereas raising (Larsson. 2010). Mammalian sirtuins (SIRT1-7) are a conserved family of NAD- - HAD' levels in old mice restores mitochondrial dependent lysine-modifying acylases that control physiological function to that of a young mouse in a SIRT1-depen- responses to diet and exercise (Haigis and Sinclair. 2010). The dent manner. Thus, a pseudohypoxic state that expression of SIRT1, an NAD•-dependent deacetylase, is disrupts PGC-1O3-independent nuclear-mitochon- elevated in a number of tissues following calorie restriction (CR) drial communication contributes to the decline in (Cohen et al.. 2004), an intervention that extends lifespan in mitochondrial function with age, a process that is diverse species. Overexpression or pharmacological activation apparently reversible. of SIRT1 reproduces many of the health benefits of CR, including protection from metabolic decline, cardiovascular disease, can- INTRODUCTION cer. and neurodegeneration (Haigis and Sinclair, 2010: Libert and Guarente. 2013). Some of the health benefits of SIRT1 have also One of the most conserved and robust phenomena in biology is been linkedto improved mitochondria' function (Baur et al., 2006: a progressive decline in mitochondrial function with age, leading Gerhart-Hines et al.. 2007: Price et al.. 2012: Rodgers et al., to a loss of cellular homeostasis and organismal health (Lanza 2005). Indeed, increased expression of neuronal SIRT1 extends 1624 Col 155. 1624-1638, December 19, 2013 *2013 Elsevier Inc. CrossMatk EFTA00611136  Cell mouse lifespan (Satoh et al.. 2013), though its role in aging in under these conditions there was no evidence of a mtUPR at lower organisms has been challenged (Burnett et al., 2011). 22 months of age (Figure SIA available online). A hallmark of cancer is a shift away from OXPHOS toward anaerobic glycolysis that provides cells with sufficient substrates Knockout of SIRT1 Mimics Aging by Decreasing for biomass. This metabolic reprogramming, known as the Mitochondria!, but Not Nuclear-Encoded, OXPHOS Warburg effect (Warburg. 1956), is driven by several different Components pathways, including mTOR, c-Myc, and hypoxia-inducible factor We wondered whether the specific decline in mitochondrially 1 (HIF-1a) (Deng, 2012). Interestingly, SIRT1 increases HIF-1a encoded OXPHOS components in aged mice might be due. in transcriptional activity (Lim et al.. 2010), SIRT3 destabilizes part, to a loss of SIRT1 activity. To test this, we utilized an HIF-la protein (Bell et al.. 2011; Finley et al., 2011), and SIRT6 adult-inducible SIRT1 knockout mouse (SIRTHKO) (Price functions as a HIF-la corepressor (Thong et al.. 2010), raising et al.. 2012), which circumvents the developmental abnormal- the possibility that HIF-1a may also be relevant to aging. Consis- ities of germline SIRT1 knockouts. VAT, was deleted at tent with this, in C. slogans, Hif-1 regulates lifespan and the 2-4 months of age, and skeletal muscle was analyzed response to CR (Leiser and Kaeberlein. 2010). A role for HIF-la 2-6 months later. As expected, the mRNA levels of all 13 mito- in mammalian aging, however, has not been explored. chondrially encoded OXPHOS genes and the two rRNAs were In this study, we provide evidence for a PGC-1a/11-indepen- reduced in the SIRT1 iKO mice compared to wild-type controls dent pathway of mitochondria' regulation that plays a role in (Figures 1G and SIB). Strikingly. there was no decrease in the the aging process. Activity of this pathway declines during aging expression of any of the nuclear-encoded components under due to changes in nuclear NAD' levels, causing a pseudo- fed conditions (Figure 1G). Again, protein levels of mitochondri- hypoxia-driven imbalance between nuclear- and mitochondrially ally encoded COX2 were significantly decreased, whereas the encoded OXPHOS subunits —a process that is prevented by CR nuclear-encoded COX4 was unaltered (Figure 1I-1), coincident and is reversed by raising NAD', with implications for treating with a decline in complex IV (CO)Q, but not complex II (SDH). age-related diseases. including cancer. activity (Figures S1I3 and S1E). Similar to old mice, cellular ATP levels and mtDNA content were reduced (Figures 11 and 1J). RESULTS with no apparent induction of mtUPR (Figure S1C). Given that SIRT1 maintains mitochondrial mass by increasing Aging Leads to a Specific Decline in Mitochondrially PGC-1a activity, we were surprised to see that, under these Encoded Genes basal conditions 0.e., the fed state), there was no effect of Aging is associated with disruption of mitochondrial homeosta- SIRT1 deletion on mitochondria] mass (Figure 1K). To under- sis. but the underlying mechanisms are unclear. As in previous stand why, we cultured SIRT1 iKO primary myoblasts and reports (Lanza and Nair. 2010), we observed a progressive. induced Cre-mediated deletion of the SIRT1 catalytic core age-dependent decline in OXPHOS efficiency with age in skel- ex vivo. After 12 hr, only the mitochondrially encoded OXPHOS etal muscle (Figures 1A and 1B). By 22 months of age. ATP mRNAs decreased (Figure 11..). Again, mtDNA content and mito- content and complex IV (CO4 activity were decreased, even chondria! membrane potential declined, with no change in mito- more so by 30 months of age. Although mtDNA content declined chondria! mass (Figures 1M, S2A, and S2B). By 48 hr, mRNA at both ages. the integrity of mtDNA was only lower in the from both the nuclear- and mitochondrially encoded genes had 30 month olds (Figures 1C and 1D). Together with previous decreased, with a loss of mitochondria' mass and a further reports (Lapointe and Hekimi. 2010), this suggested an aging decrease in membrane potential (Figures 1L, 1M, S2A, and mechanism that disrupts OXPHOS prior to the accumulation of S2B). These data suggested that loss of SIRT1 results in a significant mtDNA damage. biphasic disruption of mitochondria' homeostasis. A clue came from observations that the activity of OXPHOS complexes I, Ill, and IV decline with age, but complex II, the Nuclear NAD' Levels Regulate Mitochondrially Encoded only complex composed exclusively of nuclear-encoded sub- Genes units, does not (Kwong and Sohal. 2000). Thus, we tested Because there was no decline in SIRT1 protein with age (Fig- whether OXPHOS decline might be due to the specific loss of ure S2E), we hypothesized that SIRT1 activity might be com- mitochondrially encoded transcripts. Mitochondrially encoded promised in old mice due to a paucity of NAD'. Recent studies OXPHOS mRNAs (ND1, Cylb, COX1, AW6) were all significantly show that NAD' levels are regulated independently in different lower at 22 months relative to 6 month olds, whereas those cell compartments and that overall NAD' levels decline during encoded by the nuclear genome (NDUFS8, SDHb, Uqcrcl, aging (Braidy et al.. 2011; Massudi et al., 2012; Yang et al.. COX5,ATP5a) remained unchanged: but by 30 months of age, 2007). However, it is not clear in which cellular compartment(s) both the nuclear- and the mitochondrially encoded mRNAs is NAD` relevant to aging (Canto and Auwenc. 2011). Consistent were lower (Figures 1E). Protein levels of the mitochondrially en- with other reports (Braidy et al., 2011; Massudi et al.. 2012), coded COX2 gene were decreased at 22 months, but COX4, a there was less total NAD' in the skeletal muscle of elderly nuclear-encoded subunit, was only slightly lower. By 30 months, mice (Figure 2A). To determine which compartment might be both proteins were reduced relative to young mice (Figure 1F). responsible, we manipulated NAD' levels in the different com- The mitochondrial unfolded protein response (mtUPR) has partments by independently knocking down isoforms of nico- been recently linked to longevity (Durieux et al., 2011: tinamide mononucleotide adenylyltransferase, which regulate Houtkooper et al., 2013: Mouchiroud et al.. 2013); however, NAD' levels in the nucleus (NMNAT1). golgi/cytoplasm Cell 155.1624-1638. December 19. 2013 *2013 Elsevier Inc. 1625 EFTA00611137 Cell A C 10 1.2 1.2 ATP (pmolimg protein) 8 - E 1.0 1.0 - E Er'd 1.0 0.8 g0.8- 6 - 0.8 E • Z.' 0.6 Z ci o 0.6 4 0.4 is 0.4 2 •O 0.2 0.2 0.2 0 0.0 0.0 0.0 6 30 6 22 30 6 22 30 6 22 30 Age (months) Age (months) Age (months) Age (months) E F G H 1.2 6 mo 22 mo O WT 30 mo 2.0 • SIRT1 iKO 1.0 WT SIRT I iKO COX 2 1.5 0.8 win COX 2 M.M110111,1= (mt) 0.6 COX4 COX4 0.4 0.5 (nc) 0.2 enc-encoded emt-encoded Tubulin Tubulin 0.0 0.0 10 20 30 40 nc-encoded mt-encoded Age (months) J K 6.0 1.2 WT SIRT1 iKO 1.4 .5 so 1.2 ra 4.0 To — 1.0 3.0 9 is 0.6 - 0.8 E E .? a 2.0 .g 0.6 a 0.4 a. 0.4 l— 1.0 0.2 0.2 0.0 0.0 WT SIRT1 iKO WT SIRT1 iKO 0.0 WT SIRT1 iKO L M 1.2 enc-encoded 60 emt-encoded i 0.8 1.0 a c 1 50 •a Fl 3 rn 40 I. 1 L6) cc E co 0.6 I i 30 i 0.4 10 20 rp- 0.2 E1 10 0.0 0 0 20 40 60 12 24 48 Time of SIRT1 excision (hours) Time of SIRT1 excision (hours) Figure 1. Aging and Loss of SIRT1 Leads to a Specific Decline in Mitochondrial-Encoded Genes and Impairment in Mitochondria' Homeostasis in Skeletal Muscle (A) ATP content of 6-. 22-. and 30-month-old mice (n = 5. <0.05 versus 6-month-old mice). (8) Cytochrome c oxidase (COX) activity (n = 5. 'p < 0.05 versus 6-month-old animals). (C and 0) Mitochondria' DNA content (C) and DNA integrity (D) (n = 5. < 0.05 versus 6-incoth-old animals). (legend continued on next page) 1626 Cell 155, 1624-1638. December 19, 2013 O2013 Elsevier Inc. EFTA00611138  Cell (NMNAT2), and mitochondria (NMNAT3) (Berger et al.. 2005). target genes were considerably higher in the SIRT1 iKO (Figures Knockdown of NMNAT2 or NMNAT3 had no effect on OXPHOS 3C and S3A). Despite being cultured under normoxic conditions, genes. whereas knockdown of NMNAT1 resulted in a specific primary myoblasts deleted for SIRT1 also had increased HIF-12 reduction in the expression of mitochondrially encoded protein levels and activity of a HIF-1a reporter (Figures 3C and OXPHOS. mtDNA content, and ATP levels (Figures 2B-29. S3B). Reducing NAD' levels. either by knocking down NMNAT1 These results indicated that increasing the production of NAD' or by treating cells with lactate (which decreases the NAD'/ within the nuclear pool might stimulate mitochondria. Overex- NADH ratio), also caused HIF-12 protein stabilization (Figures pression of NMNAT1 in skeletal muscle of 10- to 12-month-old 3D, 3E, and S3C). mice dramatically increased the expression of mitochondrially HIF-12 has been studied extensively in cancer and during encoded OXPHOS genes (Figure 2G). Overexpression of hypoxia; however, its role in normal physiology remains largely NMNAT1 in primary myoblasts produced a similar effect that unknown. To better understand this, HIF-la was stabilized was SIRT1 dependent (Figure 2H). Together, these data indi- ectopically in vivo by deleting the EgIN1 gene encoding HIF cated that mitochondria are regulated by nuclear NAD' and prolyl hydroxylase 2 (PHD2) (Minamishima et al.. 2008). Upon that the impairment in OXPHOS function during aging may be EgIN1 deletion and HIF-la stabilization in muscle. there was a precipitated by depletion of the nuclear NAD pool. specific decline in mtDNA content and decreased levels of mitochondrially encoded, but not nuclear-encoded, OXPHOS SIRTI Can Regulate Mitochondria through a PGC-1a/ mRNA, paralleling the effects of SIRTI deletion and normal aging (3-Independent Pathway (Figures 3F-3H). Pharmacological stabilization of HIF-la inPGC- A central dogma in the sirtuin field is that SIRTI promotes mito- 1a/I3 knockout myotubes reduced expression of mitochondrially chondria' function in response to fasting and CR by deacetylat- encoded genes (Figures 31 and S3D), whereas treating PGC- ing PGC-la (Gerhart-Hines et al., 2007: Rodgers et al.. 2005). /gip KO cells with pyruvate (to increase NAD' levels) up- Consistent with this, SIRTI iKO animals failed to upregulate regulated mitochondrially encoded genes, an effect that was both nuclear- and mitochondrially encoded OXPHOS genes in prevented by stabilization of HIF-1a (Figure S3E). Stabilization response to fasting (Figure S2C). However, our findings in fed of HIF-la in primary cells and transgenic mice blocked the ability animals (see Figure 1) indicated that SIRTI can regulate mito- of SIRT1 to upregulate mitochondrially encoded genes and chondria' genes independently of PGC-12. To test this. we increase ATP levels, with a specific loss of mitochondrially examined primary myotubes from PGC-1a/II knockout (KO) encoded mRNAs (Figures 31-3L and S3F-SFI). Overexpression mice (Zechner et al., 2010) and from PGC-1a muscle-specific of a stabilized mutant version of the related factor HIF-2a did null mice (Handschin et al.. 2007), and we saw no defect in the not have the same effect (Figures 3J-3L and S3I), demonstrating ability of SIRT1 and NMNAT1 to induce mitochondrially encoded that the inhibition of OXPHOS and mitochondrially encoded OXPHOS genes (Figures 21 and 24 Thus. SIRT1 can induce genes is HIF-1a specific. In primary myoblasts lacking HIF-la, OXPHOS genes in the absence of PGC-12/p (Figure S2D). deletion of WATT had no effect on mtDNA content, mitochond- rially encoded gene expression, or ATP levels (Figures 3M-3P). SIRTI Regulates Mitothondrially Encoded Genes Together, our results show that HIF-12, but not HIF-2a, regulates through HIF-1 mitochondria in response to SIRT1 activity, which is under the Next. we sought to understand how SIRT1 regulates mitochon- control of nuclear NAD' levels. dria independently of PGC-17.43. Analysis of SIRTI iKO animals indicated that genes involved in glycolysis were upregulated, SIRT1 Stabilizes HIF-1a via VHL with increased lactate levels (Figures 3A and 3B) and a switch HIF-la can be stabilized by ROS originating from complex ill from slow-twitch oxidative fibers (MyHCIla) to fast-twitch glyco- of the ETC as part of retrograde response (Bell et al., 2007). lytic fibers (MyHCllb) (Figure S1F). These metabolic changes Six hours after inducing SIRTI deletion in primary myoblasts, were reminiscent of Warburg remodeling of metabolism in HIF-la levels increased (Figure 59, and by 12 hr, mitochondria] cancer cells, which is known to be mediated, in part, by the homeostasis was impaired (Figures 1L, S2A, and S2B). Yet, ROS stabilization of the transcription factor HIF-12 (Majmundar levels did not increase until the 24 hr time point (Figure 54A). et al.. 2010). The levels of HIF-12 and the expression of HIF-1a Myoblasts depleted of mitochondria' DNA (rho0), which are (0 Expression of nuclear- and mitochcothialy encoded genes (n = 5. p <0.05 versus 6-month-old animals). (F) Imrnunobbt foe COX2 and COX4 in 6-. 22-. and 30-month-old mite. (0) Expression of nuclear- WOUFS8.NOUFAS.SDHb.SDHd.Uqcrcl. Uqcrc2. COX5b. Cox641. ATPS41. and ATPcfland mitocnondzialty encoded genes WOI. ND2. ND3. N04. NO4 NOS. ND6. Cytb. COX, COX2. COX3. A7P6. and ATP8) in WT and SIRTI il<0 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 iK0 mice (n = S. < 0.05 versus WT). (J) MilochcadrialDNA content of WT and SAT, iK0 mice (1= 5.'p <0.05 versus WT). (K) Electron microscopy of gastrocnerrius from WT and SIR'", il<0 mice and mitochondrial area in = 4). (L) Expression of nuclear- and ntoctondrially encoded genes in SlAT? flog/Sox Cre-ERT2 primary myoblasts treated with vehicle (0 h0 or tamoxifen (Offl) to induce SIRT1 excision for 6. 12.24. and 48 hr (n = 4.'p < 0.05 versus vehicle). (M) Mitochondria! mass by NAO fluorescence riSIRT7 floxfflox Cre-ERT2 primary myoblasts treated with veNcle (0 h0or OHT to induce SIRT1 excision for & 12. 24. and 48 hr (n = 4. p< 0.05 versus vehicle). Nuclear- and mitochondrially encoded genes were ND). Cytb. COX1. ATP6 and NDUFSS. SOHb. Uqcrcl. COX5b. ATPSal. respectively. Tissue samples are gastrocnemius unless otherwise stated. Values are expressed as mean x SEM. See also Ftgise Si. Cell 155.1624-1838. December 19. 2013 02013 Elsevier Inc. 1627 EFTA00611139 Cell A C NANAT2 1.4 Tubtin 250 3.0 •••••• 2 1.2 yi 2.5 S 200 .2 2 .2 S 10 g 2.0 ge 150 • < ■ shNT 0.8 O shNT 1.5 • shNMNAT1 #1 re ro 0.6 ❑ shNMNAT2 x1 E 100 • E e Eg Ea 1.0 O shNMNAT1 #2 O shNMNAT2 P*2 a 0.2 11! 0 .4 50 - as 0 0.0 0.0 6 22 nc-encoded mt-encoded nc-e coded mt-en oded Age (months) D E F O shNT 9 shNMNAT1 #1 shNMNAT1 N2 1.6 1.2 1.4 _ 1.0 1.2 1.0 ... 0.8 0 0.10 a shNT a 0.8 Z (12 0.6 E a shNMNAT3 #1 0 m 0.6 O shNMNAT3 #2 t ia OA 10.05 0.4 a. 2 0.2 0.2 I 0.0 0.0 0.00 nc-encoded mt-encoded G H EmOy MfflaTIOE • WT Empty • PGC-1a/6 KO Empty MINATI ▪ WT SIRT1 OE • PGC-le) KO SIRTI OE PGC-1aIS KO myotubes LI Taal, 2.0 O Empty 2.5 20 2.5 • NMNATI OE <2 2.0 - < 2.0 z 2 15 re r, E .,.° 1.5 • E o v a x 10 N. to • 1 1.5 - v o c., c. .17.5 1105 1.0 - 5 05 • 0.5 • E E •e 0.5 0.0 r. 0.0 0 0.0 Vehicle NMNAT1 OE Vehide SIRT1 KO nc-encoded mt-e coded Empty NMNAT1 OE Figure 2. Nuclear NAIY Levels Regulate Mitochondrial-Encoded Genes and Mitochondrial Homeostasis through SIRT1, Independently of PGC-1,./(1 (A) MAD' levels in gastrocnemius of 6-. 22-. and 30-month-old mice (n = 5.13 < 0.05 versus 6-month-old mice). (3—D) Expression of nuclear- and mitochondrially encoded genes in primary myoblaststransduced withNMIVAT1(3).NMNAT2 (C).NMNATS (D). or nontargeting shRNA (n = 4.13 < 0.05 versus shNT). (E and F) Mitochondrial DNA content and (H)ATP content (I) in primary myoblaststransduced with NMNATI or nontargeting shRNA(n = 4.13 <0.05 versus shNT). (G) Expression of rritochondrially encoded genes in tibialis of 10- to 12-month-old mice overexpressing NMNATI compared to the contraleteral tibialis muscle treated with vehicle (n = 4.13 < 0.05 versus vehicle). (H) Expression of mitochondrially encoded genes in Sail flox/flox Cie-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRTI excision infected with adenovirus overexpressing NMNATI a empty vector (n = 4.13 < 0.05 versus vehicle empty vector). (I and J) Expression of nuclear-and mitochondrially encoded genes in WT and PGC-7w6 knockout myotubes treated with adenovirus overexpressing &ATI (I) or NMNATI (J) (n = 4. 'p < 0.05 versus WT empty: tip < 0.05 versus P3C-1710 KO empty). Nuclear- and mitochandrially encoded genes were NO1. Cyrb. COX1. A7P6 and NDUFSS. SOHb. Uqorl. COXSb. A7P581. respecWely. Tissue samples are gastrocnemius muscle unless otherwise stated. Values are expressed as mean * SEM. See also Fgure S2. 1628 Cell 155, 1624-1638, December 19, 2013 ic)2013 Elsevier Inc. EFTA00611140  Cell A C Skeletal muscle WT • SIRT1 KO WT SIRT1 iKO 5.0 O WT • SIRTI iKO HIF-la E" 4.0 2 3.0 Tubulin 1 2.0 Primary myoblasts E -E- 1.0 HIF-la 0.0 Tubulin D shNT sh•MNAT1 F G H wr • EgIN1 KO VVT t4 1.2 ■ EgIN1 KO HIF-la 1.2 - - 1.0 WT EgIN1 KO Tubulin 1.0 • HIF-lo Ea 2 0.8 • g 02 0.6 E Vehicle Pyruvate Lactate E 0 0.6 - Ea 0.4 • i 0.4 Tubulin ; -IIF-1a 0.2 - 0.2 0.0 1 0.0 Tubulin no -encoded mt-e coded J K L PGC-1a/p KO myotubes • Empty • HIF-la DPA O I-IIF-2o DPA 3.5 1.4 7 • D DMSO o oQP g é 3.0- .2r7 03 1 1.2 0 <2 6 y • DMOG sN 4, É 2.5 4 11- • Empty i I 5 •2 9. 2.0 - g I. 0.8 rc 0 -1 4 • SIRT1 OE ° 1s - HA E >CD 0.6 — O HIF-la DPA §i 41 m 1.0 - :§" OA .§ : 3 (7) f 5 2 + SIRTI OE Tubulin O HIF-2a DPA É$ ED, -7- 0.2 E .?_. 1 + SIRT1 OE 0.0 Iì i 0.0 • 0 Empty SIRT1 OE nc-encoded mt-encoded M N P • shNT + Vehide • shNT + SIRTI iKO O shHIF-lo + SIRTI iKO 1.4 1.2 12 1.2 rg . 1.0 1.0 ••••••. shNT shHIF-1a 1.0 eta o E e 0.8 a 0.8 0.8 HIF-1a 0.6 ,p 0.6 o 0.6 Tubulin 0.4 0.4 ci 0.4 0.2 0.2 u- 0.2 0.0 0.0 00 (legend on next page)