DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging

doi:10.1016/j.cmet.2017.04.008

. 2017 May 2;25(5):1135-1146.e7.

doi: 10.1016/j.cmet.2017.04.008.

DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging

Sung-Jun Park¹, Oksana Gavrilova², Alexandra L Brown¹, Jamie E Soto³, Shannon Bremner⁴, Jeonghan Kim¹, Xihui Xu¹, Shutong Yang¹, Jee-Hyun Um¹, Lauren G Koch⁵, Steven L Britton⁶, Richard L Lieber⁴, Andrew Philp⁷, Keith Baar⁷, Steven G Kohama⁸, E Dale Abel³, Myung K Kim¹, Jay H Chung⁹

Affiliations

¹ Laboratory of Obesity and Aging Research, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
² Mouse Metabolism Core, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
³ Program in Molecular Medicine and Division of Endocrinology, Metabolism and Diabetes, University of Utah School of Medicine, Salt Lake City, UT 84112, USA; Fraternal Order of Eagles Diabetes Research Center and Division of Endocrinology and Metabolism, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
⁴ Department of Orthopedic Surgery, University of California and V.A. Medical Centers, San Diego, La Jolla, CA 92093, USA.
⁵ Department of Anesthesiology, The University of Michigan, Ann Arbor, MI 48109, USA.
⁶ Department of Anesthesiology, The University of Michigan, Ann Arbor, MI 48109, USA; Department of Molecular & Integrative Physiology, The University of Michigan, Ann Arbor, MI 48109, USA.
⁷ Department of Physiology and Membrane Biology, University of California Davis, Davis, CA USA 95616.
⁸ Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Sciences University, Portland, OR 97239, USA.
⁹ Laboratory of Obesity and Aging Research, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: chungj@nhlbi.nih.gov.

PMID: 28467930
PMCID: PMC5485859
DOI: 10.1016/j.cmet.2017.04.008

DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging

Sung-Jun Park et al. Cell Metab. 2017.

. 2017 May 2;25(5):1135-1146.e7.

doi: 10.1016/j.cmet.2017.04.008.

Authors

Affiliations

¹ Laboratory of Obesity and Aging Research, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
² Mouse Metabolism Core, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
³ Program in Molecular Medicine and Division of Endocrinology, Metabolism and Diabetes, University of Utah School of Medicine, Salt Lake City, UT 84112, USA; Fraternal Order of Eagles Diabetes Research Center and Division of Endocrinology and Metabolism, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
⁴ Department of Orthopedic Surgery, University of California and V.A. Medical Centers, San Diego, La Jolla, CA 92093, USA.
⁵ Department of Anesthesiology, The University of Michigan, Ann Arbor, MI 48109, USA.
⁶ Department of Anesthesiology, The University of Michigan, Ann Arbor, MI 48109, USA; Department of Molecular & Integrative Physiology, The University of Michigan, Ann Arbor, MI 48109, USA.
⁷ Department of Physiology and Membrane Biology, University of California Davis, Davis, CA USA 95616.
⁸ Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Sciences University, Portland, OR 97239, USA.
⁹ Laboratory of Obesity and Aging Research, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: chungj@nhlbi.nih.gov.

PMID: 28467930
PMCID: PMC5485859
DOI: 10.1016/j.cmet.2017.04.008

Erratum in

DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging.
Park SJ, Gavrilova O, Brown AL, Soto JE, Bremner S, Kim J, Xu X, Yang S, Um JH, Koch LG, Britton SL, Lieber RL, Philp A, Baar K, Kohama SG, Abel ED, Kim MK, Chung JH. Park SJ, et al. Cell Metab. 2017 Aug 1;26(2):447. doi: 10.1016/j.cmet.2017.07.005. Cell Metab. 2017. PMID: 28768182 No abstract available.

Abstract

Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases, including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function, and decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity.

Keywords: AMPK; DNA-PK; HSP90α; aging; calorie restriction; exercise; insulin sensitivity; mitochondria; obesity; skeletal muscle; type 2 diabetes.

Published by Elsevier Inc.

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Figures

**Figure 1. Aging increases DNA DSBs and DNA-PK activity**
(A) H2AX phosphorylation (γ-H2AX) in skeletal muscle of young (3 mo) and old (25 mo) mice (in arbitrary units) (n=10 each group). Quantification is shown on the right. All values are expressed as mean ± s.e.m. Mann-Whitney Test: *, p<0.05 between the two age groups. (B) DNA-PK autophosphorylation (S2056) in skeletal muscle of rhesus (in arbitrary units) (n=5 each age group). Quantification is shown on the right. Mann-Whitney Test: **, p<0.01 compared to the youngest group. (C) p-HSP90α in skeletal muscle of 1 and 15 yr old rhesus (in arbitrary units) (n=6 per group). Quantification is shown on the right. Mann-Whitney Test: **, p<0.01 compared to the youngest group. (D) p-HSP90α in mice skeletal muscle according to age (in arbitrary units) (n=5 each group). Quantification is shown on the right. Mann-Whitney Test: *, p<0.05 compared to the youngest group.

**Figure 2. DNA-PK-mediated phosphorylation of HSP90α triggers release of clients**
(A) T5,7 are phosphorylated in both cytoplasmic and nuclear HSP90α in response to DNA DSBs induced by IR (15 Gy) in C2C12 myotubes. (B) C2C12 myotubes were transfected with vectors encoding Flag-tagged WT or mutant HSP90α. Interaction between mutant HSP90α and endogenous LKB1 was visualized by immunoprecipitating with anti-LKB1 antibody one hr after IR and immunoblotting with anti-Flag antibody. (C) Interaction between Flag-tagged WT or mutant HSP90α and endogenous GR one hr after IR was visualized by immunoprecipitating with anti-GR antibody and immunoblotting with anti-Flag antibody. (D) Interaction between Flag-tagged HSP90β and GR one hr after IR was visualized by immunoprecipitating with anti-GR antibody and immunoblotting with anti-Flag antibody. (E) C2C12 myotubes transfected with siRNA for either Ctrl or DNA-PKcs were treated with IR (4Gy) and the expression level of p-HSP90α and DNA-PK were visualized by immunoblotting. (F) C2C12 myotubes transfected with a vector encoding WT HSP90α and siRNA for either Ctrl or DNA-PKcs were treated with IR and harvested one hour later. Interaction between GR and HSP90α is shown. (G and H) The effect of IR on the interaction between HSP90α mutants and either endogenous eNOS (G) or AR (H) in C2C12 myotubes is shown.

**Figure 3. Inhibition of DNA-PK activates AMP-activated protein kinase**
(A) C2C12 myotubes were transfected with a siRNA for either control (Ctrl) or HSP90α and the expression level of HSP90α and AMPK were visualized by immunoblotting. Quantification is shown on the bottom left. All values are expressed as mean ± s.e.m. Mann-Whitney Test: *, p<0.05. (Bottom Right) C2C12 myotubes were transfected with a siRNA for either Ctrl or HSP90α and AMPKα mRNA was measured by using real-time PCR. All values are given as mean ± s.e.m. (B) C2C12 myotubes were treated with HSP90 inhibitor (1 μM 17-AAG) and the expression level of AMPK was visualized by immunoblotting. (C) C2C12 myotubes were transfected with vectors encoding Flag-tagged WT or T5,7A HSP90α. Interaction between mutant HSP90α and endogenous AMPK was visualized by immunoprecipitating with Flag antibody. (D) C2C12 myotubes were transfected with vectors encoding either WT or T5,7A HSP90α and the expression levels of Flag-tagged HSP90α and p-AMPK (T172) were visualized by immunoblotting. (E) AMPK phosphorylation (p-AMPK) in resting muscle isolated from young (3–4 months old) and middle aged (12–14 mo) WT and SCID mice. Three representative samples for each group are shown. (F) Quantification of p-AMPK in young (n=3 per genotype) and middle aged muscle (n=6 per genotype) from WT and SCID mice. Mann-Whitney Test: **, p<0.01. (G) HSP90α phosphorylation in resting skeletal muscle isolated from young and middle aged WT and SCID mice. (H) Quantification of p-HSP90α in young and middle aged muscle (n=7 per genotype). Mann-Whitney Test: *, p<0.05. (I) AMPK activation (p-AMPK, p-ACC1) and HSP90α phosphorylation in resting skeletal muscle isolated from young (5 months old) and old (17 months old) fl/fl and muscle specific DNA-PK knockout (MDPKO) mice. Quantification is shown on the right. Mann-Whitney Test: *, p<0.05: **, p<0.01.

**Figure 4. DNA-PK inhibition increases skeletal muscle mitochondrial biogenesis**
(A) Correlation between mitochondrial content and DNA-PKcs autophosphorylation in skeletal muscle of middle aged (14–16 yr) rhesus monkeys (r=−0.93, p=0.008). The correlation values were calculated by Pearson correlation. 95% confidence interval was −0.99 to −0.46. (B) Skeletal muscle was isolated from resting young (Y, 3–4 mo), obese (Ob, 5–6 mo) and middle aged (MA) WT and SCID mice. The mRNA levels (in arbitrary units) of genes important for mitochondrial biogenesis were measured by using real-time PCR (n=3–4 per group). All values are given as mean ± s.e.m. *, p<0.05, **, p<0.01 between WT and SCID values. (C) PGC-1α expression level in resting muscle isolated from old (17 months old) fl/fl and muscle specific DNA-PK knockout mice (MDPKO). (D) (Left) A representative electron micrograph of gastrocnemius isolated from middle aged WT and SCID mice (×10,000 magnification). Black bar indicates 500 nm. White arrowhead indicates mitochondria. (Right) Mitochondrial volume, expressed as percentage of total muscle volume, is shown for 3 mo and 14 mo WT and SCID mice. *, p<0.05 between WT and SCID values. (E) Relative mtDNA levels in skeletal muscle of middle aged WT and SCID mice (n=5–6 per genotype). *, p<0.05 between WT and SCID values. (F) Relative mtDNA levels in skeletal muscle of young (5 mo) and old (17 mo) fl/fl and MDPKO mice (n=5 per genotype). #, p=0.06. (G) Serum IgG levels of C57BL6/J mice treated with DNA-PK inhibitor for 10–12 week. Serum IgG was undetectable (U.D.) in SCID mice (n=5–13 per group). (H) Relative mtDNA levels in skeletal muscle of WT and AMPKα2 knockout mice fed either vehicle or DNA-PK inhibitor NU7441 (n=4–5 per genotype). #, p=0.06.

**Figure 5. DNA-PK inhibition increases physical fitness in obese and middle aged animals**
Gastrocnemius muscle fiber composition in middle aged WT and SCID mice (A) and in WT middle aged mice fed vehicle or the DNA-PK inhibitor for 10–12 wk (B) (n=5 per group). All values are given as mean ± s.e.m. ***, p<0.001 (Two-way ANOVA) between genotype and treatment groups. (C) Resting serum lactate levels in middle-age WT and SCID mice. Mann-Whitney Test: *, p<0.05 between WT and SCID values. (D) The distance (in meters) young (Y, 4 months old) and middle aged (MA, 12 mo) WT and SCID mice ran on the treadmill before exhaustion. For young, n=8; middle aged, n=8, for each genotype. Mann-Whitney Test: **, p<0.01 between WT and SCID values. (E) The distance middle aged (MA) and obese (Ob) mice treated with either vehicle or DNA-PKcs inhibitor ran on the treadmill before exhaustion (n=10 per treatment group). Mann-Whitney Test: **, p<0.01 between the treatment groups. (F) The distance obese WT and AMPKα2 knockout mice treated with either vehicle or DNA-PKcs inhibitor ran on the treadmill before exhaustion (n=4–6 per treatment group). Mann-Whitney Test: *, p<0.05 between the treatment groups.

**Figure 6. Inhibition of DNA-PK protects against obesity and type-2 diabetes**
(A) Weight gain of WT mice fed HFD (±NU7441) (n=10 per treatment group). All values are expressed as mean ± s.e.m. ***, p<0.001 (Two-way repeated measures ANOVA) between the treatment groups. Glucose tolerance (B) and insulin sensitivity (C) of mice on HFD (±NU7441) for 10–12 wk are shown (n=10 per treatment group). ***, p<0.001 (Two-way repeated measures ANOVA) between the treatment groups. (D) Plasma concentrations of GLP-1 in mice fed HFD (±NU7441) (n=7–8 per treatment group). Mann-Whitney Test: **, p<0.01 between the treatment groups. A hyperinsulinemic-euglycemic clamp study was performed on mice fed HFD (±NU7441) (n=7–8 per treatment group) to measure (E) glucose infusion rate (GIR), (F) glucose disposal rate (GDR), (G and H) glucose uptake (GU) in skeletal muscle (gastrocnemius) and WAT, respectively, and (I) endogenous glucose production (EGP) from the liver. N.S., not significant; *, p<0.05 between the treatment groups.

**Figure 7. Calorie restriction and aerobic fitness are associated with decreased DNA-PK activity**
(A) (Left) DNA-PKcs autophosphorylation and p-HSP90α in skeletal muscle of *ad libitum* (AL) fed and calorie-restricted (CR, for 3.4 yr) middle aged rhesus macaques (n=4 per group). (Right) Quantification of DNA-PKcs autophosphorylation and p-HSP90α. All values are expressed as mean ± s.e.m. Mann-Whitney Test: *, p<0.05; **, p<0.01. (B) (Left) Total DNA-PKcs level and p-HSP90α in skeletal muscle of LCR (Low Capacity Runners), HCR (High Capacity Runners) rats. (Right) Quantification of DNA-PKcs expression level and p-HSP90α in skeletal muscle of LCR and HCR rats (n=10 per group). Mann-Whitney Test: **, p<0.01. (C) Schematic diagram showing how DNA-PK links aging-associated DNA DSBs to diminished AMPK activity and metabolic functions in skeletal muscle.

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Comment in

Turning back the clock.
Shoemaker AH. Shoemaker AH. Sci Transl Med. 2017 May 17;9(390):eaan4290. doi: 10.1126/scitranslmed.aan4290. Sci Transl Med. 2017. PMID: 28515340

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References

1. Anderson CW, Carter TH. The DNA-activated protein kinase – DNA-PK. Curr Top Microbiol Immunol. 1996;217:91–111. - PubMed
1. Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology. 2007;132:2131–2157. - PubMed
1. Bailey SM, Meyne J, Chen DJ, Kurimasa A, Li GC, Lehnert BE, Goodwin EH. DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proc Natl Acad Sci U S A. 1999;96:14899–14904. - PMC - PubMed
1. Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem. 2000;275:3343–3347. - PubMed
1. Blunt T, Gell D, Fox M, Taccioli GE, Lehmann AR, Jackson SP, Jeggo PA. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci U S A. 1996;93:10285–10290. - PMC - PubMed

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[1] Anderson CW, Carter TH. The DNA-activated protein kinase – DNA-PK. Curr Top Microbiol Immunol. 1996;217:91–111. - PubMed

[2] Anderson CW, Carter TH. The DNA-activated protein kinase – DNA-PK. Curr Top Microbiol Immunol. 1996;217:91–111. - PubMed

[3] Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology. 2007;132:2131–2157. - PubMed

[4] Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology. 2007;132:2131–2157. - PubMed

[5] Bailey SM, Meyne J, Chen DJ, Kurimasa A, Li GC, Lehnert BE, Goodwin EH. DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proc Natl Acad Sci U S A. 1999;96:14899–14904. - PMC - PubMed

[6] Bailey SM, Meyne J, Chen DJ, Kurimasa A, Li GC, Lehnert BE, Goodwin EH. DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proc Natl Acad Sci U S A. 1999;96:14899–14904. - PMC - PubMed

[7] Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem. 2000;275:3343–3347. - PubMed

[8] Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem. 2000;275:3343–3347. - PubMed

[9] Blunt T, Gell D, Fox M, Taccioli GE, Lehmann AR, Jackson SP, Jeggo PA. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci U S A. 1996;93:10285–10290. - PMC - PubMed

[10] Blunt T, Gell D, Fox M, Taccioli GE, Lehmann AR, Jackson SP, Jeggo PA. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci U S A. 1996;93:10285–10290. - PMC - PubMed

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DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging

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DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging

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