Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine

Journal name:
Nature Medicine
Volume:
19,
Pages:
83–92
Year published:
DOI:
doi:10.1038/nm.3014
Received
Accepted
Published online

Abstract

Despite growing interest and a recent surge in papers, the role of autophagy in glucose and lipid metabolism is unclear. We produced mice with skeletal muscle–specific deletion of Atg7 (encoding autophagy-related 7). Unexpectedly, these mice showed decreased fat mass and were protected from diet-induced obesity and insulin resistance; this phenotype was accompanied by increased fatty acid oxidation and browning of white adipose tissue (WAT) owing to induction of fibroblast growth factor 21 (Fgf21). Mitochondrial dysfunction induced by autophagy deficiency increased Fgf21 expression through induction of Atf4, a master regulator of the integrated stress response. Mitochondrial respiratory chain inhibitors also induced Fgf21 in an Atf4-dependent manner. We also observed induction of Fgf21, resistance to diet-induced obesity and amelioration of insulin resistance in mice with autophagy deficiency in the liver, another insulin target tissue. These findings suggest that autophagy deficiency and subsequent mitochondrial dysfunction promote Fgf21 expression, a hormone we consequently term a 'mitokine', and together these processes promote protection from diet-induced obesity and insulin resistance.

At a glance

Figures

  1. Decreased muscle and fat mass in Atg7[Delta]sm mice.
    Figure 1: Decreased muscle and fat mass in Atg7Δsm mice.

    (a) Immunoblots of gastrocnemius (Gas) muscle, soleus (Sol) muscle, epididymal WAT and the liver of fasted Atg7Δsm or Atg7f/f (control) mice, using antibodies specific for Atg7, Lc3, p62 and ubiquitin. β-actin, a loading control. (b) Representative confocal images of p62 and ubiquitin (Ub) aggregates that were colocalized (arrows) in gastrocnemius muscle of Atg7Δsm mice. Scale bars, 50 μm. (c) Body weight of male Atg7f/f and Atg7Δsm mice fed chow diet (n = 11–14). Right, representative picture of 16-week-old mice. (d) Body composition of 24-week-old male Atg7f/f and Atg7Δsm mice (n = 4 or 5). (e) Gross image of calf muscle (top) and weight of gastrocnemius muscle (bottom) from Atg7f/f and Atg7Δsm mice (n = 5 or 6). (f) H&E staining of gastrocnemius muscle (left) and quantification of fiber area (right) in Atg7f/f and Atg7Δsm mice (n = 3). Scale bars, 50 μm. (g) Gross image and weight of epididymal fat from Atg7f/f and Atg7Δsm mice (n = 5 or 6). (h) H&E staining of fat sections from Atg7f/f and Atg7Δsm mice. Scale bars, 50 μm. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

  2. Increased energy expenditure and ameliorated insulin resistance in Atg7[Delta]sm mice fed HFD.
    Figure 2: Increased energy expenditure and ameliorated insulin resistance in Atg7Δsm mice fed HFD.

    (a) Body weight of male Atg7f/f and Atg7Δsm mice fed HFD for 13 weeks (n = 16–23). (b) Energy expenditure (EE) plotted against body mass with fitted lines of linear regression (left) and EE adjusted for body mass by ANCOVA (right) in male Atg7f/f and Atg7Δsm mice fed a short-term HFD for 1 week (n = 9 or 10). (c) Nonfasting blood glucose concentration in HFD-fed Atg7f/f and Atg7Δsm mice (n = 16–23). (d) Fasting blood glucose (n = 10–15) and insulin concentrations (n = 6–13), and HOMA-IR (n = 6–13) in male Atg7f/f and Atg7Δsm mice fed HFD for 13 weeks. (e) GTT in male Atg7f/f and Atg7Δsm mice fed HFD for 13 weeks (n = 6–10). (f,g) Hyperinsulinemic-euglycemic clamp. Glucose infusion rate (f) and whole-body glucose uptake, glycolysis or glycogen synthesis (normalized to body mass (g) in male Atg7f/f and Atg7Δsm mice fed HFD for 6 weeks (n = 7 or 8). (h) 2-Deoxyglucose (2-DG) uptake in gastrocnemius muscle of male Atg7f/f and Atg7Δsm mice fed HFD for 6 weeks (n = 5–7). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

  3. Increased [beta]-oxidation, lipolysis and browning of WAT in HFD-fed Atg7[Delta]sm mice.
    Figure 3: Increased β-oxidation, lipolysis and browning of WAT in HFD-fed Atg7Δsm mice.

    (a) In vivo β-oxidation of infused [1-14C]oleic acid in HFD-fed Atg7f/f and Atg7Δsm mice (n = 7–10). (b) Ex vivo β-oxidation in epididymal WAT (n = 5–7) and BAT (n = 4–6) of HFD-fed Atg7f/f and Atg7Δsm mice. NS, not significant. (c,d) Relative expression of genes related to β-oxidation, lipolysis and thermogenesis in perirenal WAT and BAT of HFD-fed Atg7f/f and Atg7Δsm mice (n = 3). (e) Relative mRNA levels from genes associated with fatty acid and triacylglycerol (TG) synthesis, β-oxidation or inflammation in the liver of HFD-fed Atg7f/f and Atg7Δsm mice (n = 4). (f) H&E (left) and Oil Red O staining (right) of the liver from HFD-fed Atg7f/f and Atg7Δsm mice. Arrows indicate infiltrating immune cells. Scale bars, 50 μm. (g) H&E staining of perirenal WAT (left) and BAT (right) from HFD-fed Atg7f/f and Atg7Δsm mice. Scale bars, 100 μm. (h) 2-Deoxyglucose (2-DG) uptake in BAT of Atg7f/f and Atg7Δsm mice fed HFD for 6 weeks (n = 7 or 8). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

  4. Atf4-dependent Fgf21 induction is responsible for improved metabolic profile in Atg7[Delta]sm mice.
    Figure 4: Atf4-dependent Fgf21 induction is responsible for improved metabolic profile in Atg7Δsm mice.

    (a) Microarray (left) and real-time RT-PCR analysis (middle) of myokine genes such as Fgf21, adiponectin (Adipoq), interleukin-6 (Il6), Il15, insulin-like growth factor binding protein 2 (Igfbp2) or tumor necrosis factor α (Tnf) in gastrocnemius muscle of chow-fed Atg7f/f and Atg7Δsm mice (n = 3). Inset, representative standard RT-PCR data for Fgf21. Right, serum Fgf21 concentration in chow-fed mice in fed (n = 13) or fasted state (n = 5 or 6). (be) Body weight (n = 6–9; b), GTT (n = 6–9; c), ITT (n = 6–9; d) and fat weight (n = 5 or 6; e) in male Fgf21+/+Atg7f/f, Fgf21−/−Atg7f/f, Fgf21+/+Atg7Δsm and Fgf21−/−Atg7Δsm mice fed HFD for 12 weeks. (f) Immunoblotting for Atf4, phosphorylated Eif2α (p-Eif2α), Eif2α and Fgf21 in gastrocnemius muscle of Atg7f/f and Atg7Δsm mice. Hsp90, a loading control. (g) Relative Fgf21 mRNA expression in C2C12 myotubes infected with adenovirus (Ad) expressing GFP or Atf4 for 36 h. (h) Luciferase activity of reporters with deletion and point mutations of the Fgf21 promoter in C2C12 myotubes transfected with a mock vector or Atf4 for 24 h. (i) Luciferase activity of Fgf21 promoter in Atg7+/+ or Atg7−/− MEFs transfected with indicated vectors for 24 h. WT, wild-type Fgf21 promoter. (j) Relative Atf4 (left) and Fgf21 mRNA expression (right) in Atg7+/+ or Atg7−/− MEFs transfected with control siRNA (siCON) or Atf4 siRNA (siAtf4) for 48 h. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

  5. Impairment of mtOxPhos in autophagy deficiency is responsible for Atf4-dependent Fgf21 induction.
    Figure 5: Impairment of mtOxPhos in autophagy deficiency is responsible for Atf4-dependent Fgf21 induction.

    (a) Representative electron micrographs of gastrocnemius muscle from Atg7f/f and Atg7Δsm mice. Scale bars, 1 μm. (b) Mitochondrial state 3 and 4 respiration in skeletal muscle of Atg7f/f and Atg7Δsm mice (n = 5). (c) Representative Cox staining of gastrocnemius muscle from Atg7f/f and Atg7Δsm mice. Scale bars, 100 μm. (d) Relative Fgf21 mRNA expression in C2C12 myotubes after treatment with DMSO (Veh), rotenone (Rot; 0.5 μM) or antimycin A (AA; 4 μM) for 8 h. (e) Immunoblotting for Atf4, phosphorylated Eif2α (p-Eif2α), Eif2α and β-actin in C2C12 myotubes treated with DMSO or rotenone (0.5 μM) for 30 min or 1 h. (f) Relative Fgf21 mRNA expression in siCON- or siAtf4-transfected C2C12 myotubes after treatment with rotenone (0.5 μM) for 8 h. (g,h) Relative Fgf21 mRNA expression in Atf4+/+ or Atf4−/− MEFs (g) and Eif2aS/S (wild type) or Eif2aA/A MEFs (mutant) (h) after treatment with DMSO, rotenone (0.5 μM) or antimycin A (4 μM) for 8 h. (i) Left, standard RT-PCR (upper two lanes) and immunoblotting for Fgf21 (lower two lanes) in muscle of 9-week-old Mfn1/2f/f (control) and Mfn1/2Δsm mice. Right, real-time RT-PCR of Fgf21 in muscle of Mfn1/2f/f and Mfn1/2Δsm mice. (j) Immunoblotting for Atf4, phosphorylated Eif2α (p-Eif2α) and Eif2α in muscle of Mfn1/2f/f and Mfn1/2Δsm mice. Hsp90, a loading control. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

  6. Atg7[Delta]hep mice are protected from HFD-induced obesity and insulin resistance.
    Figure 6: Atg7Δhep mice are protected from HFD-induced obesity and insulin resistance.

    (a) Body weight of Atg7f/f and Atg7Δhep mice fed chow diet (n = 3 or 4). (b) Gross image and weight of epididymal fat from 20-week-old mice (n = 4). (c) GTT in 16-week-old Atg7f/f and Atg7Δhep mice fed chow diet (n = 6). (d) Representative Oil Red O staining of liver from fasted 16-week-old mice fed chow diet. Scale bars, 50 μm. (e) Representative Cox staining of liver from 16-week-old mice fed chow diet. Scale bars, 50 μm. (f) Left, relative Fgf21 mRNA expression in primary hepatocytes from chow-fed mice (n = 4). Right, serum Fgf21 concentration in fed mice on chow diet (n = 8). (g) Body weight of Atg7f/f and Atg7Δhep mice fed HFD (n = 6). (h) Fasting blood glucose and insulin concentrations and HOMA-IR in mice fed HFD for 13 weeks (n = 5). (i) GTT in mice fed HFD for 13 weeks (n = 5). (j) H&E staining of liver from mice fed HFD for 13 weeks. Scale bars, 100 μm. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

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Author information

  1. These authors contributed equally to this work.

    • Yeon Taek Jeong &
    • Hyunhee Oh

Affiliations

  1. Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

    • Kook Hwan Kim,
    • Yeon Taek Jeong,
    • Jae Min Cho,
    • Do Hoon Kim,
    • Kyu Yeon Hur &
    • Myung-Shik Lee
  2. Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, Seoul, Korea.

    • Kook Hwan Kim,
    • Seong Hun Kim &
    • Myung-Shik Lee
  3. Korea Mouse Metabolic Phenotyping Center, Lee Gil Ya Cancer and Diabetes Institute, Gachon University Graduate School of Medicine, Incheon, Korea.

    • Hyunhee Oh,
    • Yo-Na Kim,
    • Su Sung Kim &
    • Cheol Soo Choi
  4. National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, FIRST Mitochondrial Research Group, Inje University, Busan, Korea.

    • Hyoung Kyu Kim,
    • TaeHee Ko &
    • Jin Han
  5. Integrative Research Support Center, College of Medicine, The Catholic University of Korea, Seoul, Korea.

    • Hong Lim Kim
  6. Department of Anatomy and Cell Death Disease Research Center, College of Medicine, The Catholic University of Korea, Seoul, Korea.

    • Jin Kim
  7. School of Biological Sciences, University of Ulsan, Ulsan, Korea.

    • Sung Hoon Back
  8. Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.

    • Masaaki Komatsu
  9. Division of Biology, California Institute of Technology, Pasadena, California, USA.

    • Hsiuchen Chen &
    • David C Chan
  10. Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA.

    • David C Chan
  11. Department of Microbial Chemistry, Kobe Pharmaceutical University, Kobe, Japan.

    • Morichika Konishi
  12. Department of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto, Japan.

    • Nobuyuki Itoh
  13. Department of Internal Medicine, Gil Medical Center, Gachon University Graduate School of Medicine, Incheon, Korea.

    • Cheol Soo Choi

Contributions

K.H.K., C.S.C. and M.-S.L. designed the study, analyzed data and wrote the manuscript. K.H.K. conducted all experiments except the portions indicated below, assisted by S.H.K., J.M.C., D.H.K. and K.Y.H. Y.T.J. analyzed the metabolic profiling of Atg7Δhep mice. H.O., Y.-N.K. and S.S.K. performed measurements of body composition, indirect calorimetry, the hyperinsulinemic-euglycemic clamp study and the fatty acid oxidation experiments. H.K.K., T.K. and J.H. measured the mitochondrial oxygen consumption. H.L.K. and J.K. performed the electron microscopy. S.H.B., M. Komatsu, H.C., D.C.C., M. Konishi and N.I. provided reagents, tissues, cells or mice and commented on the manuscript.

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The authors declare no competing financial interests.

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