Adiponectin and AdipoR1 regulate PGC-1α and mitochondria by Ca2+ and AMPK/SIRT1

Journal name:
Date published:
Published online


Adiponectin is an anti-diabetic adipokine. Its receptors possess a seven-transmembrane topology with the amino terminus located intracellularly, which is the opposite of G-protein-coupled receptors. Here we provide evidence that adiponectin induces extracellular Ca2+ influx by adiponectin receptor 1 (AdipoR1), which was necessary for subsequent activation of Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMPK and SIRT1, increased expression and decreased acetylation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and increased mitochondria in myocytes. Moreover, muscle-specific disruption of AdipoR1 suppressed the adiponectin-mediated increase in intracellular Ca2+ concentration, and decreased the activation of CaMKK, AMPK and SIRT1 by adiponectin. Suppression of AdipoR1 also resulted in decreased PGC-1α expression and deacetylation, decreased mitochondrial content and enzymes, decreased oxidative type I myofibres, and decreased oxidative stress-detoxifying enzymes in skeletal muscle, which were associated with insulin resistance and decreased exercise endurance. Decreased levels of adiponectin and AdipoR1 in obesity may have causal roles in mitochondrial dysfunction and insulin resistance seen in diabetes.

At a glance


  1. Decreased mitochondria, oxidative type I myofibres and exercise capacity in skeletal muscle of muscle-R1KO mice.
    Figure 1: Decreased mitochondria, oxidative type I myofibres and exercise capacity in skeletal muscle of muscle-R1KO mice.

    ak, Phosphorylation and amount of AMPK (a), Ppargc1a, Esrra, Nrf1, Tfam, Cycs, mt-Co2, Mef2c, Acadm, Sod2, Cat and Slc2a4 mRNA levels (b), PGC-1α protein levels (c), mitochondrial content as assessed by mitochondrial DNA copy number (d), amounts of troponin I (slow) protein (e), ATPase (pH 4.3 for type I fibres) staining of soleus muscles (scale bars, 100μm) (f), quantification of type I fibres (g) based on fibre-type analyses (f), exercise endurance (h), β oxidation (i), triglyceride content (j), and TBARS (k) in skeletal muscle (ag, ik) obtained from control or muscle-R1KO after 5h fasting. All values are presented as mean±s.e.m. n = 5–12, *P<0.05 and **P<0.01 compared to control mice.

  2. Mechanisms of abnormal glucose and insulin homeostasis in muscle-R1KO mice.
    Figure 2: Mechanisms of abnormal glucose and insulin homeostasis in muscle-R1KO mice.

    af, Plasma glucose (a, c) and plasma insulin (b) during an oral glucose tolerance test (OGTT) (1.5g glucose per kg body weight) (a, b) or during an insulin tolerance test (ITT) (0.25U insulin per kg body weight) (c), endogenous glucose production (EGP) (d), rates of glucose disposal (Rd) (e) and glucose infusion rate (GIR) (f) during a hyperinsulinaemic-euglycaemic clamp study in control and muscle-R1KO mice. gl, Phosphorylation of tyrosine (pTyr) (g), Ser302 (k) and Ser636/639 (l) in IRS-1, phosphorylation and amount of Akt (h), S6K1 (i) and JNK (j) in skeletal muscle treated with or without insulin (0.3U per kg body weight) for 7.5min in control and muscle-R1KO mice after 5h fasting. IB, immunoblot; IP, immunoprecipitate. All values are presented as mean±s.e.m. n = 6–15 from 3–5 independent experiments, *P<0.05 and **P<0.01 compared to control or as indicated. NS, not significant.

  3. Adiponectin/AdipoR1 increase PGC-1[agr] expression and activity, and mitochondrial biogenesis in C2C12 myocytes.
    Figure 3: Adiponectin/AdipoR1 increase PGC-1α expression and activity, and mitochondrial biogenesis in C2C12 myocytes.

    ai, Mitochondrial content as assessed by mitochondrial DNA copy number (a, e, f), Ppargc1a mRNA levels (b), acetyl-lysine (Ac-Lys) levels checked on PGC-1α or Flag immunoprecipitates (c, d, h), NAD+/NADH ratio (g, i) in C2C12 myocytes treated with adiponectin for the times indicated (g), in C2C12 myocytes transfected with the indicated siRNA duplex (ac), in C2C12 myocytes transfected with the wild-type or the 2A mutant form of PGC-1α (d, e) or the R13 mutant form of PGC-1α (f) treated with 10μgml-1 adiponectin for 48h (a, e, f) or for 1.5h (b) or 2h (c, d), or in skeletal muscle from control or muscle-R1KO mice treated with or without adiponectin (h, i). The supernatant was blotted against GAPDH as an input control (c, d, h). C2C12 myocytes were used after myogenic differentiation in all experiments. All values are presented as mean±s.e.m. n = 5–10, *P<0.05 and **P<0.01 compared to control or unrelated siRNA or as indicated.

  4. Adiponectin-induced Ca2+ influx by AdipoR1 in C2C12 myocytes and Xenopus oocytes.
    Figure 4: Adiponectin-induced Ca2+ influx by AdipoR1 in C2C12 myocytes and Xenopus oocytes.

    a, Pseudocoloured images of changes in fura-2 before and after 1min stimulation with adiponectin (30μgml-1). Red corresponds to the greatest response. The bottom trace demonstrates the average calcium response of C2C12 myocytes to 1-min stimulation with adiponectin along with application of 5mM EGTA (black bar). The shaded region around the trace represents s.e.m. bd, Adipor1 mRNA levels (b), fura-2 calcium response (c) and their magnitude (d) of C2C12 myocytes transfected with unrelated siRNA duplex or AdipoR1 siRNA duplex to stimulation with 30μgml-1 adiponectin for 1min. eg, The amounts of AdipoR1 protein (e), representative Ca2+-activated Cl- current traces (f) before (left) and after (right) 30-s application of adiponectin, and their magnitude (g) in Xenopus oocytes injected with or without Adipor1 cRNA in response to adiponectin (30μgml-1), and with or without application of 5mM EGTA with depolarizing pulses of +100mV. [Ca2+]i and [Ca2+]e, intracellular and external Ca2+ concentration, respectively. All values are presented as mean±s.e.m. n = 6–14, *P<0.05 and **P<0.01 compared to unrelated siRNA cells or control cells or as indicated.

  5. Adiponectin-induced Ca2+ influx is required for CaMKK and AMPK activation and PGC-1[agr] expression.
    Figure 5: Adiponectin-induced Ca2+ influx is required for CaMKK and AMPK activation and PGC-1α expression.

    a, b, Phosphorylation and amount of AMPK in C2C12 myocytes preincubated for 20min with or without 5mM EGTA and then treated for 5min with adiponectin (30μgml-1) or ionomycin (1μM), or for 1h with AICAR (1mM) (a), or C2C12 myocytes transfected with the indicated siRNA duplex and then treated with 30μgml-1 adiponectin for 5min (b). c, Amount of Ppargc1a mRNA in C2C12 myocytes preincubated for 1h with AraA (0.5mM) or for 6h with STO-609 (1μgml-1) or for 20min with EGTA (5mM), and then treated for 1.5h with or without adiponectin (10μgml-1). d, Representative pseudocoloured images of changes in the fura-2 calcium response before and after 5min stimulation with adiponectin (30μgml-1) in a soleus muscle from control mice (top) and muscle-R1KO mice (bottom). Red corresponds to the greatest response. Scale bars, 100μm. e, Trace demonstrates the calcium response of soleus muscle in the fields presented in d. Adiponectin was applied during the indicated period. f, The magnitude of fura-2 calcium response signals by 160-s adiponectin stimulation to soleus muscles. ΔF ratio indicates the change in the fluorescence ratio after adiponectin application. All values are presented as mean±s.e.m. n = 5–10, *P<0.05 and **P<0.01 compared to control or unrelated siRNA cells or as indicated.

  6. The effect of exercise on muscle-R1KO mice.
    Figure 6: The effect of exercise on muscle-R1KO mice.

    ad, The insulin resistance index (a), area under the curves (AUC) of plasma glucose levels during the ITT (b), mitochondrial content as assessed by mitochondrial DNA copy number (c), and citrate synthase (CS) enzyme activity (d) in skeletal muscle of control and muscle-R1KO mice after 2weeks exercise. The results are expressed as the percentage of the value in control littermates (a, b). e, Scheme illustrating the signal transduction of adiponectin/AdipoR1 in muscle cells. Both CaMKKβ and LKB1 are necessary for adiponectin-induced full AMPK activation. AMPK and SIRT1 are required for adiponectin/AdipoR1-induced PGC-1α activation. CaMKKβ activation by adiponectin-induced Ca2+ influx via AdipoR1 is required for adiponectin-induced increased PGC-1α expression. PGC-1α is required for mitochondrial biogenesis stimulated with adiponectin/AdipoR1. From these data, we conclude that adiponectin and AdipoR1 increase PGC-1α expression and activity by Ca2+ signalling and by AMPK and SIRT1, leading to increased mitochondrial biogenesis. We focused on the molecules that we have obtained direct evidence by both gain-of-function and loss-of-function experiments in vitro and in vivo, except for CaMK, which has already been reported to increase PGC-1α expression by other researchers39. AC, acetylation. All values are presented as mean±s.e.m. n = 5–8, *P<0.05 and **P<0.01 compared to control mice or as indicated.


  1. Scherer, P. E., Williams, S., Fogliano, M., Baldini, G. & Lodish, H. F. A novel serum protein similar to C1q, produced exclusively in adipocytes. J. Biol. Chem. 270, 2674626749 (1995)
  2. Hu, E., Liang, P. & Spiegelman, B. M. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J. Biol. Chem. 271, 1069710703 (1996)
  3. Maeda, K. et al. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1). Biochem. Biophys. Res. Commun. 221, 286289 (1996)
  4. Nakano, Y., Tobe, T., Choi-Miura, N.-H., Mazda, T. & Tomita, M. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 120, 803812 (1996)
  5. Hotta, K. et al. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler. Thromb. Vasc. Biol. 20, 15951599 (2000)
  6. Fruebis, J. et al. Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc. Natl Acad. Sci. USA 98, 20052010 (2001)
  7. Yamauchi, T. et al. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nature Med. 7, 941946 (2001)
  8. Berg, A. H., Combs, T. P., Du, X., Brownlee, M. & Scherer, P. E. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nature Med. 7, 947953 (2001)
  9. Kubota, N. et al. Disruption of adiponectin causes insulin resistance and neointimal formation. J. Biol. Chem. 277, 2586325866 (2002)
  10. Maeda, N. et al. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nature Med. 8, 731737 (2002)
  11. Yamauchi, T. et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nature Med. 8, 12881295 (2002)
  12. Tomas, E. et al. Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation. Proc. Natl Acad. Sci. USA 99, 1630916313 (2002)
  13. Kahn, B. B., Alquier, T., Carling, D. & Hardie, D. G. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 1, 1525 (2005)
  14. Kersten, S., Desvergne, B. & Wahli, W. Roles of PPARs in health and disease. Nature 405, 421424 (2000)
  15. Yamauchi, T. et al. Globular adiponectin protected ob/ob mice from diabetes and apoE deficient mice from atherosclerosis. J. Biol. Chem. 278, 24612468 (2003)
  16. Yamauchi, T. et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423, 762769 (2003)
  17. Wess, J. G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J. 11, 346354 (1997)
  18. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y. & Shimizu, T. A. G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387, 620624 (1997)
  19. Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G. & Cotecchia, S. Constitutively active mutants of the α1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. EMBO J. 15, 35663578 (1996)
  20. Yamauchi, T. et al. Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nature Med. 13, 332339 (2007)
  21. Petersen, K. F. et al. Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N. Engl. J. Med. 350, 664671 (2004)
  22. Wu, Z. et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98, 115124 (1999)
  23. Mootha, V. K. et al. Errα and Gabpa/b specify PGC-1α-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Proc. Natl Acad. Sci. USA 101, 65706575 (2004)
  24. Berchtold, M. W. et al. Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. Physiol. Rev. 80, 12151265 (2000)
  25. Wu, H. et al. MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type. EMBO J. 19, 19631973 (2000)
  26. Hotamisligil, G. S. Inflammation and metabolic disorders. Nature 444, 860867 (2006)
  27. Um, S. H. et al. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 431, 200205 (2004)
  28. Hawley, S. A. et al. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J. Biol. Chem. 271, 2787927887 (1996)
  29. Hawley, S. A. et al. Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab. 2, 919 (2005)
  30. Woods, A. et al. Ca2+/calmodulin-dependent protein kinase kinase-β acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab. 2, 2133 (2005)
  31. Jäger, S., Handschin, C., St-Pierre, J. & Spiegelman, B. M. AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1α. Proc. Natl Acad. Sci. USA 104, 1201712022 (2007)
  32. Rodgers, J. T. et al. Nutrient control of glucose homeostasis through a complex of PGC-1α and SIRT1. Nature 434, 113118 (2005)
  33. Guarente, L. Sirtuins as potential targets for metabolic syndrome. Nature 444, 868874 (2006)
  34. Tokumitsu, H. et al. STO-609, a specific inhibitor of the Ca2+/calmodulin-dependent protein kinase kinase. J. Biol. Chem. 277, 1581315818 (2002)
  35. Tóth, A. et al. Quantitative assessment of [Ca2+]i levels in rat skeletal muscle in vivo . Am. J. Physiol. Heart Circ. Physiol. 275, H1652H1662 (1998)
  36. Shkryl, V. M. & Shirokova, N. Transfer and tunneling of Ca2+ from sarcoplasmic reticulum to mitochondria in skeletal muscle. J. Biol. Chem. 281, 15471554 (2006)
  37. Anderson, K. A. et al. Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta. J. Biol. Chem. 273, 3188031889 (1998)
  38. Soderling, T. R. The Ca-calmodulin-dependent protein kinase cascade. Trends Biochem. Sci. 24, 232236 (1999)
  39. Handschin, C. & Spiegelman, B. M. The role of exercise and PGC1α in inflammation and chronic disease. Nature 454, 463469 (2008)
  40. Handschin, C. et al. Skeletal muscle fiber-type switching, exercise intolerance, and myopathy in PGC-1α muscle-specific knock-out animals. J. Biol. Chem. 282, 3001430021 (2007)
  41. Mootha, V. K. et al. PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature Genet. 34, 267273 (2003)
  42. Patti, M. E. et al. Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1. Proc. Natl Acad. Sci. USA 100, 84668471 (2003)
  43. Wang, C. et al. Adiponectin sensitizes insulin signaling by reducing p70 S6 kinase-mediated serine phosphorylation of IRS-1. J. Biol. Chem. 282, 79917996 (2007)
  44. Houstis, N., Rosen, E. D. & Lander, E. S. Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature 440, 944948 (2006)
  45. St-Pierre, J. et al. Suppression of reactive oxygen species and neurodegeneration by the PGC-1transcriptional coactivators. Cell 127, 397408 (2006)

Download references

Author information

  1. These authors contributed equally to this work.

    • Masato Iwabu,
    • Toshimasa Yamauchi &
    • Miki Okada-Iwabu


  1. Department of Diabetes and Metabolic Diseases, Graduate School of Medicine,

    • Masato Iwabu,
    • Toshimasa Yamauchi,
    • Miki Okada-Iwabu,
    • Masaaki Funata,
    • Mamiko Yamaguchi,
    • Ryo Nakayama,
    • Naoto Kubota,
    • Iseki Takamoto,
    • Hironori Waki,
    • Kohjiro Ueki &
    • Takashi Kadowaki
  2. Department of Integrated Molecular Science on Metabolic Diseases, 22nd Century Medical and Research Center

    • Masato Iwabu,
    • Toshimasa Yamauchi &
    • Miki Okada-Iwabu
  3. Department of Neurobiology, Graduate School of Medicine,

    • Shigeyuki Namiki &
    • Kenzo Hirose
  4. Department of Pathology, Graduate School of Medicine,

    • Naoko Yamauchi &
    • Masashi Fukayama
  5. Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan

    • Satoshi Ishii &
    • Takao Shimizu
  6. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

    • Koji Sato &
    • Kazushige Touhara
  7. Department of Integrated Biosciences, The University of Tokyo, Chiba 277-8562, Japan

    • Tatsuro Nakagawa &
    • Kazushige Touhara
  8. Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan

    • Mitsuhisa Tabata &
    • Yuichi Oike
  9. Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan

    • Hitomi Ogata &
    • Kumpei Tokuyama
  10. Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan

    • Yukiko K. Hayashi &
    • Ichizo Nishino


M.I., M.O.-I., T.Y., K.S., T.N., M.F., M.Y., S.N., R.N., M.T., H.O., N.K., I.T., Y.K.H. and N.Y. performed experiments. T.K. and T.Y. conceived and supervised the study. K.T., T.S. and K.H. supervised the study. T.Y., T.K., M.I. and M.O-I. wrote the paper. All authors interpreted data.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Information (1.4M)

    This file contains Supplementary Results, Supplementary Methods, Supplementary References and Supplementary Figures 1-20 with legends.

Additional data