A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity

Journal name:
Nature
Volume:
503,
Pages:
493–499
Date published:
DOI:
doi:10.1038/nature12656
Received
Accepted
Published online

Abstract

Adiponectin secreted from adipocytes binds to adiponectin receptors AdipoR1 and AdipoR2, and exerts antidiabetic effects via activation of AMPK and PPAR-α pathways, respectively. Levels of adiponectin in plasma are reduced in obesity, which causes insulin resistance and type 2 diabetes. Thus, orally active small molecules that bind to and activate AdipoR1 and AdipoR2 could ameliorate obesity-related diseases such as type 2 diabetes. Here we report the identification of orally active synthetic small-molecule AdipoR agonists. One of these compounds, AdipoR agonist (AdipoRon), bound to both AdipoR1 and AdipoR2 in vitro. AdipoRon showed very similar effects to adiponectin in muscle and liver, such as activation of AMPK and PPAR-α pathways, and ameliorated insulin resistance and glucose intolerance in mice fed a high-fat diet, which was completely obliterated in AdipoR1 and AdipoR2 double-knockout mice. Moreover, AdipoRon ameliorated diabetes of genetically obese rodent model db/db mice, and prolonged the shortened lifespan of db/db mice on a high-fat diet. Thus, orally active AdipoR agonists such as AdipoRon are a promising therapeutic approach for the treatment of obesity-related diseases such as type 2 diabetes.

At a glance

Figures

  1. Small-molecule AdipoR agonist AdipoRon binds to both AdipoR1 and AdipoR2, and increases AMPK activation, PGC-1[agr] expression and mitochondrial biogenesis in C2C12 myotubes.
    Figure 1: Small-molecule AdipoR agonist AdipoRon binds to both AdipoR1 and AdipoR2, and increases AMPK activation, PGC-1α expression and mitochondrial biogenesis in C2C12 myotubes.

    a, Chemical structure of AdipoRon. bi, l, m, Phosphorylation and amount of AMPK (bf, l, m), Ppargc1a mRNA levels (g, h), and mitochondrial content as assessed by mitochondrial DNA copy number (i), in C2C12 myotubes after myogenic differentiation (bi), in skeletal muscle (l) or in liver (m) from wild-type (WT) or Adipor1−/−Adipor2−/− double-knockout mice, treated with indicated concentrations of AdipoRon (b, di) or adiponectin (d, 15μgml−1; e, 50μgml−1; i, 10μg ml−1), for 5min (b, df), 1.5h (g, h) and 48h (i), with or without EGTA (f, h), 25μM AdipoRon, compound 112254 and 165073, 30μgml−1 adiponectin for 5min or 1mM AICAR for 1h and transfected with or without the indicated siRNA duplex (c), or AdipoRon (l, m). j, k, Surface plasmon resonance measuring AdipoRon binding to AdipoR1 and AdipoR2. AdipoR1 and AdipoR2 were immobilized onto a sensor chip SA. Binding analyses were performed using a range of AdipoRon concentrations (0.49–31.25μM). All values are presented as mean±s.e.m. b, c, eI, n = 4 each; d, l, m, n = 3 each; *P<0.05 and **P<0.01 compared to control or unrelated siRNA or as indicated. NS, not significant.

  2. AdipoRon improved insulin resistance, glucose intolerance and dyslipidaemia via AdipoR.
    Figure 2: AdipoRon improved insulin resistance, glucose intolerance and dyslipidaemia via AdipoR.

    ag, Plasma AdipoRon concentrations (a), body weight (b), food intake (c), plasma glucose (d, e, g), plasma insulin (d, e) and insulin resistance index (f) during oral glucose tolerance test (OGTT) (1.0 g glucose per kg body weight) (d, e) or during insulin tolerance test (ITT) (0.5U insulin per kg body weight) (g) in wild-type (WT) and Adipor1−/−Adipor2−/− double-knockout mice, treated with or without AdipoRon (50mg per kg body weight). h, i, Glucose infusion rate (GIR), endogenous glucose production (EGP) and rates of glucose disposal (Rd) during hyperinsulinaemic euglycaemic clamp study in wild-type and Adipor1−/−Adipor2−/− double-knockout mice, treated with or without AdipoRon (50mg per kg body weight). j, k, Plasma triglyceride (j) and free fatty acid (FFA) (k) in wild-type and Adipor1−/−Adipor2−/− double-knockout mice, treated with or without AdipoRon (50 mg per kg body weight). All values are presented as mean±s.e.m. a, n = 12–32; bg, j, k, n = 10 each; h, i, n = 5 each; *P<0.05 and **P<0.01 compared to control or as indicated. NS, not significant.

  3. AdipoRon increased mitochondria biogenesis in muscle, reduced tissue triglyceride content in liver and decreased oxidative stress and inflammation in liver and WAT.
    Figure 3: AdipoRon increased mitochondria biogenesis in muscle, reduced tissue triglyceride content in liver and decreased oxidative stress and inflammation in liver and WAT.

    ah, Ppargc1a, Esrra, Tfam, mt-Co2, Tnni1, Acadm and Sod2 mRNA levels (a), mitochondrial content as assessed by mitochondrial DNA copy number (b) in skeletal muscle, exercise endurance (c), Ppargc1a, Pck1, G6pc, Ppara, Acox1, Ucp2, Cat, Tnf and Ccl2 mRNA levels (d), tissue triglyceride content (e), TBARS (f) in liver and Tnf, Il6, Ccl2, Emr1, Itgax and Mrc1 mRNA levels (g) and TBARS (h) in WAT, from wild-type and Adipor1−/−Adipor2−/− double-knockout (DKO) mice treated with or without AdipoRon (50mg per kg body weight). All values are presented as mean±s.e.m. a, b, dh, n = 10 each; c, n = 5 each; *P<0.05 and **P<0.01 compared to control or as indicated. NS, not significant.

  4. AdipoRon ameliorated insulin resistance, diabetes and dyslipidaemia in db/db mice.
    Figure 4: AdipoRon ameliorated insulin resistance, diabetes and dyslipidaemia in db/db mice.

    a, Plasma glucose levels after intraperitoneal injection of adiponectin (30µg per 10g body weight) (left) or after oral administration of AdipoRon (50mg per kg body weight) (middle). The area under the curve (AUC) of left and middle panels is shown on the right. bi, Body weight (b), food intake (c), liver weight (d), WAT weight (e), plasma glucose (f, left, g), plasma insulin (f, middle) and insulin resistance index (f, right) during oral glucose tolerance test (OGTT) (1.0g glucose per kg body weight) (f) or during insulin tolerance test (ITT) (0.75U insulin per kg body weight) (g), plasma triglyceride (h) and free fatty acid (FFA) (i) in db/db mice under normal chow conditions, treated with or without AdipoRon (50mg per kg body weight). All values are presented as mean±s.e.m. a, n = 6-7; bi, n = 10 each from 2–3 independent experiments, *P<0.05 and **P<0.01 compared to control or as indicated. NS, not significant.

  5. AdipoRon increased mitochondria biogenesis in muscle, reduced tissue triglyceride content and oxidative stress in muscle and liver, and decreased inflammation in liver and WAT of db/db mice.
    Figure 5: AdipoRon increased mitochondria biogenesis in muscle, reduced tissue triglyceride content and oxidative stress in muscle and liver, and decreased inflammation in liver and WAT of db/db mice.

    ah, Ppargc1a, Esrra, Tfam, mt-Co2, Tnni1, Acadm and Sod2 mRNA levels (a), and mitochondrial content as assessed by mitochondrial DNA copy number (b), tissue triglyceride content (c) and TBARS (d) in skeletal muscle, Ppargc1a, Pck1, G6pc, Ppara, Acox1, Ucp2, Cat, Tnf and Ccl2 mRNA levels (e), tissue triglyceride content (f) and TBARS (g) in liver, and Tnf, Il6, Ccl2, Emr1, Itgax and Mrc1 mRNA levels (h) in WAT from db/db mice on a normal chow diet, treated with or without AdipoRon (50mg per kg body weight). All values are presented as mean±s.e.m. n = 10, *P<0.05 and **P<0.01 compared to control or as indicated. NS, not significant.

  6. AdipoRon increased insulin sensitivity and glucose tolerance, and at the same time contributed to longevity of obese diabetic mice.
    Figure 6: AdipoRon increased insulin sensitivity and glucose tolerance, and at the same time contributed to longevity of obese diabetic mice.

    a–c, Kaplan–Meier survival curves for wild-type, Adipor1−/−, Adipor2−/− and Adipor1−/−Adipor2−/− knockout mice on a normal chow diet (a) (n = 50, 32, 29 and 35, respectively) or high-fat diet (b) (n = 47, 33, 35 and 31, respectively), or for db/db mice treated with or without AdipoRon (30mg per kg body weight) on a normal chow or high-fat diet (n = 20 each) (c). P values were derived from log-rank calculations. d, Scheme illustrating the mechanisms by which AdipoR1 and AdipoR2 agonist increases insulin sensitivity and glucose tolerance, and at the same time lifespan. (See also main text.)

  7. Phosphorylation of AMPK in C2C12 myotubes.
    Extended Data Fig. 1: Phosphorylation of AMPK in C2C12 myotubes.

    Phosphorylation of AMPK normalized to the amount of AMPK in C2C12 myotubes treated for 5min with 15µgml−1 adiponectin or the indicated small-molecule compounds (10μM). #, AdipoRon; ##, no. 112254; ###, no. 165073.

  8. Distribution curves showing Z scores.
    Extended Data Fig. 2: Distribution curves showing Z scores.

    a, Distribution curve showing Z scores representing AMPK activity for all compounds tested in C2C12 myotubes shown in Extended Data Table 1 and Extended Data Fig. 1. The dashed line indicates the Z score cut-off for compounds scored as hits, which showed higher activity than 80% of that seen with adiponectin. b, Distribution curve showing Z scores representing AdipoR dependency of AMPK activation for 39 compounds tested in C2C12 myotubes shown in Extended Data Table 2. Indicated are the location of AdipoRon, another hit (no. 112254), and non-hit (no. 165073).

  9. The effect of AdipoRon on complex I activity, and expression of Adipor1 and Adipor2 mRNA in C2C12 myotubes transfected with the indicated siRNA duplex.
    Extended Data Fig. 3: The effect of AdipoRon on complex I activity, and expression of Adipor1 and Adipor2 mRNA in C2C12 myotubes transfected with the indicated siRNA duplex.

    a, Complex I activities were measured with the indicated concentrations of rotenone or AdipoRon. b, c, Adipor1 (b) and Adipor2 (c) mRNA levels were analysed by RT–qPCR. All values are presented as mean±s.e.m. a, n = 3–7; b, c, n = 3 each; *P<0.05 and **P<0.01 compared to control or unrelated siRNA cells. NS, not significant.

  10. AdipoRon binding to AdipoR1 and AdipoR2.
    Extended Data Fig. 4: AdipoRon binding to AdipoR1 and AdipoR2.

    a–d, Binding and Scatchard analyses of [3H]AdipoRon to primary hepatocytes from wild-type (a), Adipor2−/− knockout (b), Adipor1−/− knockout (c) and Adipor1−/−Adipor2−/− double-knockout (d) mice. eh, Concentration-dependent competitive [3H]AdipoRon binding to primary hepatocytes from wild-type (e), Adipor2−/− knockout (f), Adipor1−/− knockout (g) and Adipor1−/−Adipor2−/− double-knockout (h) mice. Binding analyses were performed using the indicated concentrations of AdipoRon. c.p.m., counts per minute.

  11. Raw data of Fig. 2 and time course of glucose-lowering effect of AdipoRon.
    Extended Data Fig. 5: Raw data of Fig. 2 and time course of glucose-lowering effect of AdipoRon.

    am, Raw data of Fig. 2a (a), Fig. 2d, left (b, c), Fig. 2d, right (d, e), Fig. 2e, left (f, g), Fig. 2e, right (h, i), Fig. 2g, left (j, k) and Fig. 2g, right (l, m). n, Time course of glucose-lowering effect of AdipoRon. Data are calculated from data in Fig. 4a. The glucose-lowering effect of AdipoRon was obtained by the following equation and expressed as %: (vehicle plasma glucoseAdipoRon plasma glucose)/vehicle plasma glucose. All values are presented as mean±s.e.m.

  12. The effects of compounds 112254 and 165073 on insulin resistance and glucose intolerance via AdipoR.
    Extended Data Fig. 6: The effects of compounds 112254 and 165073 on insulin resistance and glucose intolerance via AdipoR.

    a, b, Chemical structures of compounds 112254 (a) and 165073 (b). cj, Plasma glucose (c left, d left, f, g left, h left, j), plasma insulin (c right, d right, g right, h right) and insulin resistance index (e, i) during oral glucose tolerance test (OGTT) (1.0g glucose per kg body weight) (c, d, g, h) or during insulin tolerance test (ITT) (0.5U insulin per kg body weight) (f, j), in wild-type and Adipor1−/−Adipor2−/− double-knockout mice, treated with or without compounds 112254 or 165073 (50mg per kg body weight). All values are presented as mean±s.e.m. cf, n = 10 each; gj, n = 7 each from 2, 3 independent experiments, *P<0.05 and **P<0.01 compared to control or as indicated. NS, not significant.

  13. The effects of AdipoRon on glucose metabolism in Adipor1-/-, Adipor2-/- and Adipor1-/-[thinsp]Adipor2-/- mice.
    Extended Data Fig. 7: The effects of AdipoRon on glucose metabolism in Adipor1−/−, Adipor2−/− and Adipor1−/−Adipor2−/− mice.

    a, Triglyceride content (a) and TBARS (b) in skeletal muscle from wild-type or Adipor1−/−Adipor2−/− double-knockout mice treated with or without AdipoRon (50mg per kg body weight). cg, The effects of AdipoRon on glucose metabolism in Adipor1−/−, Adipor2−/− and Adipor1−/−Adipor2−/− mice. Plasma glucose (cf, left panels), plasma insulin (cf, right panels) and insulin resistance index (g) during oral glucose tolerance test (OGTT) (1.0g glucose per kg body weight). All values are presented as mean±s.e.m. ad, f, n = 10 each; e, n = 7 each; g, n = 7–10; *P<0.05 and **P<0.01 compared to vehicle mice. NS, not significant.

  14. Chemical structures and AdipoR dependency of AMPK activation.
    Extended Data Fig. 8: Chemical structures and AdipoR dependency of AMPK activation.

    ad, Chemical structures of AdipoRon (a), compound 168198 (b), compound 112254 (c) and compound 103694 (d). Within the 1-benzyl 4-substituted 6-membered cyclic amine moiety, the cyclic amine moiety is surrounded by a dashed red circle, and the aromatic ring is surrounded by a light green circle. Cyan and dark green circles surround the carbonyl group and the terminal aromatic ring, respectively, located on the opposite side from the benzyl cyclic amine. e, Phosphorylation and amount of AMPK in C2C12 myotubes treated for 5min with the indicated small-molecule compounds. Phosphorylation and amount of AMPK in C2C12 myotubes, treated for 5min with the indicated small-molecule compounds (10μM) (% relative to adiponectin). f, AdipoR dependency of AMPK activation. Phosphorylation and amount of AMPK in C2C12 myotubes and transfected with or without the AdipoR1 siRNA duplex, treated for 5min with the indicated small molecule. AdipoR-dependency ratios were obtained by the following equation: 100(ratio for those transfected with the AdipoR1 siRNA duplex/ratio for those transfected without the AdipoR1 siRNA duplex)×100 (%).

Tables

  1. Values of phosphorylation of AMPK in C2C12 myotubes
    Extended Data Table 1: Values of phosphorylation of AMPK in C2C12 myotubes
  2. Phosphorylation of AMPK in AdipoR knock-down C2C12 myotubes
    Extended Data Table 2: Phosphorylation of AMPK in AdipoR knock-down C2C12 myotubes

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Author information

  1. These authors contributed equally to this work.

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

Affiliations

  1. Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan

    • Miki Okada-Iwabu,
    • Toshimasa Yamauchi,
    • Masato Iwabu,
    • Ken-ichi Hamagami,
    • Koichi Matsuda,
    • Mamiko Yamaguchi,
    • Kohjiro Ueki &
    • Takashi Kadowaki
  2. Department of Integrated Molecular Science on Metabolic Diseases, 22nd Century Medical and Research Center, The University of Tokyo, Tokyo 113-0033, Japan

    • Miki Okada-Iwabu,
    • Toshimasa Yamauchi,
    • Masato Iwabu &
    • Takashi Kadowaki
  3. Department of Molecular Medicinal Sciences on Metabolic Regulation, 22nd Century Medical and Research Center, The University of Tokyo, Tokyo 113-0033, Japan

    • Miki Okada-Iwabu,
    • Toshimasa Yamauchi &
    • Takashi Kadowaki
  4. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan

    • Teruki Honma,
    • Hiroaki Tanabe,
    • Tomomi Kimura-Someya,
    • Mikako Shirouzu,
    • Akiko Tanaka &
    • Shigeyuki Yokoyama
  5. Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan

    • Hitomi Ogata &
    • Kumpei Tokuyama
  6. Open Innovation Center for Drug Discovery, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

    • Tetsuo Nagano &
    • Akiko Tanaka
  7. Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

    • Shigeyuki Yokoyama

Contributions

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

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Phosphorylation of AMPK in C2C12 myotubes. (430 KB)

    Phosphorylation of AMPK normalized to the amount of AMPK in C2C12 myotubes treated for 5min with 15µgml−1 adiponectin or the indicated small-molecule compounds (10μM). #, AdipoRon; ##, no. 112254; ###, no. 165073.

  2. Extended Data Figure 2: Distribution curves showing Z scores. (51 KB)

    a, Distribution curve showing Z scores representing AMPK activity for all compounds tested in C2C12 myotubes shown in Extended Data Table 1 and Extended Data Fig. 1. The dashed line indicates the Z score cut-off for compounds scored as hits, which showed higher activity than 80% of that seen with adiponectin. b, Distribution curve showing Z scores representing AdipoR dependency of AMPK activation for 39 compounds tested in C2C12 myotubes shown in Extended Data Table 2. Indicated are the location of AdipoRon, another hit (no. 112254), and non-hit (no. 165073).

  3. Extended Data Figure 3: The effect of AdipoRon on complex I activity, and expression of Adipor1 and Adipor2 mRNA in C2C12 myotubes transfected with the indicated siRNA duplex. (145 KB)

    a, Complex I activities were measured with the indicated concentrations of rotenone or AdipoRon. b, c, Adipor1 (b) and Adipor2 (c) mRNA levels were analysed by RT–qPCR. All values are presented as mean±s.e.m. a, n = 3–7; b, c, n = 3 each; *P<0.05 and **P<0.01 compared to control or unrelated siRNA cells. NS, not significant.

  4. Extended Data Figure 4: AdipoRon binding to AdipoR1 and AdipoR2. (247 KB)

    a–d, Binding and Scatchard analyses of [3H]AdipoRon to primary hepatocytes from wild-type (a), Adipor2−/− knockout (b), Adipor1−/− knockout (c) and Adipor1−/−Adipor2−/− double-knockout (d) mice. eh, Concentration-dependent competitive [3H]AdipoRon binding to primary hepatocytes from wild-type (e), Adipor2−/− knockout (f), Adipor1−/− knockout (g) and Adipor1−/−Adipor2−/− double-knockout (h) mice. Binding analyses were performed using the indicated concentrations of AdipoRon. c.p.m., counts per minute.

  5. Extended Data Figure 5: Raw data of Fig. 2 and time course of glucose-lowering effect of AdipoRon. (210 KB)

    am, Raw data of Fig. 2a (a), Fig. 2d, left (b, c), Fig. 2d, right (d, e), Fig. 2e, left (f, g), Fig. 2e, right (h, i), Fig. 2g, left (j, k) and Fig. 2g, right (l, m). n, Time course of glucose-lowering effect of AdipoRon. Data are calculated from data in Fig. 4a. The glucose-lowering effect of AdipoRon was obtained by the following equation and expressed as %: (vehicle plasma glucoseAdipoRon plasma glucose)/vehicle plasma glucose. All values are presented as mean±s.e.m.

  6. Extended Data Figure 6: The effects of compounds 112254 and 165073 on insulin resistance and glucose intolerance via AdipoR. (251 KB)

    a, b, Chemical structures of compounds 112254 (a) and 165073 (b). cj, Plasma glucose (c left, d left, f, g left, h left, j), plasma insulin (c right, d right, g right, h right) and insulin resistance index (e, i) during oral glucose tolerance test (OGTT) (1.0g glucose per kg body weight) (c, d, g, h) or during insulin tolerance test (ITT) (0.5U insulin per kg body weight) (f, j), in wild-type and Adipor1−/−Adipor2−/− double-knockout mice, treated with or without compounds 112254 or 165073 (50mg per kg body weight). All values are presented as mean±s.e.m. cf, n = 10 each; gj, n = 7 each from 2, 3 independent experiments, *P<0.05 and **P<0.01 compared to control or as indicated. NS, not significant.

  7. Extended Data Figure 7: The effects of AdipoRon on glucose metabolism in Adipor1−/−, Adipor2−/− and Adipor1−/−Adipor2−/− mice. (241 KB)

    a, Triglyceride content (a) and TBARS (b) in skeletal muscle from wild-type or Adipor1−/−Adipor2−/− double-knockout mice treated with or without AdipoRon (50mg per kg body weight). cg, The effects of AdipoRon on glucose metabolism in Adipor1−/−, Adipor2−/− and Adipor1−/−Adipor2−/− mice. Plasma glucose (cf, left panels), plasma insulin (cf, right panels) and insulin resistance index (g) during oral glucose tolerance test (OGTT) (1.0g glucose per kg body weight). All values are presented as mean±s.e.m. ad, f, n = 10 each; e, n = 7 each; g, n = 7–10; *P<0.05 and **P<0.01 compared to vehicle mice. NS, not significant.

  8. Extended Data Figure 8: Chemical structures and AdipoR dependency of AMPK activation. (124 KB)

    ad, Chemical structures of AdipoRon (a), compound 168198 (b), compound 112254 (c) and compound 103694 (d). Within the 1-benzyl 4-substituted 6-membered cyclic amine moiety, the cyclic amine moiety is surrounded by a dashed red circle, and the aromatic ring is surrounded by a light green circle. Cyan and dark green circles surround the carbonyl group and the terminal aromatic ring, respectively, located on the opposite side from the benzyl cyclic amine. e, Phosphorylation and amount of AMPK in C2C12 myotubes treated for 5min with the indicated small-molecule compounds. Phosphorylation and amount of AMPK in C2C12 myotubes, treated for 5min with the indicated small-molecule compounds (10μM) (% relative to adiponectin). f, AdipoR dependency of AMPK activation. Phosphorylation and amount of AMPK in C2C12 myotubes and transfected with or without the AdipoR1 siRNA duplex, treated for 5min with the indicated small molecule. AdipoR-dependency ratios were obtained by the following equation: 100(ratio for those transfected with the AdipoR1 siRNA duplex/ratio for those transfected without the AdipoR1 siRNA duplex)×100 (%).

Extended Data Tables

  1. Extended Data Table 1: Values of phosphorylation of AMPK in C2C12 myotubes (640 KB)
  2. Extended Data Table 2: Phosphorylation of AMPK in AdipoR knock-down C2C12 myotubes (357 KB)

Supplementary information

PDF files

  1. Supplementary Information (719 KB)

    This file contains Supplementary Results, Text and Data and additional references.

Additional data