A monoclonal antibody activating AdipoR for type 2 diabetes and nonalcoholic steatohepatitis

Figures

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  Fig. 1.. AdipoRaMab bound to AdipoR1 and AdipoR2. AdipoRaMab was evaluated for binding to AdipoR1 or AdipoR2 in AdipoR1- or AdipoR2-expressing cells or their parent strain using flow cytometry, and the results are presented as histograms [Parent strain (A), AdipoR1-expressing cells (B), AdipoR2-expressing cells (C)]. Solid histograms represent results for isotype control monoclonal antibody (Mab) (10 μg/ml). Solid line denotes AdipoRaMab (10 μg/ml), dashed line denotes AdipoRaMab (3 μg/ml), and dotted line denotes AdipoRaMab (1 μg/ml). APC-A, Allophycocyanin area.

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  Fig. 2.. AdipoRaMab increased AMPK phosphorylation at Thr172 in C2C12 myotubes. Phosphorylation and amount of AMPK in C2C12 myotubes after myogenic differentiation. C2C12 myotubes were treated (A) with or without AdipoRaMab, (B) with or without AdipoRaMab, or recombinant human adiponectin at the concentration as indicated for 10 min. AMPK phosphorylation was calculated at Thr172 as the ratio of the expression of phosphorylated AMPKα (pAMPK) to that of AMPKα and quantified by Western blotting. Results are expressed as ratios without AdipoRaMab. All values are presented as means ± SEM. n = 3. *P < 0.05 and **P < 0.01 compared with no AdipoRaMab [analysis of variance (ANOVA) followed by the Dunnett multiple comparison test].

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  Fig. 3.. AdipoRaMab improved glucose tolerance and insulin resistance index via AdipoR in high-fat diet–fed mice. Plasma glucose level (A and E), AUC (B and F), plasma insulin level (C and G), and insulin resistance index (D and H) during the OGTT (1.0 g glucose/kg body weight) in normal chow (NC)– or high-fat (HF) diet–fed, wild-type (WT), and AdipoR1 and AdipoR2 double knockout (R1·R2 DKO) mice, treated four times with AdipoRaMab or isotype control Mab at a once-weekly dose of 10 mg/kg body weight from 4 weeks after initiation of an HF diet onward. AdipoRaMab (−), isotype control Mab treatment. Results in (B), (D), (F), and (H) are expressed as ratios in normal chow–fed mice treated with isotype control Mab. All values are presented as means ± SEM. NC-fed WT or R1·R2 DKO mice treated with isotype control Mab (n = 5). HF diet–fed WT mice treated with isotype control Mab or AdipoRaMab (n = 12 or 13). HF diet–fed R1·R2 DKO mice treated with isotype control Mab or AdipoRaMab (n = 7 or 8). **P < 0.01 compared with isotype control Mab or as indicated (ANOVA followed by the Tukey-Kramer multiple comparison test). NS, not significant.

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  Fig. 4.. AdipoRaMab improved insulin tolerance via AdipoR in high-fat diet–fed mice. Plasma glucose level (A and D), AUC (B and E), and AOC (C and F) during the ITT (0.5 U insulin/kg body weight) in NC- or HF diet–fed WT and R1·R2 DKO mice, treated four times with AdipoRaMab or isotype control Mab at a once-weekly dose of 10 mg/kg body weight from 4 weeks after initiation of an HF diet onward. AdipoRaMab (−), isotype control Mab treatment. Results in (B), (C), (E), and (F) are expressed as ratios in normal chow–fed mice treated with isotype control Mab. All values are presented as means ± SEM. NC-fed WT or R1·R2 DKO mice treated with isotype control Mab (n = 5). HF diet–fed WT mice treated with isotype control Mab or AdipoRaMab (n = 12 or 13). HF diet–fed R1·R2 DKO mice treated with isotype control Mab or AdipoRaMab (n = 7 or 8). **P < 0.01 compared with isotype control Mab or as indicated (ANOVA followed by the Tukey-Kramer multiple comparison test).

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  Fig. 5.. AdipoRaMab activated AdipoR1-AMPK pathway in the skeletal muscle and AdipoR1-AMPK and AdipoR2-PPARα pathway in the liver of high-fat diet–fed mice. AMPK phosphorylation at Thr172 (A) in the skeletal muscle of HF diet–fed WT and R1·R2 DKO mice, treated for 10 min after intravenous injection of AdipoRaMab (10 mg/kg body weight). mt-Co2 and Slc2a4 mRNA levels (B and D) in the skeletal muscle (Muscle) and G6pc, Ucp2, Ccl2 mRNA levels in the liver (C and E) of NC- or HF diet–fed WT and R1·R2 DKO, treated four times with AdipoRaMab or isotype control Mab at a once-weekly dose of 10 mg/kg body weight from 4 weeks after initiation of an HF diet onward. AMPK phosphorylation at Thr172 was calculated as the ratio of the expression of phosphorylated AMPKα to that of AMPKα and quantified by Western blotting. AdipoRaMab (−), isotype control Mab treatment. Results are expressed as ratios in NC-fed mice treated with isotype control Mab. All values are presented as means ± SEM. HF diet–fed WT and R1·R2 DKO mice treated with isotype control Mab or AdipoRaMab [(A), n = 3]. NC-fed WT or R1·R2 DKO mice treated with isotype control Mab [(B) to (E), n = 5]. HF diet–fed WT mice treated with isotype…

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  Fig. 6.. AdipoRaMab did not affect NAS and total area of lipid droplets per each size in the liver of TFC diet–fed ob/ob mice. NAS (A) and individual lesion scores; steatosis score (B), lobular inflammation score (C), hepatocellular ballooning score (D), and total area of lipid droplets per each size (E) in the liver of TFC diet-fed ob/ob mice, treated four times with AdipoRaMab or isotype control Mab at a once-weekly dose of 30 mg/kg body weight from 4 weeks after initiation of a TFC diet onward. Each parameter was measured using images of the hematoxylin and eosin–stained liver sections. All values were presented as means ± SEM. n = 12. The statistical analysis between the two groups was performed using the Wilcoxon test for (A) to (D) using unpaired two-tailed t test for (E), and there were no significant differences in either variable.

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  Fig. 7.. AdipoRaMab suppressed Kupffer cell/macrophage infiltration in the liver of TFC diet–fed ob/ob mice. F4/80-immunostained images of a representative single mouse from TFC diet–fed ob/ob mice, treated four times with isotype control Mab (A) or AdipoRaMab (B) at a once-weekly dose of 30 mg/kg body weight from 4 weeks after initiation of a TFC diet onward. The F4/80-positive area (%) (C) represents the mean of five fields derived from each mouse. All values are presented as means ± SEM. n = 12. *P < 0.05 compared with isotype control Mab (unpaired two-tailed t tests).

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  Fig. 8.. AdipoRaMab suppressed the expression of molecules involved in inflammation, fibrosis and fatty acid synthesis, and PPARα in the liver of TFC diet-fed ob/ob mice. Il6 (A), Ccl2 (B), Timp1 (C), Acc (D), and Ppara (E) mRNA levels in the liver of TFC diet–fed ob/ob mice, treated four times with AdipoRaMab or isotype control Mab at a once-weekly dose of 30 mg/kg body weight from 4 weeks after initiation of a TFC diet onward. Results are expressed as ratios in mice treated with isotype control Mab. All values are presented as means ± SEM. n = 12. *P < 0.05 and **P < 0.01 compared with isotype control Mab (unpaired two-tailed t tests).