Figure 5. Compd. 23 increases liver cell insulin signaling, is orally bioavailable and reverses diabetes in obese mice.
(a–b) HepG2 hepatocytes were incubated overnight with 10 μM compound (Compd.) or dimethylsulfoxide (DMSO) and stimulated with 10 nM insulin for 5 min or left unstimulated (Unst.). Insulin receptor (IR) tyrosine phosphorylation was assessed by (a) Western blotting of anti-IR immunoprecipitations (representative of 2 independent experiments) and (b) phosphoIR (pIR) ELISA (data from 5 independent experiments, Compd. 23: p=0.0079, Compd. 28: p=0.1667). (c–g) Diet-induced obese (DIO) male B6 mice were treated with 0.05% w/w Compd. 23 in high-fat diet (HFD) or HFD alone for 2 weeks. (c) Body weight during treatment (HFD, n=17; Compd. 23, n=13). (d) Intraperitoneal glucose tolerance test (IPGTT), p=0.0162 (HFD, n=8; Compd. 23, n=6). (e) Fasting plasma insulin levels, relative to littermates fed HFD, p=0.0235 (HFD, n=4; Compd. 23, n=4). (f–g) Mice were injected intraperitoneally with insulin and livers harvested after 10 min. (f) Liver IR tyrosine phosphorylation, p=0.0002 (HFD, n=7; Compd. 23, n=6). (g) Western blot of liver homogenates (HFD, n=3; Compd. 23, n=3). (h–i) DIO liver-specific LMPTP KO mice were treated with Compd. 23 (HFD, n=6; Compd. 23, n=6) as in (c–g). (h) IPGTT, p=0.6120. (i) Liver IR tyrosine phosphorylation, p=0.2791. (b–f,h–i) Mean±SEM. *, p<0.05; NS, non-significant (p≥0.05): (b) Kolmogorov-Smirnov test, (d,h) Two-Way ANOVA, (e) two-tailed unpaired t-test with Welch’s correction, (f,i) one-tailed unpaired t-test with Welch’s correction. (a,c) Full gels are shown in Supplementary Figure 18