Combination of cmpd1 with insulin in normoglycemic mice extends glucose lowering duration of action but does not increase hypoglycemia risk.

<p>(A) Glucose lowering in normoglycemic C57BL/6 mice. Cmpd1 (10 or 30 mg/kg) and insulin (1 or 3 U/kg; only 3 U/kg data are shown) were dosed at time 0. Glucose was measured hourly for 4 hours. ** p<0.01 and *** p<0.001 vs. vehicle; # p<0.05 and ## p<0.01 vs. insulin alone. The data are means ± SEM with n = 8 in each group. Individual animal glucose values and their means of 3 U/kg insulin alone (B), 3 U/kg insulin plus 10 mg/kg Cmpd1 (C) or 3 U/kg insulin plus 30 mg/kg Cmpd1 (D) are shown. No significant increase of hypoglycemia was detected in the combination groups.</p

Cmpd1 potentiates insulin-mediated glycemic control.

<p>(A) Glucose lowering in STZ-diabetic mice. Cmpd1 (30 mg/kg) and insulin (0.3 or 1 U/kg) were dosed at time 0. Glucose was measured hourly for 4 hours. *p < 0.05 vs. insulin 1 U/kg. (B) Glucose AUC (0–4 h) calculated according to data in (A). *p < 0.05 and **p<0.01 vs. comparators as indicated. (C) Glucose lowering in db/db mice were conducted similarly as in (A). Cmpd1 (30 mg/kg) and insulin (0.6 or 2 U/kg) were dosed and AUC (0–4 h) calculated accordingly. **p<0.01 and ***p<0.001 vs. comparators as indicated. (D) Glucose tolerance test in normoglycemic mice. Cmpd1 (30 mg/kg) was dosed i.p.at—30 minutes and 5 g /kg glucose (p.o.) and insulin (0.5 U/kg; i.p.) were administered at time 0. Glucose was measured at specified time points up to 120 min. *p < 0.05 and **p<0.01 vs. insulin. The data are means ± SEM with n = 8 in each group.</p

Cmpd1 has differential effects on insulin signaling in diabetic and normoglycemic tissues.

<p>Mice were fasted for 4 hours and received Cmpd1 i.p. and 1 U/kg insulin administration at time 0. 30 min post injection, liver and gastrocnemius muscle were collected to examine insulin signaling. Tissue lysate phospho-protein analysis was conducted using phosphor-IR (Y1150/1151) from Cell Signaling Technology and phospho-Akt (Ser473) Assay kit from Meso Scale Technology. Fold changes of compound treated vs. basal untreated samples were calculated for pIR normoglycemic mice (A), pIR diabetic mice (B), pAkt normoglycemic mice (C) and pAkt diabetic mice (D). Basal liver pIR and pAkt levels in STZ-diabetic mice were ~3 fold higher than that in normoglycemic mice but overall magnitude of stimulation by insulin or Cmpd1 insulin combination are comparable in the two models. Basal muscle pIR and pAkt levels are similar in the two models. Results shown are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. comparators as indicated; n = 4 per condition.</p

<i>In vivo</i> inhibition of hepatic ¹²⁵I-glucagon binding in the hGCGR mouse following (A) acute and (B) chronic dosing with GRA1.

<p>The data are mean (±SEM) percent reductions in liver ¹²⁵I-glucagon content measured (A) 1, 3, 5, and 8 h after a single oral dose of 3 mg/kg GRA1, and (B) after treatment for 30 days with control diet or food/drug admixtures that provided 3, 6, 10, or 30 mg/kg·day GRA1. Pharmacokinetic analysis performed during the experiment in (A) determined that mean plasma GRA1 concentrations were 0.5, 0.6, 0.5, and 0.7 µM at 1, 3, 5, and 8 h postdose, respectively.</p

GRA1 lowers glucose in hGCGR HFD/STZ mice and further enhances the efficacy of a DPP-4 inhibitor.

<p>(A) Mean (±SEM) blood glucose hGCGR HFD/STZ mice treated with a single dose of 1, 3, or 10 mg/kg GRA1. (B) Non-fasted blood glucose concentrations in hGCGR HFD/STZ mice treated for 6 weeks with 10 mg/kg·day GRA1, 200 mg/kg·day des-fluoro-sitagliptin (des-F-sita), or the two agents in combination. (Additional data from this study are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049572#pone-0049572-t004" target="_blank">Table 4</a>.) *p<0.05, **p<0.01, and ***p<0.001 versus HFD/STZ controls; ^#non-significant (p>0.05) versus non-STZ controls; n = 8–15 animals per group.</p

Plasma and tissue measurements from hGCGR HFD/STZ mice treated for 6 weeks with 10 mg/kg GRA1, des-fluoro-sitagliptin (des-F-sita), or GRA1 and des-fluoro-sitagliptin in combination.

a<p>Fasting blood glucose and HbA1c were measured after 5 weeks of treatment; all other measurements were made in terminal plasma and necropsy tissue. The data are means ± SEM; *p<0.05, **p<0.01 and ***p<0.001 for comparisons made with the hGCGR HFD/STZ group fed the control (drug-free) diet.</p

The diabetic phenotype of the hGCGR.<i>ob/ob</i> mouse.

<p>The data are means ± SEM. In all comparisons, the difference between hGCGR.<i>ob/ob</i> mice and littermate controls was significant at p<0.001.</p

Fasted plasma glucagon, glucose and amino acids in rhesus monkeys treated once daily with vehicle or 30 mg/kg GRA1.

<p>Data expressed as mean ± SEM; *p<0.05, **p<0.01 and ***p<0.001 vs. vehicle-treated animals at indicated days.</p

Plasma measurements in DIO hGCGR mice treated for 10 weeks with GRA1 administered as a food admixture.

<p>All measurements were made in terminal plasma. Data are expressed as mean ± SEM; *p<0.05 and **p<0.01 in comparisons with the group on the control diet.</p

Genes involved in amino acid and glucose metabolism that were expressed differentially in rhesus monkey liver depending or whether animals received 30 mg/kg GRA1 or vehicle (yogurt without drug) twice daily for 6 days (n = 5 per group).

<p>ns = not significant (p≥0.05).</p><p>The data are expressed as fold differences between treatment groups with negative values indicating reduced expression in animals treated with GRA1.</p>*<p>p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001.</p