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

Summary of <i>in vivo</i> studies performed in mice.

<p>Summary of <i>in vivo</i> studies performed in mice.</p

Parallel comparison of PTM and CPT1 inhibitor on glucose and lipid metabolism in perfused liver of lean C57BL/6 mice.

<p>PTM and CPT1 inhibitor are used at 100 μM in the perfusion media (n = 3–5 per group). Bars represent means ± SEM. Asterisks denote statistical significance of treatment group compared to control. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Platensimycin inhibits enzymatic activity and expression of fatty acid synthase (FAS) and lipogenic genes.

<p>A: Effect of PTM on FAS enzymatic activity in the liver of eDIO mice treated with PTM (100 mpk, BID, p.o. for 4 days) determined by Malonyl-CoA dependent consumption of NADPH (n = 7–8). B–C: Protein level of FAS in the liver determined by western blot and quantitated by Li-COR. D: Relative RNA levels of FAS, sterol regulatory element-binding transcription factor 1 (SREBP-1c), liver X receptor alpha (LXRα), and carbohydrate-responsive element-binding protein (ChREBP) in the liver determined by quantitative RT-PCR. Bars represent means ± SEM. Asterisks denote statistical significance of treatment group compared to vehicle group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Efficacy of PTM in non-human primates (NHPs).

<p>A–C: Inhibition of <i>de novo</i> lipogenesis (DNL) by PTM in lean cynomolgus monkeys (A) (n = 5 for vehicle and 4 for PTM) and rhesus (B) (n = 4), and lean aged rhesus monkey (C) (n = 6). Animals were dosed with PTM (60 mpk BID p.o. in A and B, 20 and 60 mpk BID s.c. in C and blood samples were collected at 24 hrs post dosing. D: Effect of PTM on DNL of lean rhesus monkeys (60 mpk, p.o.). E–J: Effect of chronic treatment of PTM on body weight (E), fasting glucose levels for predose (F) and 2 hrs post dose at baseline, day 9 and day 22 (G) and insulin (H–I). PTM was dosed at 60 mpk mixed with yogurt for 28 days. J: Liver fat fraction was determined by MRS imaging at baseline and after 28 days of s.c. dosing of PTM (100 mpk, BID) in lean rhesus monkeys. Bars represent means ± SEM. Asterisks denote statistical significance of treatment group compared to vehicle or baseline. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Effect of Platensimycin on body weight gain and food intake in different mouse models.

<p>A–C: Subchronic treatment of PTM in eDIO mice (40, 125, and 400 μg/h, minipump for 10 days) (n = 8). D–E: Chronic treatment of PTM in <i>db/+</i> mice on high fructose diet (3, 10, 30, and 100 mpk in drinking water for 29 days) (n = 8). F–H: Subchronic treatment of PTM in <i>db/db</i> mice for 16 days (3, 10, and 30 mpk, BID, p.o. for 16 days). Bars represent means ± SEM. Asterisks denote statistical significance of treatment group compared to vehicle group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.</p

Schematic diagram of FGF21 effects in adipocytes or white adipose tissue.

<p>Schematic diagram of FGF21 effects in adipocytes or white adipose tissue.</p

Plasma levels of secreted proteins identified in WAT by Affymetrix microarray profiling.

<p>C57BL/6 mice on chow diet were treated with a single dose of vehicle or PEG30-FGF21 Q108 at one of three doses (0.25, 0.75, or 2.5 mg/kg). Plasma Ccl11, Cxcl2, Igf1, Il1rn, and Kitl levels were measured 24 hours and 48 hours post dose (Table S8). Error bars denote standard error. Asterisks denote significant change compared to vehicle treatment within each time point (students t-test p value < 0.05*, p<0.01**, p<0.001***). Arrows on far right denote effects of PEG-FGF21 Q108 at 2.5 mg/kg at the RNA level after 24 hours of treatment (2 day time point) in WT chow-fed mice (IWAT).</p