Table 1.
Functional properties of partial agonists and AgoPAMs in human, rat, and mouse GPR40 IP accumulation assays.
Fig 1.
Chemical structures and measurement of IP accumulation in stable cell lines expressing human, rat, or mouse GPR40.
(A) Chemical structures of partial agonists MK-8666, MK-2305, TAK-875, AMG-837; and AgoPAMs AM-1638, AP1, and AP3. Dose-response curves for partial agonists (MK-8666, MK-2305, TAK-875, and AMG-837) and AgoPAMs (AM-1638, AP1, and AP3) were generated in human GPR40/CHO-K1 (B), rat GPR40/HEK293 (C), or mouse GPR40/HEK293 (D) cells. Data are expressed as a percentage of the control response of an in-house partial agonist and fitted to a standard 4-parameter non-linear regression model. EC50’s were determined using a custom in-house developed software package. Each compound was profiled multiple times (n = 2–20) with representative graphs shown. The mean parameters of these are shown in Table 1.
Table 2.
Summary of [3H]L358 and [3H]AP9 ([3H]25,) binding to WT human GPR40.
Each compound was profiled multiple times (n = 2–13). N.A. specifies that a compound augmented the binding of specified radioligand in line with the positive cooperativity between the partial agonist and AgoPAM binding sites.
Fig 2.
Acute treatment with GPR40 partial agonists and AgoPAMs in GK rats.
One hour post-acute treatment with vehicle, TAK-875 at 100 mg/kg, AP1 at 30 mg/kg, or AP3 at 10 mg/kg, blood glucose dropped to its lowest levels (A) at the same time insulin peaked (B) in GK rats. All compounds were dosed at maximal efficacious doses. Acute treatment with the AgoPAMs produced a significant 24 hr increase in glucagon, while the glucagon increase of TAK-875 was not significant and was of a shorter duration (C). Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * P < 0.05 vs. Vehicle, + P < 0.05 vs. TAK-875.
Fig 3.
GK rat glucose, body weight, and food intake are affected by 28 days treatment with AgoPAMs or partial agonist.
All compounds were dosed at maximal efficacious doses. Fed blood glucose levels were significantly reduced by GPR40 AgoPAMs compared to vehicle controls on days 10 through 28 of treatment, but only on day 28 by the partial agonist (P < 0.05, A). On day 28 of study, fed and fasted blood glucose was significantly reduced with GPR40 partial and AgoPAM treatments compared to vehicle controls (P < 0.05, B). Additionally, AP1 significantly reduced fed blood glucose compared to the partial agonist (P < 0.05, B). Decreased levels of food intake were observed with AgoPAM treatment days 10 through 28 (P < 0.05), while the partial agonist did not reduce food intake until day 17 (P < 0.05, C). The effects of AgoPAMs on food intake were associated with decreases in body weight (P < 0.05, D) whereas MK-8666 had no effect on body weight. Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * P < 0.05 vs. Vehicle, + P < 0.05 vs. MK-8666.
Fig 4.
GK rat incretin hormones are affected by 28 days treatment with AgoPAMs or partial agonist.
Both AgoPAMs significantly increased total (A) and active (B) GLP-1 throughout the 4 weeks of treatment. Other gut hormones, GIP (C) and PYY (D), were significantly elevated by the AgoPAM treatments. In contrast, MK-8666 had no effect on gut hormones. Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * P < 0.05 vs. Vehicle, + P < 0.05 vs. MK-8666.
Fig 5.
GK rat glucagon but not insulin affected by 28 days treatment with AgoPAMs or partial agonist.
Little effect on insulin was seen in any treatment group (A). Treatment with AgoPAMs caused a significant and sustained increase in glucagon, while MK-8666 showed a trend of increasing glucagon at 28 days (B). Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * P < 0.05 vs. Vehicle; + P < 0.05 vs. MK-8666.
Fig 6.
The effects of GPR40 ago-PAMs on glucagon release require GPR40.
In vivo, treatment of mice with AgoPAMs AP1 or AP3 increase circulating glucagon in WT but not GPR40 -/- mice at 1h post dose. Ex-vivo, treatment of islets with GPR40 AgoPAMs, AP1 and AP3, but not MK-8666 significantly elevated glucagon secretion from WT islets (B1). Such sustained effect was abolished in GPR40-/- islets (B2). Data are mean ± SEM with analysis via ANOVA followed by Dunnett’s posttest. * P, #P < 0.05 vs. Vehicle.
Fig 7.
GLP-1R-/- mice vs. wild type mice during an IPGTT.
After treatment with MK-2305 at 10 mg/kg or AP1 at 30 mg/kg and a dextrose challenge, blood glucose excursion was reduced to similar levels in both the knockout and wild type mice compared to the respective vehicle groups (A, B). AP1 administered at a maximally efficacious dose increased insulin following the dextrose challenge in both knockout and wild type mice, but a much greater effect was seen in the wild type mice (C, D). The partial agonist MK-2305 administered at a maximally efficacious dose showed no effect in insulin for either the knockout or wild type mice. Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. ^ P < 0.05 vs. GLP-1R-/- Vehicle; * P < 0.05 vs. WT Vehicle; + P < 0.05 vs. GLP-1R-/- AP1.
Fig 8.
Total endogenous glucose production (EGP) and contributing fluxes in GK rats following chronic treatment with GPR40 partial agonist (MK-8666) or AgoPAMs (AP1 and AP3).
Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * P < 0.05 vs. Vehicle; + P < 0.05 vs. MK-8666.
Fig 9.
Skeletal muscle 13C-glycogen levels following a 13C-GTT in GK rats chronically treated with GPR40 partial agonist (MK-8666) or AgoPAM (AP1).
Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * P < 0.05 vs. Vehicle; + P < 0.05 vs. MK-8666.