PAN-AMPK activator O304 improves glucose homeostasis and microvascular perfusion in mice and type 2 diabetes patients

Abstract

AMPK activated protein kinase (AMPK), a master regulator of energy homeostasis, is activated in response to an energy shortage imposed by physical activity and caloric restriction. We here report on the identification of PAN-AMPK activator O304, which - in diet-induced obese mice - increased glucose uptake in skeletal muscle, reduced β cell stress, and promoted β cell rest. Accordingly, O304 reduced fasting plasma glucose levels and homeostasis model assessment of insulin resistance (HOMA-IR) in a proof-of-concept phase IIa clinical trial in type 2 diabetes (T2D) patients on Metformin. T2D is associated with devastating micro- and macrovascular complications, and O304 improved peripheral microvascular perfusion and reduced blood pressure both in animals and T2D patients. Moreover, like exercise, O304 activated AMPK in the heart, increased cardiac glucose uptake, reduced cardiac glycogen levels, and improved left ventricular stroke volume in mice, but it did not increase heart weight in mice or rats. Thus, O304 exhibits a great potential as a novel drug to treat T2D and associated cardiovascular complications.

Keywords: Cardiovascular disease; Diabetes; Metabolism.

Figure 1. O304 increases p-T172 AMPK in vitro and increases p-T172 AMPK and ATP in cells.

(A and B) Representative immunoblot analysis and quantification of O304 dose-dependent suppression of PP2C-mediated dephosphorylation of p-T172 AMPK in absence (A) (n = 8 per condition) and presence (B) (n = 4 per condition) of 1.0 mM ATP. (C–E) Representative immunoblot analysis (C) and quantification of O304 dose-dependent increase of p-T172 AMPK (D) and p-S79 ACC (E) phosphorylation (n = 11 per condition) in Wi-38 human lung fibroblast cells. (F) Dose-dependent increase in ATP/protein levels in O304-treated Wi-38 human lung fibroblast cells (n = 6 per condition). Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

Figure 2. O304 prevents dysglycemia and insulin resistance in diet-induced obese mice.

(A) Timeline in weeks for B6 mice fed a high-fat diet (HFD) and oral gavaged with vehicle or O304 ± Metformin. (B and C) Fasted glucose (B) and fasted insulin (C) levels in B6 mice on HFD treated with vehicle (n = 10), O304 (n = 10), Metformin (n = 10), and O304+Metformin (n = 10) for 6w. (D) HOMA-IR calculations from B and C. (E) Representative immunoblot analysis and quantification of p-T172 AMPK levels in calf muscle of B6 mice on HFD treated with vehicle (n = 10), O304 (n = 10), Metformin (n = 10), and O304+Metformin (n = 10) for 8w. (F) Relative mRNA levels of Txnip and Glut1 in calf muscle of B6 mice on HFD treated with vehicle (n = 10), O304 (n = 10), Metformin (n = 9), and O304+Metformin (n = 10) for 8w. (G and H) Fasted glucose (G) and insulin (H) levels in B6 mice fed either a regular diet (RD) (n = 40) or a HFD for 7w (=Start; n = 10 + 10). The HFD-fed mice were then continued on HFD and oral gavaged with vehicle (n = 10) or O304+Metformin (n = 10) for an additional 4w. (I) HOMA-IR calculations from G and H. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

Figure 3. O304 prevents diabetes in hIAPPtg diet-induced obese mice.

(A) Timeline in weeks for hIAPPtg mice fed a high-fat diet (HFD) and oral gavaged with vehicle or O304. (B and C) Fasted blood glucose (B) and insulin (C) levels in hIAPPtg mice on HFD treated with vehicle (n = 25) and O304 (n = 27) for 6w. (D and E) Blood glucose, plasma insulin profiles, and AUC, during i.p. glucose tolerance test (IPGTT) (D) and oral glucose tolerance test (OGTT) (E) in hIAPPtg mice on HFD treated with vehicle (IPGTT, n = 13; OGTT, n = 7) and O304 (IPGTT, n = 16; OGTT, n = 7) for 6w. (F) Plasma insulin profiles and AUC during IPGTT of 16w-old, hIAPPtg mice on HFD treated with O304 for 6w (from D, n = 16) compared with that of 10w-old hIAPPtg mice on regular diet (RD) (n = 7). (G) HOMA-IR calculations from glucose and insulin levels from B and C. (H) Matsuda index calculations from IPGTT (D) and OGTT (E) in vehicle and O304-treated hIAPPtg mice. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01 (Student’s t test).

Figure 4. O304 dose-dependently averts dysglycemia in diet-induced obese mice and reverts diabetes in hIAPPtg diet-induced obese mice.

(A) Representative immunoblot analysis and quantification of p-T172 AMPK levels in calf muscle of CBA mice on high-fat diet (HFD) (n = 10) and O304-HFD with 0.4 (n = 5), 0.8 (n = 10), and 2 mg/g (n = 10) O304 for 7w. (B–D) Fasted blood glucose (B) and fasted insulin (C) levels, as well as HOMA-IR (D; from B and C), in CBA mice on HFD (n = 10) and O304-HFD with 0.4 (n = 5), 0.8 (n = 10), and 2 mg/g (n = 10) O304 for 6w. (E) Timeline in weeks for hIAPPtg mice fed HFD for 9w and then either continued on HFD or switched to O304-HFD (2 mg/g in F–H; 0.8 mg/g in I–M) for an additional 7w. (F–H) Fasted blood glucose (F) and insulin (G) levels, as well as HOMA-IR (H; from F and G), in hIAPPtg mice at start, at 9w, and 15w on HFD (n = 10), and in hIAPPtg mice at start, at 9w on HFD, and at 9w HFD+6w O304-HFD (2 mg/g) (n = 12). (I and J) Body weight (I) and body fat (J) change in hIAPPtg mice on HFD for 15w (n = 12) or HFD for 9w + 6w O304-HFD (0.8 mg/g) (n = 7). (K–M) Fasted blood glucose (K) and insulin (L) levels, and HOMA-IR (M; from K and L) at start, 9w, and 15w in hIAPPtg mice on HFD for 15w (n = 12) and in hIAPPtg mice at start, at 9w on HFD, and at 9w + 6w O304-HFD (0.8 mg/g) (n = 7). Data are presented as mean ± SEM, **P < 0.01, **P < 0.01, ***P < 0.001 (Student’s t test [A–D, I, and J]; paired 2-tailed t test.

Figure 5. O304 increases glucose uptake in skeletal muscle.

(A) 2-deoxy-D-glucose (2-DG) uptake in rat skeletal L6 myotubes treated with O304 as indicated (vehicle, n = 8; 2.5 μM O304, n = 6; 5.0 μM O304, n = 6; and 10.0 μM O304, n = 3). (B–D) Representative immunoblot analysis (B), quantification of AMPK expression (C) (n = 6), and 2-DG glucose uptake (D) in siRNA transfected rat skeletal L6 myotubes treated with O304 as indicated (n = 8 for each condition). (E) [¹⁸F]-Fluorodeoxyglucose ([¹⁸F]-FDG) levels in calf and thigh muscle of CBA mice on high-fat diet (HFD) (n = 8) or O304-HFD (2 mg/g) (n = 6) for 2w. Data are presented as mean ± SEM, *P < 0.05, ***P < 0.001 (Student’s t test).

Figure 6. O304 reduces amyloid formation in hIAPPtg diet-induced obese mice and improves arginine-induced insulin secretion in diet-induced obese mice.

Representative images (A) and quantification (B) of Thio-S⁺ amyloid deposits in hIAPPtg mice on high-fat diet (HFD) for 16w (n = 9) and in mice on HFD for 9w and then switched to O304-HFD (2 mg/g) for an additional 7w (n = 9). (C) Representative immunoblot analysis and quantification of O304 stimulation of p-T172 AMPK in INS-1 insulinoma cells (vehicle, 2.5 μM and 5 μM O304, n = 9; 10 μM O304, n = 6, per condition, respectively), mouse primary islets (n = 6 per condition), hIAPPtg mouse primary islets (n = 6 per condition), and human islets (n = 8 per condition). (D and E) Representative images (D) and quantification (E) of Thio-S⁺ amyloid deposits in hIAPPtg islets ex vivo cultured for 96 hours in 11 mM glucose (n = 36 islets), 22 mM glucose (n = 45 islets), and 22 mM glucose with 2.5 μM (n = 42 islets), 5.0 μM (n = 41 islets), and 10 μM O304 (n = 36 islets) as indicated (n = 3 experiments for each ). (F and G) Representative images (F) and quantification (G) of Thio-S⁺ amyloid deposits in hIAPPtg islets ex vivo cultured for 96 hours in 11 mM glucose (n = 43 islets), 11 mM glucose with 5.0 μM 3-MA (n = 59 islets), 22 mM glucose (n = 54 islets), 22 mM glucose with 5.0 μM O304 (35 islets), and 22 mM glucose with 5.0 μM O304 and 5.0 μM 3-MA (n = 44 islets) as indicated (n = 3 experiments for each). (H) Plasma insulin profiles and AUC following i.p. injection of arginine (1 g/kg) in CBA mice fed a HFD (n = 10) and O304-HFD (0.8 mg/g) (n = 10) for 11w. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

Figure 7. O304 reverts established obesity at thermo-neutral conditions.

(A and B) Body weight change over time (A) and food intake (B) in CBA mice switched between high-fat diet (HFD) (n = 5) and O304-HFD (2 mg/g) (n = 5) at housing and thermo-neutral conditions as indicated. (C–E) Oxygen consumption (VO₂) (C), respiratory exchange ratio (RER) (D), and energy expenditure (EE) rates (E) in CBA mice on HFD (n = 8) and O304-HFD (0.8 mg/g) (n = 8) for 11w. (F) Representative immunoblot analysis and quantification of ATGL and of p-S406 ATGL in inguinal white adipose tissue (iWAT) of CBA mice on HFD (n = 10) and O304-HFD with 0.4 (n = 5), 0.8 (n = 10), and 2 mg/g (n = 10) O304 for 7w. (G) Relative mRNA levels of Atgl, Cpt1b, Ppargc1a, and Cox8b in iWAT of 19w-old CBA mice fed HFD (n = 10) and O304-HFD and 2 mg/g O304 (n = 10) for 7w. (H) Relative mRNA levels of Cd36, Fas, Scd1, Acc1, and Cpt1b in brown adipose tissue (BAT) of 19w-old CBA mice fed HFD (n = 10) and O304-HFD and 2 mg/g O304 (n = 10) for 7w. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

Figure 8. O304 reduces heart glycogen and improves stroke volume in diet-induced obese mice but does not cause cardiac hypertrophy.

(A) Heart glycogen content in CBA mice fed high-fat diet (HFD) (n = 10) and O304-HFD with 0.8 (n = 10), and 2 mg/g (n = 10) O304 for 7w. (B) [¹⁸F]-Fluorodeoxyglucose ([¹⁸F]-FDG) levels in heart of CBA mice fed HFD (n = 8) or O304-HFD (2 mg/g) (n = 6) for 2w. (C) Heart weight in CBA mice fed HFD (n = 10) and O304-HFD with 0.8 (n = 10) and 2 mg/g (n = 10) O304 for 7w. (D–F) End-diastolic volume (EDV) (D), end-systolic volume (ESV) (E), and stroke volume (SV) (F) in 16w-old CBA mice fed regular diet (RD) (n = 9) and in 18w-old CBA mice fed a HFD (n = 9) and O304-HFD with 0.8 mg/g O304 (n = 10) or 2 mg/g (n = 10) O304 for 6w. (G) Heart rate (HR) in 16w-old CBA mice fed RD (n = 9), and 18w-old CBA mice fed HFD (n = 9) and O304-HFD with 0.8 (n = 10) mg/g or 2 mg/g (n = 10) O304 for 6w. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test).

Figure 9. O304 improves microvascular blood flow and endurance in mice.

(A and B) Representative laser Doppler image (A) and quantification (B) of peripheral blood fusion in left hind paw in vehicle- (n = 10) and O304-treated (n = 10) B6CBAF1/J (F1) mice on 8w high-fat diet (HFD). (C and D) Endurance test (C) and lactate levels (D) after endurance test in vehicle- (n = 14) and O304-treated (n = 14) aged, lean B6 mice after 30 days of O304 treatment. (E and F) Systolic (E) and diastolic (F) blood pressure in dogs single dosed with vehicle or O304 at indicated concentrations. Data are presented as mean ± SEM, *P < 0.05, ^#P < 0.05, **P < 0.01, ^##P < 0.01, ***P < 0.001, ^###P < 0.001 (Student’s t test). In E and F, * refers to vehicle versus 540 mg/kg O304 and ^# refers to vehicle versus 180 mg/kg O304.

Figure 10. O304 reduces fasting plasma glucose and blood pressure and increases microvascular perfusion in type 2 diabetes (T2D) patients on Metformin.

(A–C) Fasting plasma blood glucose (FPG) (A and B) and HOMA-IR (C) at day 1 and day 28 in placebo- (n = 24) and O304-treated (n = 25) T2D patients on Metformin with the FPG range >7 to <13.3 mmol/l (>126 to <240 mg/dl) at day 1. (D) Hyperemic microvascular perfusion assessed by dynamic T2*-quantification monitored by MRI at screening (MRI1) and at day 27–29 (MRI2) in calf muscle of the T2D patients. The O304 group and the placebo group were split in half based on the time-to-peak (TTP) at baseline, where short TTP (placebo A [n = 14], O304 A [n = 14]) and long TTP (placebo B [n = 13], O304 B [n = 14]) represent a relative higher and lower rate of hyperemic perfusion, respectively. A significant shortening of TTP (P = 0.043) and increase in Δ-T2* (P = 0.034) was observed in subjects with relative lower rate of perfusion at baseline (long TTP) in the O304 group (i.e., comparing O304 B MRI1 with O304 B MRI2) but not in subjects with short TTP, and there was no difference in subjects with either short or long TTP at baseline in the placebo group. (E) Absolute and relative change in systolic and diastolic blood pressure from day 1–28 in T2D patients on Metformin treated with placebo (n = 27) or O304 (n = 30). Data are presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 (Signed Wilcoxon’s rank sum test).