Figure 1.
Activation of human AMPK heterotrimers by ZLN024.
(A) The structure of ZLN024 (molecular weight = 325.2). (B) The activation curve for ZLN024 and recombinant human AMPK α1β1γ1 (n = 2), α2β1γ1(n = 2) (C), α1β2γ1(n = 3) (D) and α2β2γ1(n = 3) (E). (F) ZLN024 concentration-dependently stimulates AMPK and ACC phosphorylation in L6 myotubes after incubation for 3 hr. The ratio of the phosphorylation level to the protein level of AMPK and ACC was determined (n = 3). (G) The time course of the stimulation of AMPK and ACC phosphorylation by ZLN024 (20 µmol/l). Metformin (2 mmol/l) was used as a positive control. The ratio of the phosphorylation level to the protein level of AMPK and ACC was determined (n = 3). #, P<0.1,*, P<0.05, **, P<0.01 compared with the untreated control.
Figure 2.
ZLN024 activates human AMPK α1 subunits and inhibits AMPK dephosphorylation by PP2Cα.
(A) The activation curve for the effect of ZLN024 on the activity of the AMPKα1 truncations (1–394) (n = 2), (B) α1 (1–335) (n = 2) and (C) α1 (1–312) (n = 2). (D) The effect of ZLN024 on PP2Cα (n = 3). (E) ZLN024 inhibits AMPK α1β1γ1 (n = 5) and AMPK α2β1γ1 (n = 3) (F) dephosphorylation by PP2Cα. The ratio of the phosphorylation level to the protein level of AMPK was determined. NC = negative control, PP2Ca was not added. (G) The additive activation of AMPK α2β1γ1 by different concentrations of ZLN024 and 12.5 µmol/l AMP (n = 2). (H) The additive activation of different concentrations of AMP and 10 µmol/l ZLN024 (n = 2). #, P<0.1, *, P<0.05, **, P<0.01 compared with the untreated control.
Figure 3.
ZLN024 has no effect on the ADP/ATP ratio in L6 myotubes and requires an upstream kinase.
(A) ZLN024 did not affect the mitochondrial membrane potential after incubation for 3 hr in L6 myotubes; CCCP (10 µmol/l) was used as a positive control. (B) ZLN024 did not change the ADP/ATP ratio after incubation for 3 hr in L6 myotubes, CCCP (10 µmol/l) was used as a positive control (n = 3). (C) ZLN024 stimulates AMPK and ACC phosphorylation in HeLa cells in which only CaMKKβ acts as an AMPK upstream kinase. ZLN024 was incubated for 3 hr with A-769662 (20 µmol/l) as a positive control. The ratio of the phosphorylation level to the protein level of AMPK and ACC was determined (n = 2). (D) The time course of the stimulation of AMPK and ACC phosphorylation by ZLN024 (20 µmol/l). A-769662 (20 µmol/l) was used as a positive control. The ratio of the phosphorylation level to the protein level of AMPK and ACC was determined (n = 2). (E) AMPK and ACC phosphorylation by ZLN024 is blocked by the CaMKKβ inhibitor STO609. HeLa cells were incubated with ZLN024 (20 µmol/l) for 3 hr with or without prior treatment with STO-609 (10 µg/ml) for 30 min. #, P<0.1, *, P<0.05, **, P<0.01 compared with the untreated control.
Figure 4.
ZLN024 stimulates glucose uptake and fatty acid oxidation in L6 myotubes.
(A) ZLN024 increases glucose uptake in L6 myotubes after incubation for 3 hr. Insulin (100 nmol/l) was added for the final 30 min of the experiment (n = 4). (B) ZLN024 increases fatty acid oxidation in L6 myotubes after incubation for 4 hr. AICAR (1 mmol/l) was used as a positive control (n = 3). (C) Effects of AMPK α1/α2-DN adenovirus expression on ACC phosphorylation and glucose uptake (D) caused by ZLN024 (20 µmol/l) (n = 4). #, P<0.1, *, P<0.05, **, P<0.01 compared with the untreated control.
Figure 5.
The effects of ZLN024 in rat primary hepatocytes.
(A) Concentration response of the effects on AMPK and ACC phosphorylation due to ZLN024 treatment for 3 hr in rat primary hepatocytes. (B) ZLN024 did not change the ADP/ATP ratio after incubation for 3 hr in rat primary hepatocytes, CCCP (1µmol/l) was used as a positive control (n = 3). (C) Inhibition of insulin-stimulated (10 nmol/l) fatty acid synthesis by 24 hr of exposure to ZLN024. (D) The effects of treatment with ZLN024 for 21 hr on the gene expression of FAS (n = 3). (E) AMPK α1/α2-DN adenovirus expression reversed ACC phosphorylation and fatty acid synthesis (n = 2) (F) caused by ZLN024 (20 µmol/l) effectively for 24 hr treatment (n = 2). *, P<0.05, **, P<0.01 compared with the untreated control.
Figure 6.
Chronic effects of ZLN024 in db/db mice.
Eight-week-old male db/db mice were gavaged with vehicle (0.5% methylcellulose), ZLN024 (15 mg/kg/day) or metformin (250 mg/kg−/day) (n = 6–8). (A) Blood glucose levels after an intra-peritoneal glucose load (1.5 g/kg) performed after 4 weeks of treatment. The AUC were used as an indicator of glucose clearance. White circles, vehicle; black circles, ZLN024; white squares, metformin. (B) Amount of liver tissue in proportion to body weight (wt/wt) after 5 weeks of treatment. (C) Liver triacylglycerol level after 5 weeks of treatment. (D) Liver total cholesterol level after 5 weeks of treatment. (E) Relative anabolic gene expression levels in the liver. (F) Relative catabolic gene expression levels in the liver. (G) Relative gene expression levels in abdominal muscle. (H) AMPK and ACC phosphorylation levels in the abdominal muscle. (I) AMPK and ACC phosphorylation levels in the liver. *, P<0.05, **, P<0.01 compared with vehicle.