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. 2013 Apr;62(4):1297-307.
doi: 10.2337/db12-0703. Epub 2012 Dec 18.

Novel small-molecule PGC-1α transcriptional regulator with beneficial effects on diabetic db/db mice

Li-Na Zhang  1 , Hua-Yong Zhou, Yan-Yun Fu, Yuan-Yuan Li, Fang Wu, Min Gu, Ling-Yan Wu, Chun-Mei Xia, Tian-Cheng Dong, Jing-Ya Li, Jing-Kang Shen, Jia Li
Affiliations

Novel small-molecule PGC-1α transcriptional regulator with beneficial effects on diabetic db/db mice

Li-Na Zhang et al. Diabetes. 2013 Apr.

Abstract

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has been shown to influence energy metabolism. Hence, we explored a strategy to target PGC-1α expression to treat metabolic syndromes. We developed a high-throughput screening assay that uses the human PGC-1α promoter to drive expression of luciferase. The effects of lead compound stimulation on PGC-1α expression in muscle cells and hepatocytes were investigated in vitro and in vivo. A novel small molecule, ZLN005, led to changes in PGC-1α mRNA levels, glucose uptake, and fatty acid oxidation in L6 myotubes. Activation of AMP-activated protein kinase was involved in the induction of PGC-1α expression. In diabetic db/db mice, chronic administration of ZLN005 increased PGC-1α and downstream gene transcription in skeletal muscle, whereas hepatic PGC-1α and gluconeogenesis genes were reduced. ZLN005 increased fat oxidation and improved the glucose tolerance, pyruvate tolerance, and insulin sensitivity of diabetic db/db mice. Hyperglycemia and dyslipidemia also were ameliorated after treatment with ZLN005. Our results demonstrated that a novel small molecule selectively elevated the expression of PGC-1α in myotubes and skeletal muscle and exerted promising therapeutic effects for treating type 2 diabetes.

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Figures

FIG. 1.
FIG. 1.
ZLN005 increases expression of the PGC-1α gene in L6 myotubes. L6 myotubes were differentiated for 4–6 days. A: The structure of ZLN005 (molecular weight 250.3). B: Dose-dependent effect of ZLN005 on PGC-1α mRNA levels. L6 myotubes were treated for 24 h with different doses of ZLN005 or 1 mmol/L AICAR as a positive control. C: Time course of ZLN005 on PGC-1α mRNA levels. D: Effect of ZLN005 (10 μmol/L) on relative mRNA levels. E: Dose-dependent effect of ZLN005 on glucose uptake over 24 h. Insulin (100 nmol/L) was added in the last 30 min. F: Dose-dependent effect of ZLN005 on palmitic acid oxidation over 24 h. Radioactive medium containing compounds of interest was changed at the start of the last 4 h. Two millimolar AICAR was also added in the last 4 h. *P < 0.05, **P < 0.01 compared with DMSO.
FIG. 2.
FIG. 2.
ZLN005 has no effect on the expression of the PGC-1α gene in rat primary hepatocytes. A and B: Effects of ZLN005 on PGC-1α and PEPCK mRNA levels. Rat primary hepatocytes were treated for 24 h with different doses of ZLN005 using forskolin (10 μmol/L) as a positive control. C: Effect on glucose output in primary hepatocytes after 20 h of treatment. The glucose production medium was changed 4 h from the end of incubation. *P < 0.05, **P < 0.01 compared with DMSO.
FIG. 3.
FIG. 3.
AMPK is involved in the mechanism of PGC-1α induction in L6 myotubes. A and B: Effects of ZLN005 (10 μmol/L) in L6 myotubes on PGC-1α expression and palmitic acid oxidation after PGC-1α silencing. The controls were transfected with a scramble RNA (NC). C: The wild-type PGC-1α promoter with luciferase reporter or containing mutations in the MEF or CRE sites were transfected into L6 myoblasts and treated with 10 μmol/L ZLN005 after differentiation. Luciferase protein levels were detected by Western blot. The blank represents untransfected L6 that differentiated for the same time period. D: Effects of ZLN005 (10 μmol/L) on p38 mitogen-activated protein kinase, AMPK, and CREB phosphorylation in L6 myotubes at different times by Western blot. AICAR (1 mmol/L) was used as a positive control. E: Effect of ZLN005 on AMPK phosphorylation in L6 myotubes at 24 h by Western blots. F: Influence of compound C (CC) on ZLN005-induced AMPK activation. CC (5 μmol/L) was added 30 min before and during incubation with ZLN005 (20 μmol/L), DMSO, or AICAR (1 mmol/L) for 24 h. G: Influence of CC (5 μmol/L) on ZLN005-induced (20 μmol/L) increase in PGC-1α mRNA levels over 24 h. H: Influence of CCCP (10 μM) and ZLN005 on ADP/ATP ratio for 3 h. I: Effect of CCCP (4 μM) and ZLN005 on muscle mitochondria respiration. *P < 0.05, **P < 0.01 compared with DMSO.
FIG. 4.
FIG. 4.
Chronic effects of ZLN005 on RER in db/db mice. A: Mean plasma concentration time profiles of ZLN005 after a single oral dose (15 mg · kg−1 ) in db/db mice (n = 3). B: Distribution of ZLN005 in tissues harvested after a single oral dose (n = 3). C and D: For RER measurement, 8-week-old db/db mice were gavaged with vehicle (0.5% methylcellulose) or ZLN005 (15 mg · kg−1 · day−1) for 2 weeks. After a 4-h rest, mice were placed in a metabolic chamber and observed over a 21-h period (n = 6–8). Energy expenditure was evaluated by oxygen consumption (Vo2) and carbon dioxide release (Vco2). E and F: Changes in RER and heat throughout the monitoring period (white circle = vehicle, black circle = ZLN005). The adjacent bar graphs represent the average for each group. *P < 0.05, **P < 0.01 compared with vehicle.
FIG. 5.
FIG. 5.
Antidiabetic effects of ZLN005 in db/db mice. Eight-week-old db/db mice were gavaged with vehicle (0.5% methylcellulose), ZLN005 (15 mg · kg−1 · day−1), or metformin (250 mg · kg−1 · day−1) (n = 6–8) and lean mice (wt) were gavaged with vehicle (0.5% methylcellulose) and ZLN005 (15 mg · kg−1 · day−1). A: Body weight. B: Food consumption. C: Random blood glucose. D: Fasting blood glucose. E: Blood glucose levels after an intraperitoneal glucose load (1.5 g · kg−1) performed after 4 weeks of treatment. The areas under the curve are indicators of glucose clearance. F: Blood glucose levels after an intraperitoneal insulin load (1 unit insulin · kg−1) performed after 5 weeks of treatment in db/db mice. The areas under the curve are indicator of insulin clearance. G: Blood glucose levels after an intraperitoneal sodium pyruvate load (1.5 g · kg−1) given after 5 weeks of treatment in db/db mice. The areas under the curve are indicators of pyruvate clearance (white circle = db/db vehicle, black circle = db/db ZLN005, white square = db/db metformin, white triangle = wt vehicle, black triangle = wt ZLN005). *P < 0.05, **P < 0.01 compared with vehicle.
FIG. 6.
FIG. 6.
Chronic effects of ZLN005 in skeletal muscle and liver of db/db mice. A: RT-PCR analysis was used to measure mRNA levels from the gastrocnemius of animals (n = 6–8). B: RT-PCR analysis was used to measure the mRNA levels of the glucose production gene, mitochondrial biogenesis gene, and fatty acid oxidation gene in livers of animals (n = 6–8). C: Mitochondrial DNA copy number of gastrocnemius muscle and liver from ZLN005-treated and untreated db/db mice (n = 6–8). D and E: AMPK and ACC phosphorylation from the abdominal muscle (D) and liver (E) of db/db mice. The ratio of the phosphorylation level to the protein level of AMPK and ACC was determined. *P < 0.05, **P < 0.01 compared with vehicle.
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