DMC (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone) improves glucose tolerance as a potent AMPK activator.

OBJECTIVE: We investigated the effect and regulatory mechanism of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) isolated from Cleistocalyx operculatus on metabolic parameters in myotubes, adipocytes and an obese mouse model. MATERIALS AND METHODS: Myotubes and adipocytes were incubated with or without DMC. Glucose uptake, fatty acid oxidation, AMPK activation and adipocytes differentiation were investigated. To examine in vivo effect of DMC, 30mg/kg/day DMC was administered by oral gavage for 2weeks in high fat fed C57BL/6 male mice and intra-peritoneal glucose tolerance test was performed. In order to examine whether DMC directly activates AMPK, we performed cell free AMPK assay and surface plasmon resonance spectroscopy analysis. RESULT: DMC increases glucose uptake and fatty acid oxidation (FAO) in myotubes. Also, DMC inhibits adipocyte differentiation in 3T3-L1 cells. Interestingly, DMC stimulates phosphorylation of AMP-dependent protein kinase (AMPK) alpha subunit (T172) by directly binding to AMPK, which results in the activation of AMPK. Furthermore, DMC binds AMPK with a higher affinity than AMP. When AMPK was knocked down, the stimulatory effect of DMC on FAO and its inhibitory effect on adipogenesis were abolished. These results suggest that the effects of DMC were primarily mediated by AMPK activation. In addition, treating mice fed a high fat diet with DMC improved glucose tolerance and significantly increased FAO of the muscles. CONCLUSION: DMC, as a novel AMPK activator, shows anti-diabetic effects in cell culture systems, such as myotubes and adipocytes, and in a diet-induced obese mouse model.

BNIP3 is essential for mitochondrial bioenergetics during adipocyte remodelling in mice.

AIMS/HYPOTHESIS: Adipose tissue is a highly versatile system in which mitochondria in adipocytes undergo significant changes during active tissue remodelling. BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is a mitochondrial protein and a known mitochondrial quality regulator. In this study, we investigated the role of BNIP3 in adipocytes, specifically under conditions of peroxisome proliferator-activated receptor-γ (PPARγ)-induced adipose tissue remodelling. METHODS: The expression of BNIP3 was evaluated in 3T3-L1 adipocytes in vitro, C57BL/6 mice fed a high-fat diet and db/db mice in vivo. Mitochondrial bioenergetics was investigated in BNIP3-knockdown adipocytes after rosiglitazone treatment. A putative peroxisome proliferator hormone responsive element (PPRE) was characterised by promoter assay and electrophoretic mobility shift assay (EMSA). RESULTS: The protein BNIP3 was more abundant in brown adipose tissue than white adipose tissue. Furthermore, BNIP3 expression was upregulated by 3T3-L1 pre-adipocyte differentiation, starvation and rosiglitazone treatment. Conversely, BNIP3 expression in adipocytes decreased under various conditions associated with insulin resistance. This downregulation of BNIP3 was restored by rosiglitazone treatment. Knockdown of BNIP3 in adipocytes inhibited rosiglitazone-induced mitochondrial biogenesis and function, partially mediated by the 5' AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor γ, co-activator 1 α (PGC1α) signalling pathway. Rosiglitazone treatment increased the transcription level of Bnip3 in the reporter assay and the presence of the PPRE site in the Bnip3 promoter was demonstrated by EMSA. CONCLUSIONS/INTERPRETATION: The protein BNIP3 contributes to the improvement of mitochondrial bioenergetics that occurs on exposure to rosiglitazone. It may be a novel therapeutic target for restoring mitochondrial dysfunction under insulin-resistant conditions.

4-Deoxypyridoxine improves the viability of isolated pancreatic islets ex vivo.

The successful islet transplantation, for the treatment of type 1 diabetes, depends on the quantity and the quality of transplanted islets. Previously, it has reported that the significant loss of isolated islet mass could be prevented by sphingolipid metabolite, sphinogosine 1-phophate (S1P). This study was performed to elucidate whether the beneficial effects of S1P maintaining isolated pancreatic islets ex vivo are mimicked by modulation of intracellular S1P. We tested the in vitro effect of various agents that modulate intracellular S1P levels in insulinoma cell lines and isolated islets to compare their anti-apoptotic effects with that of S1P. As results, we discovered that 4-deoxypyridoxine (DOP), which inhibits the degradation of intracellular S1P by inhibiting S1P lyase (SPL) activity, minimized the chemically induced apoptosis of insulinoma cell lines as S1P did. Also, supplementation of DOP in the culture media protected the regression of isolated islets that have been maintained ex vivo at least for 18 h providing the evidence of increasing viability of isolated islets with DOP, which impaired SPL activity. In conclusion, these results suggest that the application of SPL inhibitors could be considered as a supplement for the maintenance of viable islets isolated from donor sources in the process of islet transplantation.

Mechanism for antioxidative effects of thiazolidinediones in pancreatic ß-cells.

Thiazolidinediones (TZDs) are synthetic ligands of peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear receptor superfamily. TZDs are known to increase insulin sensitivity and also to have an antioxidative effect. In this study, we tested whether TZDs protect pancreatic ß-cells from oxidative stress, and we investigated the mechanism involved in this process. To generate oxidative stress in pancreatic ß-cells (INS-1 and ßTC3) or isolated islets, glucose oxidase was added to the media. The extracellular and intracellular reactive oxygen species (ROS) were measured to directly determine the antioxidant effect of TZDs. The phosphorylation of JNK/MAPK after oxidative stress was detected by Western blot analysis, and glucose-stimulated insulin secretion and cell viability were also measured. TZDs significantly reduced the ROS levels that were increased by glucose oxidase, and they effectively prevented ß-cell dysfunction. The antioxidative effect of TZDs was abolished in the presence of a PPARγ antagonist, GW9662. Real-time PCR was used to investigate the expression levels of antioxidant genes. The expression of catalase, an antioxidant enzyme, was increased by TZDs in pancreatic ß-cells, and the knockdown of catalase significantly inhibited the antioxidant effect of TZDs. These results suggest that TZDs effectively protect pancreatic ß-cells from oxidative stress, and this effect is dependent largely on PPARγ. In addition, the expression of catalase is increased by TZDs, and catalase, at least in part, mediates the antioxidant effect of TZDs in pancreatic ß-cells.