Dihydromyricetin Ameliorates Insulin Resistance By Down-regulating The Phosphorylation Of PPAR?

Diabetes is generally recognized as one of the non-communicable diseases(NCD)and is a serious harm to human health worldwide.The prevalence of type2 diabetes remains increasing year by year in China.Insulin resistance is the most important pathogenesis of type2 diabetes.Therefore,the clinical prevention and treatment of type2 diabetes focus on improving insulin resistance.Natural plant extracts have many bioactivities,such as anti-inflammatory,anti-oxidant and anti-cancer.Flavonoids,one of the phytochemicals,are reported to decrease blood glucose with fewer side effects and have been widely used in the prevention and treatment of many diseases for decades.The anti-diabetes effect of the flavonoids has attracted widespread attention and research due to their beneficial effects.Although the mechanisms remain unclear.Peroxisome proliferator-activated receptor(PPAR)-? is a main regulator involved in glucose homeostasis and lipids metabolism and has been targeted to treat diabetes for decades.Rosiglitazone(ROSI),one of the thiazolidinediones(TZDs),improves insulin sensitivity potently.However,due to the full agonism of PPAR?,ROSI also promotes adipogenesis and causes many serious side effects.So,it's better to modulate than fully activate PPAR?.Researches found that many flavonoids improved insulin sensitivity as partial agonists of PPAR? without causing adipogenesis and weight gain.Dihydromyricetin(DHM),one of the flavonoids,possesses a broad spectrum of biological and pharmacological properties such as anti-inflammatory,anti-oxidant and anti-cancer,but the mechanism of its anti-diabetic bioactivity is yet to be elucidated.Research found that the phosphorylation of PPAR? was closely associated with insulin resistance.PPAR? ligands exert the anti-diabetic effect through inhibiting the phosphorylation of PPAR? at Ser 273.Proteomics researches found that both extracellular regulating kinase(ERK)and cyclin-dependent kinase(CDK5)could directly phosphorylate serine273 of PPAR? and subsequently induce obesity-linked insulin resistance.The activity of ERK is promoted in obese rodents,while the insulin sensitivity is increased in ERK-deficient mice.Inhibition of MEK/ERK signaling pathway could significantly improve insulin sensitivity and maintain glucose homeostasis.Previous researches found that many flavonoids such as quercetin,luteolin and kaempferol could down-regulate MEK/ERK pathway to induce the apoptosis of cancer cells.Based on the theory of structure-effect relation and the preliminary result of molecular docking finding that DHM could bind PPAR? to the ligand-binding domain(LBD).Therefore,we presume that DHM ameliorates insulin resistance by down-regulating phosphorylation of PPAR? through inhibiting the MEK/ERK pathway.Meterials and methods:There are two main parts involved in the present study: in vivo and in vitro experiments.We used the Zucker Diabetic Fatty rats as the diabetic animal model in vivo experiments and differentiated 3T3-L1 cells as adipocytes in vitro experiments.1.There are six groups in animal experiments: healthy control(Zucker Lean rats,ZL),diabetic control(ZDF control),DHM(50 mg/kg),DHM(100 mg/kg),DHM(200 mg/kg)and Rosiglitazone(4 mg/kg,ROSI).Animals were given the dose of 2ml water or DHM or ROSI daily by gavage.Body weight was measured every other day.Food intake and fasting blood glucose,insulin and glucagon levels were monitored every week.At 0,4,8 weeks of experiment,serum lipid profile,adiponectin and FGF21 levles were determined.At week 7 and week 8,oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)were performed respectively.2.At week 7,body compositions were evaluated by Quantum FX microCT Imaging System.Quantitative analysis,reconstruction,and three-dimensional visualization of adipose tissues were performed with Analyze 12.0 software3.At the end of experiment,animals under anesthesia were sacraficed.Livers,pancreas and adipose tissues were collected.Oil red O,hematoxylin & eosin(HE)and immunohistochemical staining were performed to explore the protective effects of DHM on glucose metabolic organs.Western Blot was performed to determine PPAR? protein and phosphorylation.4.We used dexamethasone(DEX)to induce insulin resistance of differentiated 3T3-L1 adipocytes and detected the effects of DHM on adipogenesis,glucose uptake and adipokines(adiponectin and FGF21)secretion in insulin-resistant adipocytes.5.Western Blot was performed to detect the phosphorylation of PPAR?,CDK5 and ERK.The inhibitor of PPAR?,GW9662 was used to block the activity of PPAR? in order to observe the effects of DHM on the insulin-resistant adipocyte.Furthermore,we used the inhibitor of MEK,PD98059 to block the activity of ERK in order to observe the effect of PD98059 on the insulin-resistant adipocyte and compare with that of DHM.Results:1.DHM decreased fasting blood glucose and improved insulin sensitivity.Compared with ZDF control,the medium and high doses of DHM(100 mg/kg and 200 mg/kg)significantly decreased fasting blood glucose.The low level of DHM(50 mg/kg)maintained the blood glucose level under 10 mM until week 7.The result of OGTT showed that after glucose administration thirty minutes,the blood glucose levels of all three doses of DHM remained significantly lower than the ZDF control.At week 8,the result of ITT showed that after thirty minutes of the insulin injection,the glucose levels of the medium and high doses of DHM remained lower than the ZDF control.2.DHM improved the serum lipid profile without causing excessive body weight gain.DHM significantly decreased the serum TG and LDL-C levels while increased HDL-C level of the ZDF rats.The food intake among all groups did not differ significantly.The body weight of DHM treatments decreased by 56-74% compared with ROSI treatment.3.DHM protected the liver and pancreas.DHM decreased the lipid droplet accumulation of hepatocytes in diabetic rats.DHM also maintained the normal construction of hepatic lobules and ameliorated hepatocellular macrovesicularsteatosis.DHM treatment preserved the beta cell mass and insulin content in pancreas islet compared with ZDF control.DHM significantly attenuated renal tubulointerstitial inflammatory cells infiltration and alleviated glomerular mesangial stromal hyperplasia.4.DHM decreased adipose tissue mass,down-regulated the size of adipocyte and promoted adiponectin secretion.Body composition results showed that DHM significantly decreased total abdominal and visceral fat masses compared with ROSI treatment.Oil red O staining results revealed that the adipocyte sizes of both subcutaneous and visceral fat tissues were increased in ZDF control rats.In DHM treated rats,the sizes of both subcutaneous and visceral adipocytes were significantly decreased compared with ZDF control while nonsignificantly different from ZL healthy control.The serum adiponectin level of week 0,4,8 were declined in ZDF control rats,while DHM increased adiponectin secretion significantly.5.Western Blot showed that,both in vivo and in vitro,DHM inhibited the phosphorylation of PPAR? at Ser273 more potently than ROSI.Furthermore,DHM markedly down-regulated the phosphorylation of ERK and CDK5 in adipose tissue and cultured adipocytes.6.DHM dose-dependently inhibited the adipogenesis and improved glucose uptake of 3T3-L1 adipocytes.After established the insulin-resistant adipocyte model using DEX,we found that DHM significantly improved glucose uptake of insulin resistant adipocytes and increased the secretion of adiponectin and FGF21.Intriguingly,the effects of PD98059,the inhibitor of MEK,were similar with that of DHM and these two together can work synergistically to improve the insulin sensitivity of adipocytes.Conclusion:1.DHM decreases fasting blood glucose,ameliorates insulin resistance,reduced lipid accumulation in hepatocyte,improves serum lipid profile and increased pancreas islet mass and insulin content,alleviates renal tubulointerstitial inflammatory cells infiltration and glomerular mesangial stromal hyperplasia.More importantly,the administration of DHM for long term will not cause the excessive body weight gain.2.In vivo,DHM diminishes adipocytes size,reduces adipose tissue masses and increases adiponectin and FGF21 secretion.In vitro,DHM improves the glucose uptake and stimulates the secretion of adiponectin and FGF21 in the insulin-resistant 3T3-L1 adipocytes.3.DHM inhibits PPAR? phosphorylation at Ser273 via MEK/ERK signaling and this mechanism may contribute to its anti-diabetic efficacy.Moreover,the inhibitor of MEK PD98059 and DHM together can work synergistically to improve the insulin sensitivity of adipocytes.4.In summary,our study further revealed the molecular mechanism of the anti-diabetic effect of DHM.For the first time,we propose that DHM inhibits the phosphorylation at Serine 273 of PPAR?.Our study lays a foundation for future researches to explore the preventive and therapeutic utilization of natural phytochemicals,such as DHM,to cope with type2 diabetes.