Dihydromyricetin Improves Insulin Resistance Via PPARγ/FGF21/AMPK Signaling Pathway

Diabetes (diabetes mellitus, DM) has become a serious global public health issue impacting human health. Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, its main characteristic was insulin sensitivity of insulin peripheral target tissue such as skeletal muscle, namely insulin resistance (IR). IR exists during the occurrence and development of T2DM, which is also the "power" of various complications of diabetes. Clinically, it is common to use oral glucose-lowering drugs such as rosiglitazone TZDs to improve IR, promote fat storage, water sodium retention and so on. However, long time using will lead to obvious side effects, thus, its clinical use is greatly limited. In recent years, finding a natural new type of hypoglycemic drugs with non-toxic side effects from dietary phytochemicals has become one of the main development direction of insulin sensitization agent class of T2DM treatment drugs.A lot of studies have shown that phytochemicals from diet has significant effect on health protection, and they are potential candidates in drug research and development. Several flavonoids widely existed in fruits and vegetables, such as luteolin, kaempferol, quercetin and so on, has been found to lower blood glucose, increase insulin sensitivity and regulate glucose metabolism. Dihydromyricetin (DHM), also known as ampelopsin (AMP), is a kind of flavonoids with high level of food and drug plant rattan tea (also called mildewed tea). DHM content in tender stem leaf (by dry weight)is over 20%. Rattan tea are widely distributed in southern mountainous area,such as guangxi, yunnan, guizhou, hunan and hubei,province. According to the survey, in China, zhuang and yao minorities have made tender stem leaf to health protection tea for a long time, its security has been fully confirmed. DHM’s molecular structure is similar to quercetin and luteolin, which has been confirmed with many kinds of pharmacological therapy efficacy such as antioxidant, liver protecting, anti-tumor and so on. Animal experiment reported that DHM can reduce the blood glucose of diabetes mice induced by alloxan, however, its specific mechanism is unclear yet.Peroxisome proliferator-activated receptor-gamma (PPARy) is a member of type Ⅱ nuclear receptor superfamily, which is related closely to the body’s glucolipid metabolic regulation. In Clinical, the common use drug rosiglitazone TZDs, a kind of oral glucose-lowering drug, is insulin sensitization agent for PPARy, which can effectively improve IR. Studies have shown that in fat cells, the PPARy agonist can induce the expression of fibroblast growth factor-21 (FGF-21), thus further increase the activity of PPARy, and FGF-21 can activate AMP dependent protein kinase (AMPK), enhance the capacity of mitochondrial function and oxidation, thus improve the glucose tolerance. Another team of our laboratory found that DHM can activate AMPK promoted mitochondria generation, thus improve physical decline in rats with acute low hypoxia condition. So we speculate that rattan tea extract and DHM may raise the expression of FGF2 through activation of PPARy, that is activate AMPK signaling pathways to achieve the improvement of insulin resistance.This study firstly establish T2DM rats model by high-fat feeding SD rats and in combination with low dose of Streptozocin (STZ) with intraperitoneal injection of STZ (30 mg/kg). After successful model, T2DM rats were randomly divided into diabetic model control group (DIA), metformin (MET,125 mg/kg), DHM intervention group(DHM,100 mg/kg), low doses of rattan tea extract intervention group (R50,50 mg/kg), middle dose of intervention group (R100,100 mg/kg) and high dose of intervention group (R200,200 mg/kg) 6 group. Each group of rats were intervened by gastric drug delivery after 4 weeks, we measured insulin, insulin C peptide in rats serum by radiation immunity analysis method; pAkt, FGF21 pIRS-1, pAMPK, PPAR gamma protein expression in muscle tissue by western blot; FGF21, PPAR gamma mRNA expression in muscle tissue by qRT-PCR method, observing the effect of the rattan tea extracts improving insulin resistance. Secondly, we established insulin resistance vitro model by palmitic acid(PA) induced L6 skeletal muscle myoblast, and further studied the effect of improving insulin resistance by DHM and its molecular mechanism, which seeking a new strategy for preventing and treating diabetes, and providing experimental basis for the research of new hypoglycemic drugs.The main results and conclusions:In vivo:rattan tea extract and DHM can improve diabete rats’ muscle fiber atrophy, degeneration of inflammatory and so on. Compared with DIA group, metformin, DHM and rattan tea extract intervention group’s blood glucose, serum insulin levels, and HOMA-IR index are decreased significantly (P<0.05), while, insulin and insulin c-peptide were increased significantly (P<0.05); In muscle tissue, pAkt, pAMPK pIRS-1, FGF21 and PPARγ protein expression level were up-regulated. At the same time, expression of FGF21 and PPARγ mRNA were increased (P< 0.05), suggesting rattan tea extract and DHM can significantly improve the insulin resistance of diabetes rats induced by STZ.In vitro:(1) DHM increased PA induced insulin resistance L6 cell’s ability of glucose uptake dose and time dependently, and up-regulated expression of the insulin signaling pathway related proteins pAkt,and pIRS-1, prompting DHM can effectively improve the insulin resistance of skeletal muscle cells induced by PA.(2) DHM activated AMPK dose and time dependently. AMPK specific inhibitor Compound C and AMPK siRNA significantly inhibited DHM’s activation of AMPK and reduced the insulin resistance L6 cells’ enhancement of glucose uptake ability,up-regulation of pAkt and IRS-1 showing that the improvement of insulin resistance L6 cells mediated by DHM may related to its activation of AMPK.(3) DHM increase the expression of FGF21 dose and time dependently in insulin resistance L6 cells, FGF21 siRNA inhibited activation of DHM to AMPK and reduced the glucose uptake ability enhancement and up-regulation of pIRS-1 and pAkt in insulin resistance L6 cells mediated by DHM, prompting DHM activates AMPK by raising the expression of FGF21, which plays a role in improving insulin resistance of L6 skeletal muscle cells.(4) DHM increased the expression of PPARγ dose and time dependently,while PPARγ GW6992 specific inhibitors and PPARγ siRNA could inhibit DHM mediated up-regulation of FGF21 and activation of AMPK,inhibit the increasing expression of pAkt and pIRS-1, then further reduce DHM enhanced glucose uptake ability of insulin resistance L6 cells, indicating that DHM raised FGF21 expression by activation of PPARγ.From what has been discussed above, DHM’s function of improving insulin resistance is related to the activation of PPARy and regulation of FGF21/AMPK signaling pathways.