The Effects And Mechanisms Of Dihydromyricetin And Supplementary Sangzhi Alkaloids On Lowering Blood Glucose

Background:Type 2 diabetes(T2DM)is a chronic metabolic disease characterized by hyperglycemia and insulin resistance,and maintaining blood glucose balance is the primary goal of T2DM treatment.Non-Camellia teas refer to the plants with a long history of application that are not in Camellia genus of Theaceae family and are still used as tea drinks in folk so far.Modern pharmacological studies have shown that nonCamellia teas has effects such as lowering blood glucose and lipid levels,making them important plant resources for the preventing chronic metabolic diseases.Vine tea,also known as Ampelopsis grossedentata(Hand.-Mazz.)W.T.Wang,is type of nonCamellia tea with various pharmacological activities,including good hypoglycemic effects.Its main active ingredient is dihydromyricetin(DHM),which as a nutritional supplement is already being used in the United States and has entered the phase II clinical trials for T2DM treatment in Mexico.Therefore,investigating the mechanism of DHM’s hypoglycemic action is crucial for the development and utilization of vine tea or DHM in preventing and treating of T2DM.To maintain normal blood glucose levels in T2DM patients,substances with different mechanisms of action are often combined for optimal glycemic control.Sangzhi alkaloids(SZ-A)is a natural antihyperglycemic drug developed in China that lowers blood glucose by inhibiting intestinal α-glucosidase activity.Mulberry leaves and vine tea,as novel food ingredients,are non-Camellia teas.Based on a basic understanding of the mechanism of DHM lowering blood glucose,investigating the combined effect of DHM and SZ-A on lowering blood glucose balance lays the foundation for the development of antihyperglycemic tea drinks or functional products mainly composed of mulberry leaf and vine tea.Objective:The main objectives of this study are:(1)To explore the main hypoglycemic mechanism of DHM;(2)To preliminarily investigate the effectiveness of DHM as an adjunct to SZ-A in glycemic control;(3)To preliminarily explore the hypoglycemic active compounds and mechanism of mulberry leaf extract.Method and results:(1)In this study,we established a high-fat diet(HFD)-induced hyperglycemia model in Sprague-Dawley(SD)rats to investigate the hypoglycemic effect of DHM.Six-week-old SD rats were randomly divided into six groups:Control,HFD,HFD+ pioglitazone(PIO)(5 mg/kg PIO),HFD+100 mg/kg DHM,HFD+200 mg/kg DHM and HFD+400 mg/kg DHM.The control group was administered a standard diet,while the other groups were fed a HFD.DHM was orally administered for 8 weeks,and an insulin tolerance test was performed in the 8th week.The results showed that DHM significantly reduced fasting blood glucose and insulin levels,improved insulin resistance,and decreased the levels of serum total TC,TG and LDL while elevating serum HDL.Overall,DHM demonstrated effective antidiabetic and lipid-lowering effects in HFD-induced hyperglycemic SD rats.(2)Moreover,6-weekold C57BL/6J mice were randomly divided into four groups:Control,DHM(200 mg/kg),HFD,and HFD+DHM(200 mg/kg).Control and DHM groups were fed with standard diet,while HFD and HFD+DHM groups were fed with HFD and orally administered DHM for 13 weeks in HFD+DHM group.Glucose tolerance test and insulin tolerance test were performed at weeks 10 and 11,respectively.The results showed that DHM did not significantly affect body weight and fasting blood glucose levels in normal diet-fed mice,but significantly reduced body weight,fasting blood glucose levels,glycated serum protein and insulin levels in high blood glucose mice.DHM intervention also alleviated hepatic lipid accumulation,improved glucose and insulin tolerance,and increased hepatic glycogen levels in hyperglycemic mice.(3)Subsequently,transcriptome analysis was performed to explore the mechanism of DHM’s hypoglycemic effect,the results uncovered that the hypoglycemic effect of DHM was closely related to "bile secretion".Therefore,targeted metabolomics technology was further used to analyze hepatic bile acid metabolism,and the results showed that HFD induced abnormal accumulation of some bile acids,including glycocholic acid(GCA),glycochenodeoxycholic acid(GCDCA),deoxycholic acid(DCA)and lithocholic acid(LCA)in the liver,and DHM intervention significantly reduce the levels of these bile acids.(4)In order to verify the relationship between these bile acids and insulin resistance,this study prepared a mixed bile acid solution(GCA:GCDCA:DCA:LCA=1:1:1:1)and stimulated human hepatocellular HepG2 cells and human normal liver L02 cells.Results revealed that 25 μmol/L of mixed bile acids significantly inhibit insulin-stimulated cellular glucose uptake.When the two types of hepatocytes were separately stimulated with different concentrations of bile acids(GCA/GCDCA/DCA/LCA),and only LCA(25 μmol/L)induced insulin resistance.This study explored whether LCA could induce insulin resistance in vivo by administrating LCA(125 mg/kg)orally or intraperitoneally to C57BL/6J mice for one week.Results showed that intraperitoneal injection of LCA for one week increased mouse blood glucose and induced insulin resistance.(5)Network pharmacology and molecular docking were then employed to identify potential targets of LCA-induced insulin resistance,which were verified by qPCR.It was found that LCA-induced insulin resistance was closely related to bile acid and lipid metabolism,with FXR,SHP,PPARa and PPARγ playing important roles,and SHP playing a critical role in LCA-induced insulin resistance.(6)In order to explore the role of SHP in LCA-induced insulin resistance,we detected the effect of LCA treatment on SHP expression in vitro hepatocytes.The results showed that LCA could induce SHP gene and protein expression in hepatocytes.By knocking down SHP expression using SHP-Cas9 plasmid in L02 cells,it was found that LCA could not induce insulin resistance when SHP is suppressed.However,overexpression of SHP using SHP-pCMV6 plasmids in L02 cells resulted in insulin resistance.These results suggest that SHP is necessary for LCAinduced insulin resistance.(7)Then,we docked LCA and DHM with SHP and found that both LCA and DHM have good binding activities with SHP,the binding sites of LCA were distributed at the N-terminus of SHP active pocket,while the binding sites of DHM were mainly distributed at the C-terminus of SHP.Therefore,we expressed the SHP N-terminal and C-terminal fusion proteins in vitro and verified the binding activities of LCA and DHM using surface plasmon resonance(SPR)technology.Furthermore,through RNA-seq,ChIP-seq analysis,we discovered two nuclear receptors,PPARα and PPARγ,which interact with SHP.Moreover,SHP overexpression significantly increase PPARG gene expression and obviously inhibit PPARA gene expression.In addition,our study revealed that DHM significantly inhibited Nr0b2 expression induced by HFD in the liver,upregulated the expression of Ppara and its target gene Cpt1α,and significantly suppressed the expression of Pparg and its target genes Cd36,Fabp4 and Scd1.In summary,DHM exerts a hypoglycemic effect by increasing hepatic lipid consumption and inhibiting hepatic lipid accumulation through the SHP/PPAR α/γ pathway.(8)Based on the above results,this study used HFDinduced hyperglycemic mice as a model to explore the hypoglycemic effect of DHMassisted SZ-A.Mice were orally administered DHM(200 mg/kg),SZ-A(200 mg/kg),DHM+SZ-A 100(100 mg/kg DHM+100 mg/kg SZ-A),and DHM+SZ-A 200(200 mg/kg DHM+200 mg/kg SZ-A)for 15 weeks.Insulin tolerance test was performed in the 12th week,and glucose tolerance test was performed in the 15th week.The results showed that DHM assisted SZ-A significantly reduced the fasting blood glucose,glycated serum protein and insulin levels in hyperglycemic mice while improving their insulin and glucose tolerance.DHM+SZ-A 100 exhibited significantly lower glycated serum protein and insulin levels compared to SZ-A,which may be related to the inhibition of PPARy and its downstream target activating transcription factor 3(Atf3)in the liver.In addition,both SZ-A and DHM assisted SZ-A remarkably decreased body weight,epididymal adipose tissue and subcutaneous adipose tissue weight,serum LDL and liver TG levels,and improved hepatic steatosis.HFD increased the levels of serum growth differentiation factor 15(GDF15),glucagon-like peptide-1(GLP-1),and fibroblast growth factor 21(FGF21)related to food intake and energy consumption,which were reversed by DHM,SZ-A,DHM+SZ-A 100 or DHM+SZ-A 200.These effects of SZ-A and DHM-assisted SZ-A were related to the inhibition of lipid synthesis-related genes(Pparg,Dgat2,Hmgcr and Acat2),and upregulation of fatty acid oxidation-related gene Ehhadh expression in the liver.(9)Finally,the hypoglycemic effect of mulberry leaf extract(MLE)was studied in ob/ob mice,a T2DM animal model.MLE(80 mg/kg)and SZ-A(50 mg/kg)were administered orally to ob/ob mice for 14 weeks.The results showed that MLE improved insulin resistance and glucose tolerance,and reduce blood glucose levels in ob/ob mice.Network pharmacology was employed to identify the potential targets and active compounds of MLE in anti-T2DM.The hypoglycemic activity and gene expression of potential targets were validated using molecular docking and insulin-resistant cell models.Here,the results showed that MLE significantly inhibited Adora1 gene expression in the liver.Kuwanon C,morusin and morusyunnansin L were the key active compounds,which respectively exerted hypoglycemic effects by targeting lipid metabolism(ADORA1 and PPARγ)and insulin signaling(AKT1 and GSK3β).Conclusion:(1)DHM can inhibit HFD-induced partial accumulations of bile acid in the liver,especially LCA,which can inhibit insulin resistance and hyperglycemia induced by LCA mainly through the inhibition of SHP expression,thereby regulating PPARα,PPARγ,and downstream target genes.(2)DHM assisted SZ-A can reduce the dosage of SZ-A,but achieve the same hypoglycemic and anti-obesity effects as the original dose of SZ-A,while being superior to SZ-A in improving certain parameters(glycated serum protein and insulin).(3)DHM assisted SZ-A intervention can not only inhibit the PPARγ pathway in the liver,but also suppress Atf3 expression to lower blood glucose.(4)SZ-A possesses weight loss and lipid-lowering effects by inhibiting hepatic lipid accumulation and inhibiting appetite by enhancing the sensitivity of GDF15,GLP1 and FGF21.(5)MLE lowers blood glucose in ob/ob mice,with its main active components including kuwanon C,morusin and morusyunnansin L.