Study On The Mechanism Of Total Bitter Melon Of Bitter Gourd On Hypoglycemic And Lipid - Regulating Effects In Type 2 Diabetic Rats

Objective:Traditional Chinese Medicine considered that Momordica charantia(Bitter gourd, MC) has the clearing heat, discharging fire andanti-diabetes effects. The modern medicine has also proved that totalsaponins of MC has anti-diabetes effects. This article’s objective isto clear total saponins of MC’s anti-diabetes mechanism in T2DM ratsthrough discussing total saponins of MC’s effects: hyperglycemicmechanism, the impact of serum lipid and adipocytokines and the regulatorymechanism, the influence of insulin signal transduction pathway.Materials and methods:Part one:8rats were randomly selected as normal control group in60SPF SDrats. Other rats were used to build the T2DM models. The T2DM rats modelswere established by eating food containing the higher lipid and sugar andby intravenous injection of a low dose of streptozotocin.。The fastingblood glucose≥11.1mmol/L was considered as the success rats models ofT2DM. After the disease models were successfully induced, the model ratswere then randomized into five groups: the model control group (n=8), themetformin group (n=8) and the three dose total saponins of MC group (n=24).The total saponins of MC group was administrated with total saponins ofMC100,200,400mg/(kg d) for8weeks, the metformin group wasadministrated with metformin50mg/(kg d) for8weeks. At the end of theexperiment, the fasting blood glucose and insulin were measured. At thesame time, a part of pancreas islet, liver and skeletal muscle werepreserved. The pancreas islet structure, the pancreatic β cell’s insulin distribution, the hepatic glycogen and Glucose transporter4(GLUT-4) expression were observed by electron microscope,immunohistochemistry, glycogen dyeing and western blot methodsrespectively.Part two:The animal models, grouping and the treat method were as same as thepart one except that the fenofibrate group was administrated withfenofibrate100mg/(kg d) for8weeks. The rats’ body weight and the serumlipid parameters were measured. The adipocyte morphology was observed byHE dyeing method. The adipocytokines, the liver PPAR-α protein and theadipose PPAR-γ protein were measured by ELISA and western blot method.Part three:The method of animal mouding, grouping and administration was as sameas the part one. The partly liver tissue was used. The factors such asAkt-2, PTP-1B, SOCS-3, JNK, PTEN, and AMPK’s mRNA and protein relatedwith insulin signal transduction pathway were measured by RT-PCR andwestern blot method.All measurement data were showed with “x-±s”. SPSS17.0statisticalsoftware was used to statistic analysis. ANOVA(one-way analysis ofvariance) and LSD(least significant difference) method was used tocomparison among and multiple comparisons.Results:Part one:1. The rats blood glucose value decreased in the MC groups and the Metgroups compared to the T2DM model group（P<0.01）.The insulin value ofT2DM model group is higher than that of the normal control group.Theinsulin values of all treatment groups were lower than that of T2DM modelgroup(MC100group’s P<0.05，other group’s P<0.01). With the increaseof total saponins of MC dose, fasting blood glucose, insulin values and HOMA-IR reduced more obviously(P<0.01).2. The pancreaticβ cell amount of T2DM group were reduced obviouslycompared to the normal control group, however, the ratio of the dyeingpositive pancreatic β cell amount occupying all pancreatic β cell amountin T2DM group increased obviously compared to the normal control group.The amount of insulin granules increased in total saponins of MC groupcompared to T2DM group, however, was less than that of control group. Theratio of insulin positive pancreatic β cell amount occupying the totalpancreatic β cell decreased in total saponins of MC group compared tothe T2DM model group and the change tendency of ratio decreased with thedosage of total sapoins of MC.3. The number of the pancreaticβ cell’s secretory granules decreasedin T2DM group by electron microscopy examination. The number of thepancreaticβ cell’s secretory granules increased obviously in MC groupsand Met group compared to T2DM group, but the number was less than thatof normal control group. The number of secretory granules increased withthe increasing of the dosage of MC.4. The number of hepatic glycogen decreased in T2DM model group comparedto the normal control group. The number of glycogen in200mg and400mgMC groups increased compared to T2DM group, and the number of glycogenin400mg MC group was closed to the normal contral group.5. The amount of the skeletal muscle GLUT-4expression decreased in T2DMgroup and increased obviously in200mg MC （P<0.05）and400mg MC (P<0.01）group compared to the normal contral group. The amount of the skeletalmuscle GLUT-4expression increased obviously in all MC groups comparedto the T2DM model group(P<0.01), and the amount of the skeletal muscleGLUT-4expression has significant difference in every MC groups(P<0.01).Part two:1. The body weight loss was found in all total saponins of MC groups compared to the T2DM model group(P<0.05or P<0.01).2. The value of plasma TG, TC and LDL-C decreased and the value of plasmaHDL-C increased obviously in200,400mg total saponins of MC groups andfenofibrate group compared to the T2DM model group（P<0.05or P<0.01）.The value recovered to normal after treatment of total saponins of MC.3. The adipocyte volume decreased obviously in all total saponins of MCgroups compared to the T2DM group. The adipocyte volume turned to normalwith the increasing dosage of total saponins of MC.4. The value of plasma leptin, resistin, TNF-α and IL-6increasedobviously in the T2DM model group compared to normal control groups(P<0.05or P<0.01). Compared with the T2DM model group, the value of leptin andresistin decreased obviously in200,400mg total saponins of total MCgroups(P<0.05or P<0.01), the value of plasma TNF-α,IL-6decreasedobviously in all total saponins of MC groups(P<0.05or P<0.01), the valueof plasma adiponectin increased obviously in400mg total saponins of MCgroup(P<0.05).5. The liver mRNA and protein’s expression amount of PPAR-α increasedobviously in all total saponins of MC group compared to the T2DM modelgroup (P<0.01). The PPAR-α’s protein expression amount increased withthe increasing dosage of total saponins of MC, however, the increasingeffect is lower than that of fenofibrate group.6. The adipocyte mRNA and protein’s expression amount of PPAR-γ increasedobviously in all total saponins of MC group compared to the T2DM modelgroup(P<0.01). The increasing effect of400mg total saponins of MC groupis as same as that of fenofibrate group(P>0.05).Part three：1. The mRNA expression amount of Akt-2and AMPK decreased obviously inthe T2DM model group compared to the normal group, and the expressionamount increased obviously in all treatment groups compared to the T2DM model group(P<0.01).2. The mRNA expression amount of PTP-1B, SOCS-3, JNK and PTEN increasedobviously in T2DM group compared to the normal group, and the expressionamount decreased obviously in all treatment groups compared to the T2DMmodel group(P<0.01), and the mRNA expression amount of SOCS-3and JNKreduced with the increasing dosage of total saponins of MC(P<0.01).3. The protein expression of p-Akt-2(Ser473) decreased obviously in theT2DM model group compared to the normal group, and the protein expressionincreased obviously in the total saponins of MC200mg and400mg groupscompared to the T2DM model group(P<0.05and P<0.01), and the proteinexpression of p-Akt-2(Ser473) increased with the increasing dosage oftotal saponins of MC. The protein expression of p-AMPK(Thr172) increasedin all treatment groups obviously compared with the T2DM modelgroup(P<0.01).4. The protein expression of SOCS-3and JNK increased obviously in theT2DM model group compared to the normal group(P<0.05and P<0.01), andthe protein expression decreased obviously in all treatment groupscompared to the T2DM model group(P<0.05and P<0.01), and the expressionlevel decreased with the increasing dosage of total saponins of MC. Therewere no statistics differences in all groups of the protein expressionof PTP-1B and PTEN.Conclusions:1. Total saponins of MC has the hypoglycemic activity. It’s mechanismsmaybe include protecting the the pancreaticβ cell， increasing thehepatic glycogen and promoting insulin sensitivity by increasingperipheral tissue’s GLUT-4expression.2. Total saponins of MC can decrease the body weight, plasma lipid,adipocyte volume and adjust the plasma adipocytokines, It’s effectsenhanced with the increasing dosage of total saponins of MC. Above effects maybe caused of up-regulating the expression of liver PPAR-α and adipocytePPAR-γ.3. The ameliorating insulin resistance mechanism of total saponins of MCmaybe caused by increasing the insulin sensitivity through the severalreasons: down-regulating the mRNA and protein’s expression of PTP-1B,SOCS-3, JNK; down-regulating the protein expression amout of SOCS-3andJNK; up-regulating the mRNA of Akt-2and AMPK; increasing the activityof Akt and AMPK.4. The above actions inhance with the increasing dose of total saponinsof MC,400mg/(kg d) is the ideal dose.