Effects Of Trigonelline On Glucolipid Metabolization And Hippocampus Neuritin Expression In Diabetic Rats

BACKGROUNDIn recent years, there is a rapidly increasing trend of diabetics. Diabetics, it was predicted, would reach129.7millions in China in2030. Loose blood glucose control can hurry the progression of diabetes related complications, such as retinopathy, nephropathy and neuropathy. Type2diabetes, a chronic heterogeneous metabolic disorder characterized by hyperglycemia resulting from defective insulin secretion, resistance to insulin action or both. Type2diabetics constitute90-95percent of total diabetics. Most patients are obese with type2diabetes, often associated with hyperlipidemia, and are prone to development of diabetic complications.At present, there are mainly sulfonylureas, thiazolidinediones, biguanides, glinides, alpha-glucosidase inhibitors, dipeptidyl peptidase IV inhibitors and insulin agents for the treatment of type2diabetes. Although different types of oral hypoglycemic agents are available for the treatment of diabetes, there is increasing demand by patients to use anti-diabetic natural products for their fewer side effects. In many countries, much attention has been paid to find novel type of natural anti-diabetic drugs from various medicinal plants. It had accumulated plenty of clinical practice in traditional Chinese medicine for the treatment of diabetes and its complications. In virtue of complex ingredients and prearations of traditional Chinese medicine often shows a variety of biological activities and different mechanisms, the monomer composition of traditional Chinese medicine on anti-diabetic is attracting more and more researchers’ attention. It is a new way to combine modern pharmacological studies and ancient literature to find the monomer composition on anti-diabetic. It also has become a hotspot study on finding hypoglycemic drugs to prevent and treat type2diabetic patients with dyslipidemia.Trigonelline is the major component of fenugreek seeds which also consists in Mirabilis jalapa roots, pumpkins, coffee beans and potatoes. Trigonelline has effects of hypoglycemic, hypocholesterolemic, enhancement in nerve regeneration, anti-cancer and tranquilizing. Trigonelline has antioxidant effectiveness in cell-free systems and human colon cell lines. Epidemiological studies showed drinking coffee could reduce the risk of type2diabetes, because the content of trigonelline reached1%in coffee beans. Trigonelline could inhibit the process of diabetes on KK-Ay mice and Goto-Kakizaki rats. Trigonelline was shown to prevent both dendritic and axonal atrophy induced by amyloid β(25-35) in a dose-dependent manner. Trigonelline also induced dendritic and axonal regeneration in cortical neurons of rats.In diabetes, it is thought that the reduction of neurotrophin contributes, in part, to the failure in axonal regeneration and pathogenesis of diabetic neuropathy. Neuritin(also known as candidate plasticity-related gene15, CPG15), a neurotrophic factor, plays an important role in neurite growth and neuronal survival. Mitogen-activated protein kinase(MAPK) pathway is one of the two main insulin signaling pathways. The MAPK family consists of extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK), and p38kinases. MAPKs are activated in diabetics. ERK are rapidly activated in the region of nerve injury to reduce nerve injury on the body, but phosphorylated JNK and p38gathered in nerve injury sites to promote nerve cell apoptosis and the process of nerve injury. MAPK pathway is closly linked with the occurrence and repair of nervous system injury.Trigonelline has hypoglycemic and nerve protection effectiveness. Neuritin is closely related to the pathogenesis of diabetic neuropathy. Hyperglycemia damages nerve and results in the occurrence of diabetic neuropathy by MAPK signaling pathways. Nerve protection and plasticity regulating by neurotrophic factors is often linked with MAPK signaling pathway.In view of the above analysis, we planned to evaluate the effects of trigonelline on anti-diabetic in streptozotocin-induced diabetic rats in this study, as well as study the effects of the expressions of hippocampus neuritin and MAPKs in diabetic rats. According to Novelty Search, there were only a few papers about the effect of trigonelline on anti-diabetic. The completion of this project would be used as the theoretical basis of trigonelline on anti-diabetic, which would promote the studies and development of trigonelline. It would also provide new train of thoughts to find novel types of natural anti-diabetic drugs from various medicinal plants.OBJECTIVES1. To induce type2diabetic rats by intraperitoneal injection40mg·kg-1streptozotocin and a high-carbohydrate/high-fat diet. To investigate the effects of trigonelline on fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol, triglyceride, fasting insulin, insulin sensitivity index levels and glycogen contents in liver and skeletal muscle in diabetic rats. To probe into hypoglycemic and hypolipidemic effects of trigonelline in diabetic rats.2. To investigate the effects of trigonelline on the expressions of hippocampus neuritin and mitogen-activated protein kinase (MAPK) signaling pathway (including extracellular signal regulated kinase (ERK), c-Jun amino-terminal kinase (JNK) and p38MAPK) in diabetic rats. To probe into beneficial effects of trigonelline on hippocampus of diabetic rats.METHODS1. To induce type2diabetic ratsAfter2weeks feed with the high-carbohydrate/high-fat diet, overnight fasted rats were injected intraperitoneally with a freshly prepared solution of40mg·kg-1streptozotocin (in0.1mM citrate-phosphate buffer, pH4.5). Normal control rats were injected an equivalent volume of citrate buffer through the same route of administration. Three days after streptozotocin injection, the diabetic rats with fasting blood glucose levels higher than16.7mmol·L-1were selected.2. Experimental group and drug treatmentThe diabetic rats were divided into5groups:diabetic model, trigonelline low dose (5mg·kg-1), trigonelline middle dose (15mg·kg-1), trigonelline high dose (45mg·kg-1) and rosiglitazone (4mg·kg-1) groups. In addition,6normal SD rats were used as normal control group. Trigonelline was administered by gavages (i.g.) to diabetic rats for8consecutive weeks. Rosiglitazone was given to diabetic rats as a positive control drug. Normal control rats and diabetic control rats received the same volume of distilled water. The fasted blood sample was collected from the tail for blood glucose measurement every2weeks, with a portable glucometer. Animal weight was measured every1week throughout the experiment and the drug dose was accordingly adjusted.3. Tissue preparationAfter8-week treatment, rats were fasted overnight and were euthanized by anesthesia(120mg·kg-1, i.p.). Blood was collected from heart and transferred immediately into microcentrifuge tubes and allowed to clot to get serum. Brain was quickly removed and stripped the hippocampus in ice-cold saline solution, liver and skeletal muscle was quickly removed and weighed from the sacrificed rat.4. Measurement of blood glucose, blood lipids and insulin levelsGlycosylated hemoglobin A1c, total cholesterol, triglyceride and fasting insulin levels were measured by commercial kits according to the manufacturer’s instructions, respectively.Insulin sensitivity index=ln[1/(Fasting blood glucosexFasting insulin)].5. Measurement of glycogen contents in liver and skeletal muscleGlycogen contents in liver and skeletal muscle were determined by spectrophoto-metry with commercial kit provided by Nanjing Jiancheng Bioengineering Institute, China.6. Determination of hippocampus neuritin and MAPK signaling pathway expressions in type2diabetic ratsThe expressions of neuritin、ERK1/2、p-ERKl/、JNK、p-JNK、p38MAPK、 p-p38MAPK were determined by Real-time PCR and Western blot.7. Statistical analysisAll statistical analysis was carried out using the SPSS13.0Statistical software package, and all data were presented as means±S.D. If the variances between groups were homogenous, groups were subjected to the multiple comparisons least significant differences (LSD) test. In case of no homogeneity variances, differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett’s T3test. Statistical significance was achieved whenP<0.05.RESULTS1. The levels of blood glucose and blood lipidsCompared with those in the normal group, fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol and triglyceride levels were significantly increased in the diabetic model group (P<0.01). Compared with those in the model group, there were no obvious differences in the levels of fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol and triglyceride in the trigonelline low dose group (P>0.05); the levels of fasting blood glucose and glycosylated hemoglobin A1c decreased significantly in the trigonelline middle, high dose group and rosiglitazone group (P<0.01); total cholesterol and triglyceride levels decreased significantly in the trigonelline middle, high dose group (P<0.01), while there were no obvious differences in the levels of total cholesterol and triglyceride in the rosiglitazone-treated group(P>0.05).2. The levels of fasting insulin and insulin sensitivity indexCompared with those in the normal group, fasting insulin levels were significantly increased, and insulin sensitivity index decreased significantly in the diabetic model group(P<0.01). Compared with those in the model group, there were no obvious differences in the levels of fasting insulin and insulin sensitivity index in the trigonelline low dose group(P>0.05); the levels of fasting insulin decreased significantly, insulin sensitivity index increased significantly in the trigonelline middle, high dose group and rosiglitazone group (P<0.01).3. The glycogen contents in liver and skeletal muscleCompared with those in the normal group, glycogen contents in liver and skeletal muscle decreased significantly in the diabetic model group(P<0.01). Compared with those in the model group, there were no obvious differences in glycogen contents in liver and skeletal muscle in the trigonelline low dose group(P>0.05); glycogen contents in liver and skeletal muscle increased significantly in the trigonelline middle, high dose group and rosiglitazone group (P<0.01).4. The expression of hippocampus neuritin Compared with those in the normal group, neuritin mRNA and protein levels decreased significantly in the diabetic model group(P<0.01). Compared with those in the model group, there were no obvious differences in neuritin mRNA and protein levels in the trigonelline low dose group(P>0.05); neuritin mRNA and protein levels increased significantly in the trigonelline middle, high dose group and rosiglitazone group (P<0.01).5. The expression of hippocampus MAPK signaling pathway5.1. The expression of ERK1/2Compared with those in the normal group, there were no obvious differences in the levels of ERKl/2protein (P>0.05), while ERKl/2mRNA and p-ERKl/2protein levels increased significantly in the diabetic model group(P<0.05). Compared with those in the model group, there were no obvious differences in the levels of ERKl/2mRNA and ERKl/2、p-ERKl/2protein levels in the trigonelline low, middle, high dose group and rosiglitazone group (P>0.05).5.2. The expression of JNKThere were no obvious differences in JNK1/2protein levels among the normal group, diabetic model group, trigonelline low, middle, high dose group and rosiglitazone group (P>0.05). Compared with those in the normal group, JNK mRNA and p-JNKl/2protein levels increased significantly in the diabetic model group (P<0.01). Compared with those in the model group, there were no obvious differences in JNK mRNA and p-JNKl/2protein levels in the trigonelline low dose group (P>0.05); JNK mRNA and p-JNK1/2protein levels decreased significantly in the trigonelline middle, high dose group and rosiglitazone group (P<0.01).5.3. The expression of p38MAPKThere were no obvious differences in p38MAPK protein levels among the normal group, diabetic model group, trigonelline low, middle, high dose group and rosiglitazone group(P>0.05). Compared with those in the normal group, p38MAPK mRNA and p-p38MAPK protein levels increased significantly in the diabetic model group(P<0.01). Compared with those in the model group, there were no obvious differences in p38MAPK mRNA and p-p38MAPK protein levels in the trigonelline low dose group(P>0.05); p38MAPK mRNA and p-p38MAPK proteins levels decreased significantly in the trigonelline middle, high dose group and rosiglitazone group (P<0.01).CONCLUSION1. After type2diabetic rats were induced by intraperitoneal injection streptozotocin40mg·kg-1and high-carbohydrate/high-fat diets. The food, water and urine in diabetic rats were significantly higher than that of the control ones, fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol, triglyceride and insulin levels in diabetic rats increased significantly. These results indicated that type2diabetic rats with hyperlipidemia were successfully induced.2. Fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol and triglyceride levels in diabetic rats were significantly higher than that of the control ones. The middle, high dose trigonelline and rosiglitazone could significantly reduce the levels of fasting blood glucose and glycosylated hemoglobin A1c in diabetic rats. The middle, high dose trigonelline could significantly reduce both total cholesterol and triglyceride levels, while low dose trigonelline and rosiglitazone could not reduce them in diabetic rats.3. Fasting insulin levels were significantly increased, and insulin sensitivity index levels were significantly decreased in diabetic rats compared with in normal control rats. The middle, high dose trigonelline and rosiglitazone could significantly reduce fasting insulin levels and increase insulin sensitivity index levels in diabetic rats. Trigonelline has beneficial effects on the regulation of insulin levels of diabetic rats. 4. Glycogen contents in liver and skeletal muscle were significantly decreased in diabetic rats compared with in normal control rats. Trigonelline increased the declined glycogen contents in liver and skeletal muscle of diabetic rats. Trigonelline has beneficial effects on glucose metabolism in liver and skeletal muscle of diabetic rats.5. Hippocampus neuritin mRNA and protein expressions were significantly decreased in diabetic rats compared with in normal control rats. Trigonelline restored the decreased neuritin mRNA and protein expressions to the control levels in hippocampus of diabetic rats, which may play protective effects on hippocampus of diabetic rats.6. The cascades of hippocampus MAPKs were activated, ERK1/2, JNK and p38MAPK mRNAs were significantly increased, p-ERKl/2, p-JNK and p-p38MAPK proteins were significantly increased in hippocampus of diabetic rats. Trigonelline declined the increased JNK and p38MAPK expressions to the control levels, but has no effect on the increased ERK1/2expression in diabetic rats. Trigonelline may have beneficial effects on hippocampus of diabetic rats by modulating MAPK signaling pathway.