| Objective1. To observe the initial effects of melatonin (MLT) on glucose and lipid metabolism in glucose intolerance rats induced by chronic stress fed with high-fat diet.2. To observe the effects of MLT on glucose and lipid metabolism in glucose intolerance rats induced by chronic stress fed with high-fat diet.3. To study the improving mechanisms of MLT on glucose and lipid metabolism in glucose intolerance rats induced by chronic stress fed with high fat diet.4. To study the molecular mechanisms of MLT on glucose and lipid metabolism in glucose intolerance rats induced by chronic stress fed with high fat diet.5. To investigate the relationship between the Hypothalamic-pituitary-adrenal (HPA) axis and MLT in type2diabetes (T2DM) patients with different level of blood glucose. To study the change of MLT level in development of T2DM.Methods1. Rats were randomly divided into six groups:control group, model group, stress and amitriptyline group (AMI,6mg·kg-1·d-1), stress and MLT-L group (10mg·kg-1·d-1), stress and MLT-M group (20mg·kg-1·d-1), stress and MLT-H group (40mg·kg-1·d-1). Except control group, other groups were fed with high-fat diet and treated with one of four stressors concluding absorbent gauze wrap secured with tape, restraint in a polyvinyl chloride tube (L=25cm, ID=5cm) closed at either end, immobilization on a board with tape, tail suspension. Except control group, others should be continuously stimulated in seven weeks. The food and water intake and body weights were recorded every day. FPG was determined per week and IPGTT was detected in the4th and7th week. After7week’s administration of MLT, rats were decapitated after6h fasted and the trunk blood was collected. Organs index of rats were measured. The levels of FINS, CORT, ACTH and CRH were assayed in the1st,4th and7th week, while the TG, TC, HDL-C, LDL-C levels were detected in the4th week, and the glycogen content was measured in the7th week.2. Grouping, stress protocol and administration are same as described above. Positive drug is fluoxetine (FLU,10mg·kg-1·d-1). The food and water intake and body weights were recorded every day. FPG was determined per week and IPGTT was detected in the2nd and7th week. After7weeks’ administration of MLT, rats were decapitated after6h fasted and trunk blood was collected. Organs index of rats were measured. TG, TC, HDL-C, LDL-C levels and glycogen content were detected in the last week.3. Grouping, stress protocol and administration are same as described above. After the1week’s administration, plasma was collected for detecting hormone. After7weeks’administration, rats were decapitated after6h fasted and trunk blood was collected. The activity of G-6-Pase in liver was determined. FINS, CORT, CRH and ACTH levels were detected in the first and last week, and MLT level in plasma was measured in the last week.4. Grouping, stress protocol and administration are same as described above. After7weeks’ administration, rats were decapitated after6h fasted. The gene transcript levels of GR in hippocampus, the gene transcript levels of TNF-a,11β-HSD1, PPAR-y, GR in liver, and the gene transcript levels of TNF-α,11β-HSD1, PPAR-y, Leptin, GLUT-4, adiponecin (Adipo) in adipose tissue were determined by the RT-PCR method, while western-blot method was used to detect GR expression level in liver.5. The basic information of90patients with T2DM and30healthy volunteers were collected. The blood pressure and body mass index (BMI) were measured. The blood samples were collected, and the serum FPG, FINS, C-P, HbA1c and plasma lipid level including TC, TG, HDL-C, LDL-C, as well as CORS, ACTH, CRH, MLT levels were assayed. All of these data were computed with statistics. According to HbA1c levels, patients were divided into three groups (Group A:HbA1c＜7.1%, Group B:7.1%≤HbA1c≤11.1%, and Group C:HbA1c＞11.1%). The relationship between endogenous MLT and HPA axis in different HbA1c levels were analyzed. The result was compared with those of non-diabetic controls. The results were compared among the four groups by using one-way analysis of variance, multiple linear correlation analysis methods and multiple linear regression, the relationship between endogenous MLT and hormones of the HPA axis in the different HbA1c levels was analyzed.Result1. The body weight of rats in model group was decreased significantly compared with control group. The FPG of model group was increased by23.38%(7.23±1.20mmol·L-1vs5.86±1.00mmol·L-1, P＜0.05) after four weeks. And the FPG in MLT-M (5.64±0.69mmol·L-1), MLT-H (5.55±0.83mmol·L-1) group was decreased by21.99%(P＜0.01) and23.24%(P＜0.01), compared with model group. The last week of IPGTT showed that plasma glucose level in30min in model group was increased by27.09%, compared with control group (18.25±2.60mmol·L-1vs14.36±3.11mmol·L-1, P＜0.01), plasma glucose level in30min of MLT-H group (15.78±3.62mmol·L-1) was decreased by13.53%, compared with model group. In the4th week, CORT levels in model group was increased by11.73%, compared with control group. The levels of CORT in MLT-M group and MLT-H group was decreased by26.01%(P＜0.05),44.84%(P＜0.01), compared with control group. In the last week, the activity of HPA axis was inhibited. The level of CORT was increased by MLT-M administration. The result shows FINS level and HOMA-IR index in model group were increased by30.09%(P＜0.05) and44.48%(P＜0.01) respectively in7th week. Compared with model group, FINS levels was decreased by26.28%and29.53%(all P＜0.01) in MLT-L and MLT-M group. HOMA-IR index of MLT-L group and MLT-M group was decreased by36.71%and35.00%respectivly (all P＜0.01), compared with model group. In addition, after4weeks of chronic stress combined with high-fat diet, lipid metabolism disorders appeared in model group. HDL-C level in model group was deceased by21.55%(P＜0.01), compared with control group. Compared with model group, HDL-C level was increased by24.18%(P＜0.05),29.67%(P＜0.01) and19.78%(P＜0.05) respectively in different dose of MLT group.2. After2weeks of chronic stress combined with high-fat diet, the body weight of model group was decreased by9.3%(P＜0.05), compared with control group. The body weight of MLT-H was increased by7.18%, compared with model group. In the2nd week, FPG level of model group was increased by23.19%, compared with control group (5.63±0.68mmol·L-1vs4.57±0.90mmol·L-1, P＜0.05). The FPG levels in MLT-M and MLT-H group were reduced significantly. IPGTT showed that30min blood glucose levels in model group was increased by40.84%,52.30%in the2nd and7th week.(all P＜0.01), compared with control group. The blood glucose was reduced significantly in MLT-M and MLT-H group. The dyslipidemia in rats was improved by MLT administration. After7weeks of chronic stress combined with high-fat diet, TG level in model group was increased by67.5%(P＜0.05), compared with control group. TG levels were decreased by51.74%(P＜0.01),59.20%(P＜0.01) and43.28%(P＜0.05) respectively in MLT-L, MLT-M and MLT-H groups, compared with model group. HDL-C levels in MLT-L, MLT-M and MLT-H group were increased by35.48%(P＜0.01),30.11%(P＜0.05) and15.05%(P＜0.05), compared with model group. Muscle glycogen content in rats was decreased by27.28%(P＜0.05) in model group, compared with control group. The muscle glycogen contents were enhanced significantly by MLT-M and MLT-H administration. The liver glycogen content was significantly reduced by MLT administration. Adrenal index in model group was increased by53.44%(P＜0.01), compared with control group. And adrenal index was decreased by14.61%in MLT-H group, compared with model group. Liver index of MLT-H was decreased by10.85%(P＜0.05), compared with model group.3. After7weeks’ chronic stress combined with high-fat diet, CORT level in model group was increased by17.92%, compared with control group. CORT levels were decreased by31.78%(P＜0.05),35.23%(P＜0.01) and33.12%(P＜0.05) after MLT-L, MLT-M and MLT-H administration, compared with model group. FINS level in model group was reduced by68.69%(P<0.01), compared with control group, and it was increased by38.52%,103.28%(P＜0.05) and143.44%(P＜0.01) in MLT-L, MLT-M and MLT-H group, compared with model group. ACTH level in MLT-H group was decreased by14.21%(P＜0.05), compared with model group. G-6-Pase activity in model group was increased by34.20%(P＜0.05), compared with control group. The activity of G-6-Pase in MLT-M group was decreased by24.77%(P＜0.05), compared with model group.4. Relevant genes in adipose tissue and liver were measured by PT-PCR method. The TNF-α transcript levels was higher than control group significantly(P<0.05) in model group, and it was decreased significantly by MLT administration (all P<0.01). The11β-HSD1transcript levels in model group was elevated significantly compared with control group (P<0.01), while it was decreased by MLT administration (all P＜0.01). In model group, PPAR-y transcript levels was lowered significantly than normal rats (P＜0.01) and it was significantly increased by MLT administration (all P＜0.05). Some relevant genes in adipose tissue were measured by PT-PCR method. Compared with model group, leptin transcript levels in MLT-L, MLT-M and MLT-H group were reduced significantly,(all P＜0.01), while GLUT-4transcript levels in MLT-M, MLT-H group were increased significantly. Adipo transcript levels in MLT-L, MLT-M and MLT-H group, GR transcript levels in hippocampal in MLT-L, MLT-M and MLT-H group were all increased significantly.(all P＜0.01).5.(1) There was no significant differences in age, BMI and pathogenesis among three groups of T2DM patients (P＞0.05). The levels of hormones related HPA axis and MLT are low in non-diabetic controls and Group A patients. The levels of hormones of HPA axis and MLT are high in patients of Group B and Group C, compared with non-diabetic controls. The levels of hormones of HPA axis and MLT were significantly different between Group B and non-diabetic controls.(2) Multiple linear correlation analysis showed that there is a positive correlation between corticotropin release hormone (CRH) and MLT in non-diabetic controls and Group A (r=0.476, P＜0.05; r=0.487, P＜0.05). There is a positive correlation between MLT and cortisol (CORS) in Group B and Group C (r=0.318, P＜0.05; r=0.359, P＜0.05).(3) Multiple linear regression showed that CRH, Adrenocorticotropic hormone (ACTH), and CORS are independent factors of MLT in T2DM patients.Conclusion1. The glucose metabolism and lipid metabolism in glucose intolerance rats induced by chronic stress fed with high fat diet were improved by MLT administration. The level of FPG was reduced, and the hyperactivity of HPA axis was inhibited by MLT.2. The glucose and lipid metabolism in glucose intolerance rats induced by chronic stressfed with high-fat diet were all improved by MLT. The levels of FPG and TG were reduced by MLT. The glucose metabolism was better ameliorated by MLT (20～40mg/kg·d-1), lipids metabolism was better regulated by MLT (10～20mg/kg·d-1). In addition, the content of muscle glycogen was increased and the content of liver glycogen was decreased in MLT groups.3. The activity of HPA axis was significantly suppressed by MLT. Administration of MLT was found to elevate FINS level. The hyperactivity of G-6-Pase in glucose intolerance rats was decreased by MLT administration.4. The levels of gene transcript and protein expression in liver, adipose tissue and hippocampus were ameliorated by MLT administration. The hyperfunction of HPA axis was suppressed by daily administration of MLT, non-active CORT was converted into an active role which was inhibited, regulation of HPA axis by the hippocampus was ameliorated, insulin sensitivity and lipid metabolism were improved by MLT administration.5. The relationship between HPA axis and MLT is various in different blood glucose levels of T2DM patients. The excessive secretion of CORS may stimulate the excretion of MLT, and which interacts with the HPA axis, then influence the development of diabetes synergistically. |
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