| Objective: To make rats insulin resistant, they were given a high-fat diet. The effects of leucine and calorie restriction on the metabolism, insulin sensitivity,oxidative stress, the expression level of insulin release relevant gene and mitochondrial biogenesis relevant gene of rats are determined. The aim is to explore the effects of leucine and calorie restriction on the islet function of insulin resistant rats and the underlying mechanisms.Method: Sixty rats were separated according to body weight randomly and then given with normal chow diet(ND), high fat diet(HFD), high fat diet and calorie restriction(HFD+CR), high fat diet supplemented with leucine(HFD+Leu) or high fat diet supplemented with leucine and calorie restriction(HFD+Leu+CR),respectively. At the 36 th week of the trial, these rats were fasted and the insulin and the glucose tolerance test were conducted. All of the rats were killed until 36 weeks.The samples of blood and pancreas tissues were gathered. Levels of insulin and glucose of serum were tested and HOMA-IR was calculated. Level of oxidative stress were measured. To determine the expression levels of GDH, ATPsyn-β and mitochondrial biogenesis related genes, real time-PCR was used.Results:1.Metabolic changes in rats:(1) Body weight: Rats on HFD were obviously heavier than rats on ND from the5 th week(P<0.05). Rats of HFD+Leu group were lighter than HFD group from the13 th week and HFD+Leu+CR group were lighter than HFD+CR group from the 15 th week significantly(P<0.05).(2) Daily food intake: Rats on HFD significantly ate less than rats on ND from the 2th week(P<0.05). HFD group was ate much more than HFD +Leu group only during the 3-4 weeks and 25-32 weeks and from the 3th week HFD+CR group was higher than HFD +Leu +CR group obviously(P<0.05).(3) Daily calorie intake: Rats on HFD significantly took more energy than rats on ND from the 3th week(P<0.05). That of HFD group was higher than HFD +Leu group only during the 3-4 weeks and 25-32 weeks but HFD+CR group was higher than HFD +Leu +CR group from the 3th week obviously(P<0.05).(4) The ratios of organs and adipose over body weight : The ratios of testis, liver and perirenal adipose over body weight of rats on HFD were larger than ND group(P<0.05). The ratios of kidney, testis, liver and perirenal adipose over body weight of rats on HFD were larger than HFD+CR group and HFD+Leu+CR group significantly(P<0.05). The ratios of kidney, testis and perirenal adipose over body weight of rats on HFD+Leu were higher than HFD+Leu+CR group(P<0.05)significantly.2. Changes on insulin sensitivity, fasting insulin and glucose level.(1) The average fasting glucose level of rats on HFD was higher than that of ND group, HFD+CR group and HFD+Leu+CR group(P<0.05). The HOMA-IR of ND group, HFD+Leu group, HFD+CR group and HFD+Leu+CR group were obviously decreased compared with rats fed on HFD(P<0.05).(2) ITT:Compared with ND group, HFD+CR group and HFD+Leu+CR group,the AUC and the glucose levels of these four time points after injecting insulin of HFD group were increased obviously. The glucose levels of these four time points and the AUC of HFD+Leu group were higher than HFD+Leu+CR group(P<0.05).(3) GTT:The glucose levels after giving glucose were higher than ND group and HFD+Leu+CR group(P<0.05). The glucose levels of 60 and120 minutes were higher than HFD+Leu group and HFD+CR group(P<0.05). The AUC of HFD group was higher than ND group, HFD+CR group and HFD+Leu+CR group significantly(P<0.05).3.Morphological changes of pancreas For the ND group, the boundary between islet and exocrine gland was clear, the cluster of islet cells were observed and distributed regularly. For the HFD group, an amount of fat accumulated and hemosiderin in pancreatic exocrine gland. Exocrine cells were enlarged, inflammatory cells infiltrated and fibrosis aggravated in the islets.The boundary between islet and exocrine gland was relatively clear and little hemosiderin accumulated in the HFD+CR group. The boundary between islet and exocrine gland was vague, fibrosis aggravated in the islets and some fat and hemosiderin accumulated in pancreatic exocrine gland in the HFD+Leu group. For the HFD+Leu+CR group, the boundary between islet and exocrine gland was clear and the cluster of islet cells were observed and distributed regularly.4. Serum oxidative and anti-oxidative levels The MDA level in rats fed with HFD was higher than that of ND group,HFD+Leu group, HFD+CR group and HFD+Leu+CR group(P<0.05).The MDA level of HFD+Leu+CR group was lower than HFD+Leu significantly. The SOD activitity in rats fed with HFD was lower than that of rats fed with ND, HFD+Leu, HFD+CR group and HFD+Leu+CR significantly. The activity of SOD of HFD+Leu+CR group was higher than HFD+Leu and HFD+CR group(P<0.05). The differences of GSH-Px activity between these groups were similar to SOD.5. The expression of GDH m RNA in pancreas: The level of GDH m RNA of HFD group was decreased as that of ND group, HFD+Leu group, HFD+CR group and HFD+Leu+CR group were higher obviously. The expression levels of GDH m RNA in rats on HFD+Leu+CR were higher than that of HFD+CR group and HFD+Leu group obviously.6. The expression of ATPsyn-β m RNA in pancreas: The level of ATPsyn-βm RNA of HFD group was increased as ND group and HFD+CR group expressed less(P<0.05). The expression level of ATPsyn-β m RNA in rats on HFD+Leu was higher than that of HFD group obviously. The level of ATPsyn-β m RNA of HFD+Leu+CR group was increased as HFD+CR group expressed less(P<0.05).7.The expression levels of genes related with mitochondrial biogenesis in pancreas: The expression levels of NRF-1, SIRT1, PGC-1α, TFAM were reduced in HFD group compared with ND, HFD+Leu, HFD+CR and HFD+Leu+CR group. The m RNA expression levels of SIRT1, NRF-1, TFAM were elevated in HFD+Leu+CR group compared with HFD+CR group. The m RNA expression levels of SIRT1 and TFAM were higher in HFD+Leu+CR group than HFD+Leu group.Conclusions:1. Leucine supplementation and calorie restriction repressed the excessive growth of body weight. Leucine intervention lowered the daily food intake of rats on HFD.2. Leucine supplementation and calorie restriction reduced the fasting glucose level and improved the glucose and insulin tolerance and pancreatic injury of rats on HFD. There were combined effects between them.3. Leucine supplementation and energy restriction decreased oxidative stress of rats fed with HFD.4. Leucine may improve islet function through increasing the expression of GDH,elevating the synthesis of ATP and the biogenesis of mitochondria. In addition, there were combined effects between leucine and calorie restriction. |
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