| High fat diet(HFD),a high-energy diet,has become a major cause of obesity-induced disorders of lipid metabolism and cognitive dysfunction.Isoleucine is a branched-chain amino acid and an essential amino acid involved in many important brain functions.It has been shown that reasonable restriction of isoleucine content can effectively alleviate HFD-induced lipid metabolism disorders,but whether it has an effect on cognitive impairment and the related mechanism of action are unclear.In this study,a high-fat obesity model was constructed in male C57BL/6J mice,and the intervention effects of isoleucine-restricted diet on brain cognition in obese mice and the possible molecular mechanisms were analyzed.It provides a theoretical reference for dietary intervention in the cognitive behavior of obese people.The main findings are as follows:(1)Effects of isoleucine-restricted diet on weight-related indexes in high-fat induced obese mice.Thirty 3-month-old C57BL/6J mice were divided equally into the following groups: Control,high-fat diet and high-fat isoleucine-restricted(HFD Low Ile,67% reduction of isoleucine in high-fat diets).Mice were fed continuously for 11 weeks,and the effects of different diets on phenotype,intake,and glucose tolerance of each group were compared by measuring body weight,food intake,water consumption,and glucose tolerance.The results showed that the HFD significantly increased the body weight of mice compared with the control group(P < 0.05),while the isoleucine-restricted treatment group significantly decreased the body weight of mice compared with the high-fat group(P < 0.05).Food intake was lower in the high-fat group and in the high-fat isoleucine-restricted group than in the control group,while energy intake was significantly higher in the high-fat and isoleucine-restricted groups compared with the control group(P < 0.05);there was no significant difference in water intake between the groups(P > 0.05).The isoleucine-restricted group significantly restored glucose tolerance in mice compared with the high-fat group(P <0.05).(2)Effects of isoleucine-restricted diet on learning memory function in high-fat induced obese mice.The cognitive function of the mice was assessed using the behavioral test Y-maze as well as the Morris water maze during the last week of the test cycle.The results showed that in the Y-maze test,there was no significant difference in total number of entries into the arm between the groups(P > 0.05),and isoleucine restriction treatment significantly increased the percentage of mice successfully alternating(P < 0.05).For the water maze test,during the1-5 days of the positioning cruise test,the isoleucine restriction-treated group used less escape latency time,which was significantly(P < 0.05)different from the high-fat group.And in the spatial exploration test,the isoleucine restriction group significantly increased the number of times the mice crossed the platform and the percentage of time and distance spent in the target quadrant(P < 0.05).(3)Effects of isoleucine-restricted diet on neuronal damage and brain-derived neurotrophic factor in high-fat-induced obese mice.Stained sections of the hippocampal region of mice brain were observed using H&E staining and immunohistochemical experiments,respectively.The results showed that the isoleucine-restricted diet significantly reduced the extent of hippocampal neuronal damage(P < 0.05)and significantly increased the expression of brain-derived neurotrophic factor compared with the high-fat group(P < 0.05).(4)Effects of isoleucine-restricted diet on the gut microbiota composition of high-fat-induced obese mice.The composition of intestinal microorganisms in each group of mice was determined and analyzed using 16 S r RNA sequencing technology.The results showed that compared with the high fat group,the diversity and richness of intestinal microorganisms in isoleucine restricted group were significantly increased(P < 0.05).In addition,at the phylum level,the isoleucine-restricted group significantly increased the proportion of Bacteroidetes,decreased the proportion of Firmicutes and Proteobacteria,and the proportion of Firmicutes/Bacteroidetes was decreased(P < 0.05).(5)Alleviation of neuroinflammation in high-fat induced obese mice by isoleucine restricted diet.LPS levels in serum and mice brain were measured using the kit,and the results showed that isoleucine restriction reduced LPS levels in serum and brain.Immunohistochemical experiments were used to observe the effects of microglia activation in the DG and CA1 regions of the hippocampus as well as the CA3 region in mice.The results showed that the number of microglia activation was significantly increased in the high-fat group compared to the control group(P < 0.05),while this was reduced in both isoleucine-restricted groups.The protein expression of TLR4/My D88/NF-κB signaling pathway was further analyzed.The results showed that isoleucine restriction reduced the protein expression of TLR4/My D88/NF-κB signaling pathway.In addition,isoleucine restriction inhibited the upregulation of m RNA and protein expression of pro-inflammatory cytokines IL-6,IL-1β and TNF-α.These results suggest that isoleucine restriction alleviates high-fat diet-induced brain inflammation in mice by regulating the TLR4/My D88/NF-κB signaling pathway.(6)Effects of isoleucine restriction diet on insulin resistance in the brain of high-fat induced obese mice.The expression of PTP1 B,a key protein linking inflammation and insulin resistance,was measured by Western Blot.The results showed that an increase in PTP1 B protein expression induced by inflammation subsequently led to impaired expression of the insulin signaling pathway p-IRS/p-AKT/p-GSK3β and the related protein GLUT4.By the interventional effects of the isoleucine-restricted diet,the results showed that isoleucine restriction significantly improved the expression of GLUT4 protein as well as p-IRS/p-AKT/p-GSK3β insulin signaling pathway(P < 0.05).(7)Effects of isoleucine-restricted diet on synaptic plasticity in high-fat-induced obese mice.The postsynaptic dense material in the hippocampal region of mice was observed and quantified using transmission electron microscopy.The results showed that a HFD reduced the length and width of postsynaptic dense material compared to controls.Isoleucine restriction significantly restored the length and width of postsynaptic dense material(P <0.05).The expression of synapse-associated proteins was subsequently measured by Western Blot,and the results showed that isoleucine restriction could improve synaptic plasticity by increasing protein expression of SNAP25,SYN and PSD95. |
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