The Role Of Endoplasmic Reticulum Stress PERK Pathway In Aerobic Exercise Improving Hypothalamic Leptin Resistance In Obese Mice

Objective:Obesity is often accompanied by leptin resistance in the hypothalamus.Reducing leptin resistance can restore the regulation of energy balance in the hypothalamus and improve obesity.Endoplasmic reticulum stress is one of the main mechanisms leading to hypothalamic leptin resistance and energy balance disorders.Protein kinase R-like ER kinase(PERK)pathway is an important downstream pathway of endoplasmic reticulum stress,which is involved in the occurrence and development of various chronic metabolic diseases.Aerobic exercise can improve leptin resistance and reduce obesity,but its mechanism has not been fully elucidated.Does the PERK pathway play a role in aerobic exercise improving hypothalamic leptin resistance in obese organisms ? Worth studying.This study used animal and cell experiments,combined with exercise or drug intervention,to explore the role of endoplasmic reticulum stress PERK pathway in aerobic exercise to improve hypothalamic leptin resistance in obese organisms.Method:Animal experiment : 72 SPF 6-week-old C57BL/6J male mice were randomly divided into two groups after one week of adaptation,and fed with normal diet(n=12)and high-fat diet(n=60)respectively.After 16 weeks of feeding,48 mice in the high-fat diet were randomly divided into four groups : high-fat group(HFD),high-fat plus exercise group(HFD+E),high-fat plus placebo group(HFD+P)and high-fat plus PERK inhibitor group(HFD+G),12 in each group.After the successful modeling,the normal diet mice were used as the quiet control group(NC)intervention method unchanged.Mice in the HFD+E group underwent 10 weeks of moderate-intensity aerobic exercise intervention;HFD+G group mice were treated with PERK inhibitor for 5 weeks.After the exercise and drug intervention,the mice in each group were placed in metabolic cages to detect the energy metabolism related indicators before and after intraperitoneal injection of leptin.Tail blood was taken to detect blood glucose concentration.Four mice in each group were randomly selected for intraperitoneal injection of leptin,and tissue samples were taken immediately after30 minutes.Serum leptin concentration was detected by ELISA,liver H&E staining and oil red O staining were used to observe the pathological changes of liver,and epididymal fat H&E staining was used to observe the morphological changes of adipocytes.Western blot was used to detect the expression levels of leptin signal transduction related proteins(STAT3,P-STAT3)and endoplasmic reticulum stress related proteins(GRP78,P-PERK,PERK,P-e IF2α,e IF2α,CHOP,IRE1α,ATF6)in hypothalamus.Cell experiments : Mouse hypothalamus cell line N43/5 was selected for in vitro experiments and divided into control group(N43),model group(N43+PA)and inhibitor group(N43+PA+GSK).PBS control group and leptin activation group were set up in the group.The leptin resistance model was induced by palmitic acid(PA).The inhibitor group was given PERK inhibitor intervention on the basis of PA to explore the role of PERK pathway in leptin resistance.Western blot was used to detect the expression levels of leptin signal transduction-related proteins(STAT3,P-STAT3)and endoplasmic reticulum stress PERK pathway-related proteins(P-PERK,PERK,P-e IF2α,e IF2α,CHOP)in hypothalamic cells.Graph Pad Prism9.0 was used to draw the chart,and SPSS26.0 was used for statistical analysis of the data.Independent sample T test was used between the two groups,single factor analysis of variance was used between multiple groups,and paired T test was used before and after the test between groups.Results:1.Aerobic exercise reduced the obesity phenotype of obese mice induced by high-fat diet and enhanced their energy metabolism : Compared with NC group,the body weight,liver weight,epididymal fat mass,blood glucose,serum leptin concentration,energy consumption and energy intake of HFD group were significantly increased,while respiratory quotient,spontaneous physical activity,unit energy consumption and unit energy intake were significantly decreased.Compared with HFD group,the body weight,blood glucose and serum leptin concentration of mice in HFD+E group were significantly decreased,and spontaneous physical activity was significantly increased.Compared with NC group,liver lipid accumulation and steatosis were significantly increased in HFD group.Compared with the HFD group,mice in the HFD+E group had less liver lipid accumulation and smaller epididymal adipocyte volume.2.Aerobic exercise improves hypothalamic leptin resistance in obese mice induced by high-fat diet : Compared with before intraperitoneal injection of leptin,the energy intake of mice in NC group and HFD+E group was significantly reduced at 12 hours after intraperitoneal injection of leptin,but there was no change in HFD group.Compared with the HFD group,the energy intake of mice in the HFD+E group was significantly reduced at 12 hours after intraperitoneal injection of leptin.The expression of P-STAT3/STAT3 in hypothalamus of NC group and HFD+E group was significantly up-regulated by intraperitoneal injection of leptin,but not in HFD group.3.Aerobic exercise alleviated endoplasmic reticulum stress in hypothalamus of obese mice induced by high-fat diet : Compared with NC group,the expression levels of endoplasmic reticulum stress-related proteins P-PERK,P-e IF2α,CHOP and IRE1αin HFD group were significantly increased,and the proportions of P-PERK/PERK and P-e IF2α/e IF2α were significantly increased.Compared with the HFD group,the expression levels of P-PERK,P-e IF2α,CHOP and IRE1α in the HFD+E group were significantly decreased,and the ratios of P-PERK/PERK and P-e IF2α/e IF2α were significantly decreased.4.PERK inhibitor improves hypothalamic leptin resistance and obesity phenotype in obese mice induced by high-fat diet : Compared with the HFD+P group,the body weight,epididymal fat mass,serum leptin concentration,energy intake,and unit energy intake of the HFD+G group were significantly reduced,and spontaneous physical activity and unit energy consumption were significantly increased.Compared with HFD+P group,liver lipid accumulation and epididymal adipocyte volume were decreased in HFD+G group.Compared with the HFD+P group,the energy intake of mice in the HFD+G group was significantly reduced 12 hours after intraperitoneal injection of leptin.Compared with HFD+P group,the expression of P-STAT3/STAT3 in HFD+G group was significantly increased after intraperitoneal injection of leptin.Compared with HFD+P group,the expression levels of P-PERK,PERK,P-e IF2α,e IF2α,IRE1α,CHOP and the ratio of P-e IF2α/e IF2α in HFD+G group were significantly decreased.5.Inhibition of PERK improves palmitic acid-induced leptin resistance in embryonic mouse hypothalamic cells : After activation with leptin,the proportion of P-STAT3/STAT3 in the control group and the inhibitor group increased significantly,and the proportion of P-STAT3/STAT3 in the model group decreased significantly;compared with the model group,the ratio of P-STAT3/STAT3 in the inhibitor group increased significantly after leptin activation.In addition,compared with the control group,the proportion of P-PERK,PERK,P-e IF2α,e IF2α and CHOP proteins in the model group increased significantly;compared with the model group,the expression of P-PERK,PERK,P-e IF2α,e IF2α and CHOP protein in the inhibitor group was significantly decreased.Conclusion:1.Aerobic exercise can inhibit the endoplasmic reticulum stress PERK pathway in obese mice and improve hypothalamic leptin resistance;PERK pathway inhibitors also have similar effects with aerobic exercise.2.The effect of aerobic exercise on improving hypothalamic leptin resistance in obese mice may be related to its inhibition of endoplasmic reticulum stress PERK pathway.