Effects Of Fasting Mimicking Diet On Motor Ability,Cognitive Phenotypes And Metabolic Health In Normal Mice

PurposeIt has been widely confirmed that fasting mimicking diet(FMD)intervention can achieve positive health benefits such as reducing fat,improving blood lipids,reducing body weight,improving cognition,preventing and treating chronic diseases in obese,overweight or chronic patients.Would FMD intervention also improve phenotypes in normal weight people(or normal laboratory animals)? There is a lack of related research.At the same time,are there potential health risks and adverse effects of long-term FMD intervention in normal-weight people? Based on the above considerations,this study conducted short-term FMD intervention experiment and long-term FMD intervention experiment in normal mice to study the effects of FMD intervention on motor ability,cognitive phenotypes and metabolic health in normal mice,so as to provide theoretical basis for whether FMD diet is suitable for weight loss and health maintenance in normal people.MethodsIn study 1,sixteen 10-week-old C57BL/6J healthy male mice were divided into two groups: normal diet control group(CON group,n = 8)and fasting mimicking diet group(FMD group,n = 8)after 2 weeks of adaptive feeding.Normal mice in the CON group were fed with normal diet(total energy: 1513 kJ/100 g,energy supply ratio: fat 12.0%,protein 20.6%,carbohydrate 67.4%).Normal mice in the FMD group were fed with FMD diet(total energy: 1526 kJ/100 g,energy supply ratio: fat 45.2%,protein 19.2%,carbohydrate 35.6%),water was freely consumed,FMD diet(energy intake was 1/3 of energy demand)was consumed on the 1st to 3rd day of each week,and ordinary diet was freely consumed on the 4th to 7th day of each week.The intervention period of both groups was 6 weeks.Treadmill exhaustion test,muscle grip test,balance beam test,rope climbing test,Morris Water Maze experiment and open field test were used to test the endurance,muscle strength,balance ability,motor coordination,spatial learning and memory ability,inquiry behavior and anxiety of mice during 4-6 weeks,in order to explore the effects of FMD dietary pattern intervention on motor ability and cognitive phenotypes in normal mice.In study 2 and study 3,twenty 11-week-old C57BL/6J healthy male mice were divided into short-term CON group(n = 5),short-term FMD group(n = 5),long-term CON group(n = 5)and long-term FMD group(n = 5)after one week of adaptive feeding in a single cage.Normal mice in the short-term CON group and the long-term CON group were fed normal diet,with food intake and water intake freely.Normal mice in the short-term FMD group and the long-term FMD group were fed FMD diet with water intake freely.They were fed FMD diet(energy intake was 1/3 of energy demand)on the 1st-3rd day of each week,and freely ingested normal diet on the4th-7th day.The intervention period of short-term CON and short-term FMD group was 2 weeks,and the intervention period of long-term CON and long-term FMD group was 2 years.After the expiration of short-term or long-term intervention,the mice were sacrificed under anesthesia,and the muscle,femur,epididymal fat,inguinal fat,liver and other tissues were taken for reserve.In study 2,real-time quantitative PCR was used to detect mRNA expression levels of genes related to glucose and lipid metabolism,myogenic regulatory factors and muscular atrophy regulatory factors in gastrocnemius of normal mice.The expression levels of autophagy marker LC3 protein,autophagy substrate protein p62 and muscle atrophy marker Trim 63(Mu RF-1)in gastrocnemius were detected by Western blot.Bone structure of femur was scanned by MicroCT and bone analysis parameters were measured.Skeletal muscle morphological structure was observed by HE staining,and comparative analysis was performed to investigate the effects of short-term and long-term fasting mimicking diet intervention on muscle metabolic health and bone health in normal mice.In study 3,electronic balance was used to measure the changes of food intake of normal mice,metabolic cage was used to detect the energy metabolism of mice,Echo MRI animal body composition analysis system was used to detect the body composition of mice,blood glucose was measured by glucose meter,serum and liver triglyceride levels were measured by colorimetry with enzyme marker,and adipose-tissue morphological changes were observed by HE staining.Real-time fluorescence quantitative PCR was used to detect the mRNA expression level of genes related to inflammation in adipose tissue,HE staining was used to observe the morphological changes of liver tissue,real-time fluorescence quantitative PCR was used to detect the mRNA expression level of genes related to glucose and lipid metabolism in liver,transcriptome sequencing was used to detect the changes in liver transcriptome,and comparative analysis was conducted to investigate the effects of short-term and long-term fasting mimicking diet intervention on body composition and glycolipid metabolism in normal mice.Results(1)Compared with the short-term CON group,the short-term fasting mimicking diet intervention significantly decreased the endurance(P<0.01)and balance ability(P<0.05)of normal mice in the short-term FMD group,maintained the relative stability of muscle strength,but did not significantly improve motor coordination in normal mice.(2)Compared with mice in short-term CON group,the number of normal mice entering the central zone in short-term FMD group significantly decreased(P<0.05),the time of staying in the central zone extremely significantly decreased(P<0.01),the times of immobiling extremely significantly increased(P<0.01),and immobile time of mice significantly increased(P<0.05)in the open field experiment.In the orientation cruise experiment on days 1-5 of the Morris Water Maze experiment,the escape latency of normal mice in the FMD group increased significantly(P<0.01)on the first day of learning,but there was no significant difference between the groups on days 2-5 of learning.In the space exploration experiment on day 6,There was no statistical difference between the two groups in the number of crossing the original platform position,the original platform quadrant swimming time,the original platform quadrant swimming time to total time ratio,and the original platform quadrant swimming distance to total distance ratio.(3)Compared with mice in the short-term CON group,the expression levels of gastrecnemius muscular atrophy regulatory factor Trim63(Mu RF1)mRNA and Fbxo32(Atrogin-1)mRNA in the short-term FMD group were significantly down-regulated(P<0.05).The mRNA expression level of myogenic regulatory factor Myh6 was significantly up-regulated(P<0.05),and the weight of gastrocnemius had a downward trend(P=0.085),but the weight of gastrocnemius per unit body weight was similar among groups.Compared with mice in the long-term CON group,the expression levels of gastnemius muscle atrophy regulatory factor Trim 63 mRNA and Fbxo32 mRNA in the long-term FMD group were significantly up-regulated(P<0.05),the expression levels of myogenic regulatory factor Myh6 mRNA were significantly down-regulated(P<0.05),and the expression of LC3-Ⅱ protein was up-regulated,the expression of P62 protein was down-regulated,indicating that autophagy level was increased,the expression level of muscle atrophy marker Trim63(Mu RF1)protein was up-regulated,and the weight of gastnemius muscle was significantly decreased(P<0.01).(4)Compared with mice in the corresponding short-term CON group,GLUT4 mRNA,CD36 mRNA,SREBP-1 mRNA,PGC-1α mRNA and FOXO1 mRNA of normal mice in the 6-week short-term FMD group had no significant changes.PEPCK mRNA expression was significantly down-regulated(P<0.05),and G6 Pase mRNA expression showed a decreasing trend.(5)Compared with long-term CON group,the bone analysis parameters bone volume(BV),relative bone volume(BV/TV)and bone trabecular number(Tb.N)of femur in long-term FMD group significantly decreased(P<0.05),and bone trabecular separation(Tb.Sp)extremely significantly increased(P<0.01).(6)At week 2,the average daily food intake and average daily energy intake of normal mice in short-term FMD group were significantly(P<0.05)or extremely significantly(P<0.01)higher than those in short-term CON group,respectively.At week 82,the average daily food intake and average daily energy intake of normal mice in the long-term FMD group were similar to those in the long-term CON group.At week 83,oxygen consumption and energy consumption of normal mice in long-term FMD group were significantly higher than those in long-term CON group,and respiratory exchange rate was significantly lower than those in long-term CON group.(7)Compared with short-term CON group,body fat weight of normal mice in short-term FMD group increased(P<0.01),BAT weight decreased(P<0.01),i WAT weight significantly increased(P<0.05),and body weight,lean body weight and eWAT weight were not significantly different among groups.The body weight,body fat weight,BAT weight and eWAT weight of normal mice in the long-term FMD group showed a decreasing trend compared with those in the long-term CON group,but there was no significant difference between groups.The weight of i WAT had decreased to a similar level to that of mice in the long-term CON group,and the lean body weight was significantly lower than that in the long-term CON group(P<0.01).Compared with mice in the long-term CON group,the long-term fasting mimicking diet intervention significantly reduced the muscle strength of normal mice in the long-term FMD group(P<0.05).(8)During short-term FMD intervention,there were no significant differences in blood glucose and serum triglyceride levels between the FMD group and the CON group.At week 38,there were no significant differences in glucose tolerance and insulin tolerance of normal mice in the long-term FMD group compared with those in the long-term CON group.At week 83,the area under glucose tolerance curve of normal mice in long-term FMD group was significantly higher than that in long-term CON group(P<0.05),and the area under insulin curve showed a increasing trend compared with that in long-term CON group,but there was no significant difference between groups.(9)Compared with the mice in the short-term CON group,the expressions of inflammatory factor IL-6 mRNA and TNF-α mRNA in the inguinal white adipose tissue of normal mice in the short-term FMD group were up-regulated(P<0.01),and the expression of inflammatory factor MCP-1 mRNA was up-regulated(P<0.05).(10)Compared with the CON group,the expressions of ATGL mRNA,CD36 mRNA and PGC-1α mRNA in liver of mice in FMD group were significantly up-regulated after 6-week fasting mimicking diet intervention(P<0.05).The expressions of HSL mRNA and FOXO1 mRNA showed a increasing trend,the expression of G6 Pase mRNA showed a decreasing trend,and liver triglyceride level was significantly decreased(P<0.01).Compared with mice in the corresponding CON group,the liver weight of normal mice in FMD group was significantly decreased after 2-week short-term fasting mimicking diet intervention(P<0.01),the liver weight of normal mice in FMD group was significantly decreased after 2-year long-term fasting mimicking diet intervention(P<0.05).(11)According to the results of gene set enrichment analysis(GSEA)on differentially expressed genes,the liver inflammation signaling pathway and cell aging signaling pathway were down-regulated after long-term fasting mimicking diet intervention,while the liver lipid metabolism signaling pathway PPAR signaling pathway was activated.Conclusions(1)The short-term fasting mimicking diet significantly reduced the endurance and balance ability of normal mice,maintained the relative stability of muscle strength,but did not significantly improve motor coordination in normal mice.Meanwhile,short-term fasting mimicking diet weakened the inquiry behavior and enhanced the anxiety of normal mice,and did not significantly improve the spatial learning and memory ability of normal mice.(2)Short-term fasting mimicking diet reduced muscle atrophy and maintained the relative stability of lean body mass in normal mice;Long-term fasting mimicking diet promoted muscle atrophy and significantly reduced lean body mass and muscle strength in normal mice,and exacerbated the destruction of bone microstructure and decreased bone mass in normal mice during aging.It is suggested that long-term implementation of fasting mimicking diet pattern in normal weight population has the potential risk of promoting muscular atrophy,reducing lean body mass,decreasing muscle strength and increasing bone mass loss.(3)Short-term fasting mimicking diet significantly increased body fat weight and i WAT weight of normal mice,which was due to the fact that normal mice did not adapt to low-energy diet during fasting period of short-term fasting mimicking diet intervention,resulting in experiencing stress-compensated high food intake during the free-feeding period,and the excess food energy intake was converted into fat deposition in the body.However,the normal mice receiving long-term fasting mimicking diet have adapted to the production and utilization of ketone bodies through a "metabolic conversion" during the alternating process of long-term fasting and free feeding,and have improved the efficiency of fat utilization,thus reducing body fat weight and i WAT weight.(4)Fasting mimicking diet significantly upregulated the mRNA expression of genes related to liver lipid metabolism in normal mice,which significantly reduced liver triglyceride level and liver weight in normal mice,significantly improved ectopic fat deposition in the liver of normal mice,and promoted metabolic health.The effect of fasting mimicking diet on the improvement of liver lipid metabolism may be partly through the regulation of PPAR signaling pathway.