Effects Of Light And Dietary Rhythm On Insulin-growth Hormone Balance And Glucose And Lipid Energy Metabolism In Male Mice

Background:Insulin and growth hormone(GH)are two important metabolic hormones that can regulate substrate and energy metabolism.These two hormones have different effects on substrate and energy metabolism under different external conditions.Clinical studies have shown that the ratio of insulin:GH may be a biomarker of energy expenditure and fat accumulation.There is a balance between the two hormones and the insulin/GH balance can reflect the body's metabolic state.Previous studies found that different light and feeding patterns can affect the clock system and metabolism through the suprachiastic nucleus(SCN).The effect of light and diet on the balance of insulin/GH and whether it will further affect metabolism is still unclear.This research plans to explore the effects of light rhythm disorder and time-restricted feeding on insulin/GH balance,circadian clock system,substrate and energy metabolism in mice,and further discuss the relationship among them.Part One The effect of rotating light on insulin-growth hormone balance and substrate,lipid and energy metabolism in wild type(WT)miceMethods:The research objects in this study are 5-week-old C57BL6/L male mice obtained from the Endocrinology Laboratory of the University of Queensland,Australia.All mice are kept in the animal facility of the School of Bioengineering,University of Queensland.Twenty mice were randomly divided into two groups(10:10):rotating light(RL)and normal light group(normal light,NL).NL was defined as normal 12h light/12h dark rhythm,turn on the light at 8am(ZT0)and turn off the light at 8pm(ZT12).The mice in the RL group have a normal 12h light/12h dark rhythm for the first three days of a week,and reverse the 12h light/12h rhythm for the next four days with lights on at 8pm(ZT12),lights off at 8am(ZT0).Then we record the body weight and food intake of mice every week and perform growth hormone pulse test in week 8.Pulsatile GH secretion test(start at 9am,take 2ul blood from the tail every 10min,last for 6h).In order to detect the influence of circadian rhythm on energy metabolism,Phenomaster instrument was used to monitor the oxygen consumption,activity level,respiratory exchange ratio and other indicators of mice in the 9th week.ELISA was used to detect the change of GH,insulin,C-peptide,glucagon,leptin,and free fatty acid in blood.After the mice were sacrificed,PCR was performed to detect the expression of clock-related genes(CLOCK,BMAL1,PER1,CRY1 and REV-ERB?),substrate metabolism-related genes(G6P,PEPCK,PGC-1?,PPARa and FASN)and GH-related genes(GH,GHRHR,GHSR and SSTR2)in the liver and pituitary gland.Results:Compared with the NL group,the number of GH bursts in the RL group increased(RL vs NL,4.22±0.36 vs 2.70±0.30,P=0.005),the release of GH each burst decreased(146±26.63 vs 71±18.49,P=0.034),the secretion regularity was weakened(0.40±0.03 vs 0.58 ± 0.05,P=0.005).There was no significant difference in pulsatile secretion,total secretion of GH and secretion mode.The expression of circulating IGF-1 decreased(21.23±2.61 vs 29.50±1.05,P=0.009).The expression of circulating insulin(1.37±0.12 vs 1.99±0.1.9,P=0.014)and C-peptide(1.24±0.13 vs 2.01±0.16,P=0.003)decreased in fasting state.There was no significant difference in insulin secretion in feeding state.Compared with the NL group,the RER of the mice in the RL group was lower in the dark phase.There was no significant difference on RER in the light state.The oxygen consumption of mice in the RL group was reduced in the dark phase(P<0.05),but there was no difference in the light phase,which may be the reason for the increase in fat mass in the RL group.Regardless of in the dark or light phase,there was no significant difference in activity between the two groups.The eating rhythm of the mice in the RL group was disturbed with more food intake in the light phase,and decreased food intake in the dark state(P<0.05).There was no significant difference in the total food intake between the two groups.We further analyzed the daily changes in energy metabolism of the two groups of mice,and found that the RL group mice only reduced oxygen consumption,RER,and food intake(P<0.05)on the first day after light disturbance.There was no significant difference in activity level.There was no significant difference in energy metabolism between the two groups of mice over time,indicating that the mice in the RL group gradually adapted to the disordered light on the first day after switching the light rhythm.The change of RER from the feeding state to the fasting state reflects metabolic flexibility.We recorded the energy metabolism for 12 hours after the conversion.The results found that the RER of the RL group did not decrease rapidly compared with the NL group,indicating reduced metabolic flexibility.Insulin resistance and FFA play an important role in metabolic flexibility.Circulating FFA in the RL group was significantly increased(P<0.05),but there was no significant difference in ITT.Compared with the NL group,the expression of IGF-1 and GHR genes decreased in the RL group in the liver.The expression of GH-related genes GH and GHRHR in the pituitary decreased.There was no significant difference in the expression of GHSR and SSTR2.The expression of peripheral clock system related genes CLOCK,BMAL1,PER1,CRY1,and REV-ERBa decreased.The expressions of fat oxidation-related gene PPAR?,fat synthesis-related gene FASN,gluconeogenesis-related genes G6P,PEPCK,and PGC1-? decreased.There was no significant difference in the expression of fat synthesis-related gene SREBF1.Conclusions1.Disturbance of light rhythm can change the GH secretion mode of WT male mice and disturb the insulin/GH balance.Light disturbances only cause metabolic damage to mice in the first day after changing the light rhythm.It also could disrupt the balance of energy metabolism,glucose and lipid metabolism,and insulin/GH balance may involved in it.Part 2 The effect of time-restricted feeding on insulin-growth hormone balance and substrate and energy metabolism in MC4RKO obese miceMethodsThe research objects in this study are 8-week-old MC4RKO male mice obtained from the Endocrinology Laboratory of the University of Queensland,Australia.All mice are kept in the animal room of the School of Bioengineering,University of Queensland.In order to verify the effect of time-restricted feeding(TRF)on the metabolism of obese mice,14 MC4RKO male mice were randomly divided into two groups(7:7):TRF group and normal food Group(NF).The NF group have access to food for 24 hours,and the TRF group mice have access to food(ZT13-ZT22-24)during the 9-12h of the day when the lights are turned off.Both groups of mice have access to water freely.The two groups of mice have normal 12h light/12h dark rhythm,turn on the light at 8pm(ZT0),and turn off the light at 8am(ZT12).Then we record the weight and food intake of the mice every week,and perform the pulsatile growth hormone secretion test with the lights off in the 5th week(start at 9am,take 2ul blood from the tail every 10 minutes,and last for 6h),and ITT,GTT were performed in 8-9 weeks.The pulsatile growth hormone release test was performed in the 10th week with the lights on(starting at 8pm,taking 2ul blood from the tail every 10 minutes,and last for 6 hours).In order to detect the effect of TRF on energy metabolism,the Phenomaster instrument was used to monitor the oxygen consumption,activity level,respiratory exchange ratio(RER)and other indicators of mice in the 6th week.ELISA was performed to detect the changes of GH,insulin,C-peptide,glucagon,leptin,and free fatty acid(FFA)in blood.After the mice were sacrificed,PCR clock system related genes(BMAL1 and PER1),glucose and lipid metabolism related genes(G6P,PEPCK,PGC-1?,PPAR? and FASN)Express changes.the liver,fat,and pituitary.ResultsIn the dark state,the total GH secretion(TRF vs NF,225.2±38.08 vs 106.9±12.10,P=0.009),pulsatile GH secretion increased(204.3±30.89 vs 101.3±10.04,P=0.006)in the TRF group compared with those in the NF group.There was no significant difference in the number of bursts and secretion patterns.But the secretion of GH each burst increased(55.13 ± 11.03 vs 24.39±6.18,P=0.028),and the secretion pattern was more regular(0.78 ±0.09 vs 1.026±0.04,P=0.020)in TRF group compared those in NF group.Regardless of in feeding or fasting state,TRF reduces the levels of insulin and C peptide(P<0.05).The insulin/GH ratio is related to energy and lipid metabolism.TRF can significantly reduce the insulin/mean GH ratio and the insulin/GH pulse amplitude ratio(P<0.05).In the light phase,there was no significant difference in the total GH secretion,pulsatile GH secretion,and secretion mode between two groups.There was no significant difference circulating IGF-1 level between the two groups.Compared with the NF group,TRF reduced the body weight,subcutaneous fat and visceral fat of mice(P<0.05).The NMR results showed that the ratio of fat mass decreased(ZT14,ZT23),lean mass ratio(ZT14)increased(P<0.05).There was no significant difference in lean mass ratio at ZT23.Results show that TRF reduces RER in light phase and increases oxygen consumption and activity in dark phase(P<0.05).There was no significant difference in oxygen consumption and activity volume in light phase and RER in dark phase.There was no significant difference in the total food intake between two groups.The above results suggested that TRF can increase energy consumption independently of changes in food intake.There was no significant difference in RER between the two groups when switching from the feeding state to the fasting state.When switching from the fasting state to the feeding state,the RER of the TRF group increased rapidly,suggesting an increase in metabolic flexibility.TRF also increased the oxygen consumption and activity.Insulin resistance and FFA are usually associated with metabolic flexibility.It was found that TRF significantly improved insulin sensitivity and reduced the levels of FFA and triglycerides in the blood and liver(P<0.05).In the 10th week,random blood glucose levels(ZT0,6,13,19 and 23)were tested at 5 time points in a 24-hours time range.Results showed that TRF significantly reduced the blood glucose fluctuation and fasting blood glucose level(P<0.05).Glucose tolerance test result showed that TRF improved the glucose tolerance.Compared with the NF group,TRF decreased the expression of fat synthesis genes FASN and SREBF1,fat storage gene CD36,fat oxidation gene PPAR?,gluconeogenesis genes G6P and PEPCK in the liver.The increase of IGF-1 expression confirmed the increase of GH release.Although there was no significant difference in circulating IGF-1 level.At the same time,there was no significant difference in the expression of genes PGC1-? and GHR in the liver.The expression of white fat browning markers UCP-1 and CIDEA in fat increased,and there was no significant difference in the expression of genes TMEM26,PPARy and HSL.In order to explore whether TRF has an effect on the peripheral clock system,we detected the expression of clock genes PER1 and BMAL1 in the liver.Results showed that TRF significantly reduced the expression of PER1 in the liver,but there was no significant difference in BMAL1.Conclusions:1.Time-restricted diet restores GH secretion in MC4RKO obese mice,improves hyperinsulinemia,and restores growth hormone-insulin balance.It also could improve energy metabolism,glucose and fat metabolism,and GH may play a role in it.