The Regulation And Underlying Mechanisms Of Hydrogen Sulfide And Allicin On Glucose Uptake And Utilization By Skeletal Muscle Of Broilers

As a gas transmitter,the physiological effects of hydrogen sulfide（H₂S）have attracted increasing attention.Skeletal muscle is the largest organ in body,accounting for approximately 40%of broilers weight.The body weight gain,pectoral muscle weight and feed conversion efficiency of broilers are improved by selection in commercial genetic breeding system.From 1994 to 2018,the energy consumption of broilers reaching market weight is reduced by 16%.Glucose was an important energy source in the body.Muscle fibers are tissue-specific in glucose utilization due to the discrepant metabolic regulation,which further affectes the feed conversion efficiency.The pectoralis major（PM）muscle of broilers mainly consistes of fast-twitch glycolytic fibers,whereas,the biceps femoris（BF）muscle mainly consistes of slow-twitch oxidative fibers.This study aimed to investigate the effects and mechanisms of H₂S and allicin,a natural H₂S donor,on glucose uptake in different muscle fibers of broilers,which provides basis for regulating glucose homeostasis and improving the feed conversion efficiency.1.Effects of H₂S on glucose utilization in skeletal muscle of broilersThe effects of H₂S on growth performance of broilers were preliminarily investigated with Na HS（Sodium hydrosulfide,H₂S donor）intraperitoneal injection.Forty-eight 1-day-old AA（Arbor Acres）male broilers with similar body weight were randomly subjected to 2treatments.The Na HS treatment group was subjected to intraperitoneal injection of Na HS（50μmol/kg BW/day）at 8:00 am and 8:00 pm,and the control group was injected with the same amount of saline.Each treatment has 3 replicats,and each replicate had 8 chicks.After continuously injected for 7 days,the blood,PM muscle and BF muscle samples were collected.Results showed that Na HS had no significant effect on body weight and the weight of breast muscle and thigh muscle（P>0.05）,while significantly decreased the blood glucose and insulin level（P<0.05）of broilers.The glycogen content was increased in PM muscle（P<0.05）,but showed a tendency to be decreased in BF muscle（P=0.06）by Na HS treatment.In order to further investigate the regulatory effects of Na HS on glucose homeostasis of broilers,128 1-day-old AA male broilers treated with Na HS or saline were subjected to oral glucose（2 g/kg BW）treatment.Results showed that the glucose level was lowered in Na HS+glucose group when compared with the glucose group（P<0.05）.S-sulfhydrated and phosphorylated adenosine monophosphate activated protein kinase（AMPK）were then detected in PM and BF muscles of broilers.Results showed that S-sulfhydrated and phosphorylated AMPK were not significantly changed in PM muscle（P>0.05）,but activated in BF muscle by Na HS treatment（P<0.05）,which indicating that intraperitoneal injected Na HS enhanced the glucose homeostasis of broilers,and may specifically promote glucose utilization in muscle fiber depemdent way.2.Effects and mechanisms of H₂S on glucose uptake and utilization in myoblastsTo thoroughly investigate the regulatory effects of H₂S on glucose uptake in different muscle fibers,myoblasts from chicken embryonic PM and BF muscles were isolated and cultured.In myoblasts from PM muscle,results showed that glucose uptake（P<0.05）,intracellular H₂S level（P<0.05）,cell viability（P<0.01）and the production of adenosine triphosphate（ATP）（P<0.05）were significantly increased by Na HS（100μM）.The protein expression of AMPK S-sulfhydration and AMPK phosphorylation in myoblasts from PM muscle were significantly activated by Na HS（P<0.05）.DL-dithiothreitol（DTT）and Compound C（AMPK inhibitor）were then used to block the protein expression of AMPK S-sulfhydration and AMPK phosphorylation.The glucose uptake,S-sulfhydrated AMPK and phosphorylated AMPK promoted（P<0.05）by Na HS were significantly suppressed（P<0.05）in the present of DTT or Compound C in myoblasts from PM muscle,indicating that Na HS may promote glucose uptake via AMPK S-sulfhydration and AMPK phosphorylation in myoblasts from PM muscle.Moreover,phosphorylated AMPK activated by Na HS was significantly suppressed by Na HS+DTT（P<0.05）,suggesting that phosphorylated AMPK may be regulated by S-sulfhydrated AMPK in the upstream.Therefore,Na HS activated AMPK phosphorylation via AMPK S-sulfhydration,and thus promoted glucose uptake in myoblasts from PM muscle.Phosphatidylinositol-3-kinase（PI3K）/Akt and mitogen activated protein kinase（MAPK）are two main insulin pathways in poultry.Results showed that in myoblasts from PM muscle,Na HS（100μM）did not stimulate the PI3K/Akt pathway（P>0.05）,but significantly activate extracellular signal regulated kinase（ERK）,c-Jun N-terminal kinase（JNK）and P38 signaling pathway（P<0.05）.However,in myoblasts from PM muscle,the P38 inhibitor SB203580suppressed the glucose uptake increased by Na HS（P<0.01）,indicating that Na HS promoted glucose uptake via P38 MAPK pathway.Furthermore,the protein expression of phosphorylated P38 was suppressed in the present of DTT and Compound C in myoblasts from PM muscle（P<0.05）,indicating that P38 signaling pathway maybe regulated by AMPK pathway.Results in mammalian target of rapamycin（m TOR）pathway showed that Na HS（100μM）promoted protein synthesis via m TOR/p70S6K pathway in myoblasts from PM muscles（P<0.05）and may be regulated by S-sulfhydrated and phosphorylated AMPK.Rapamycin was used to block the expression of m TORC1.The glucose uptake and protein expression of p-AMPK were not changed in Na HS+Rapamycin group when compared with the Na HS group（P>0.05）,suggesting that maybe m TOR was not involved in regulating Na HS induced glucose uptake.Therefore,the PI3K/Akt pathway did not involve in the Na HS regulated glucose uptake in myoblasts from PM and BF muscles.Na HS promoted glucose uptake via activating AMPK/P38 pathway in myoblasts from PM muscle.Na HS activated AMPK signaling pathway thereby stimulated m TOR/p70S6K pathway to promote protein synthesis but not glucose uptake in myoblasts from PM and BF muscles.In myoblasts from BF muscle,Na HS（100μM）significantly decreased the glucose uptake（P<0.05）,but increased the intracellular H₂S level（P<0.05）,cell viability（P<0.01）,ATP level（P<0.05）and the reactive oxygen species（ROS）level（P<0.05）.The protein expression of AMPK S-sulfhydration and AMPK phosphorylation（P<0.05）were significantly activated（P<0.05）by Na HS.However,the inhibitory effects of Na HS on BF myoblasts were not related to the activation of AMPK.Na HS（100μM）significantly suppressed the protein expression of p-P38（P<0.05）,while had no effects on PI3K/Akt,ERK and JNK signaling pathway（P>0.05）.U46619 was used to activate P38 MAPK signaling patway.Na HS+U46619 significantly promoted the protein expression of phosphorylated AMPK（P<0.01）and glucose uptake（P<0.05）when compared with Na HS treatment.Collectively,the PI3K/Akt pathway did not involve in the Na HS regulated glucose uptake in myoblasts from BF muscle.Na HS suppressed glucose uptake via suppressing P38MAPK pathway.Na HS（100μM）promoted protein synthesis via m TOR/p70S6K pathway in myoblasts from BF muscle（P<0.05）.The glucose uptake and protein expression of p-AMPK were not changed in Na HS+Rapamycin group when compared with the Na HS group（P>0.05）.Therefore,Na HS activated AMPK signaling pathway thereby stimulated m TOR/p70S6K pathway to promote protein synthesis but not glucose uptake in myoblasts from BF muscle.3.Effects of allicin on glucose utilization in skeletal muscle of broilersAllicin is a natural organic sulfur compound,which acts as H₂S donor to regulate energy homeostasis.A total of 180 1-day-old AA male broilers with similar body weight were randomly divided into 3 groups.Each group has 4 replicates and 15 broilers per replicate.The control group was fed a basal diet,and the allicin group was fed allicin diet which supplemented with allicin 150 mg/kg and 300 mg/kg,respectively.At 21-day-old and42-day-old of broilers,the body weight,feed intake,fasting blood glucose,muscle glycogen and plasma H₂S level were detected.Effects of allicin on blood glucose homeostasis of broilers were tested by glucose gavage test under feeding and fasting state.S-sulfhydrated and phosphorylated AMPK in skeletal muscle were detected.At day 42,broilers were subjected to intraperitoneal injection of PAG（D,L-propargylglycine）30 mg/kg BW consecutively for 3days to explore the effects of endogenous H₂S.Results showed that allicin（150,300 mg/kg）significantly elevated the production performance of broilers（P<0.05）,had no effect on fasting blood glucose（P>0.05）,and decreased the glycogen content in PM and BF muscles（P<0.05）.At day 21,allicin（300mg/kg）significantly suppressed the plasma H₂S level（P<0.05）.The glucose oral test showed that the glucose level in allicin group was significantly lower than that in control group either in feeding or fasting state（P<0.05）.Dietary allicin significantly decreased S-sulfhydrated AMPK in PM and BF muscles（P<0.05）,decreased the protein expression of p-AMPK in PM muscle（P<0.05）,but increased the protein expression of p-AMPK in BF muscle（P<0.05）.The fasting blood glucose was significantly decreased by PAG（P<0.05）,while restored by allicin+PAG treatment（P<0.05）.Compared with allicin treatment group,allicin+PAG had certain combined effects on glycogen content,S-sulfhydrated AMPK and phosphorylated AMPK.Therefore,the production performance,glucose uptake and utilization,and the protein expression of p-AMPK in BF muscle of broilers were elevated by dietary allicin.Endogenous CSE/H₂S system at least partly regulated the effects of allicin on glucose utilization and AMPK pathway in skeletal muscle of broilers.4.Effects and mechanisms of allicin on glucose uptake and utilization in myoblastsTo further investigate the effects of allicin on glucose uptake and utilization,myoblasts from chicken embryo PM and BF muscles were cultured and treated with allicin（0.1,1μg/m L）in vitro.In myoblasts from PM muscle,allicin significantly increased（P<0.05）glucose uptake,intracellular H₂S level,glutathione（GSH）level and the production of ATP,while had no significant effects（P>0.05）on ROS level and glycogen content.Allicin still inducded（P<0.05）the protein expression of S-sulfhydrated AMPK and phosphorylated AMPK.However,the glucose uptake（P<0.01）,S-sulfhydrated AMPK（P<0.05）and phosphorylated AMPK（P<0.05）increased by allicin were suppressed in the present of DTT and Compound C,indicating that allicin may regulate glucose uptake via S-sulfhydrated AMPK and phosphorylated AMPK in myoblasts from PM muscle.However,in myoblasts from BF muscle,the glucose uptake,intracellular H₂S level and GSH level were not affected（P>0.05）by allicin.Meanwhile,the ATP content,ROS level and glycogen content were not influenced（P>0.05）by allicin.Allicin still did not affect（P>0.05）the protein expression of S-sulfhydrated AMPK and phosphorylated AMPK in myoblasts from BF muscle.Results suggested the differences of different muscle fibers on glucose uptake and utilization by allicin treatment.Cystathionineγlyase（CSE）was the main endogenous enzyme to produce intracellular H₂S.Results showed that allicin significantly increased the expression of CSE in myoblasts from PM muscle（P<0.05）,whereas had no effects on myoblasts from BF muscle（P>0.05）.PAG was the inhibitor of CSE.In myoblasts from PM muscle,the increased（P<0.05）intracellular H₂S level and glucose uptake induced by allicin were significantly decreased in the present of PAG（P<0.05）.AMPK S-sulfhydration and phosphorylation activated（P<0.05）by allicin were also suppressed by allicin+PAG（P<0.05）.Collectively,allicin may activate AMPK signaling pathway via CSE/H₂S system to promote glucose uptake in myoblasts from PM muscle but not BF muscle.In conclusion:（1）dietary allicin or intraperitoneal injected Na HS could promote glucose uptake and utilization of broilers;（2）allicin increased the production of endogenous H₂S through CSE/GSH level,thereby activated S-sulfhydrated and phosphorylated AMPK,and then promoted the glucose uptake in myoblasts from PM muscle;（3）Na HS stimulated S-sulfhydrated and phosphorylated AMPK in myoblasts from PM and BF muscle,promoted glucose uptake via AMPK/P38 pathway in myoblasts from PM muscle,and suppressed glucose uptake via P38 MAPK pathway in myoblasts from BF muscle;（4）Na HS activated the m TOR pathway through AMPK pathway to promote protein synthesis in myoblasts from skeletal muscles of broilers.