Effects Of Exogenous Alpha-ketoglutaric Acid On Protein Synthesis And Degradation Of Skeletal Muscle And Its Mechanisms

Protein is an important material basis in life,and it can directly affect the growth development and skeletal muscle protein deposition when protein is metabolized and turnover in animals.The amount of protein deposition mainly depends on a comprehensive effect between protein synthesis and protein degradation in skeletal muscle.?-ketoglutarate acid?AKG?is the key intermediate products in TCA cycle,and is also a metabolism of glutamate acid?Glu?.AKG has the ability in anti-oxidation and anti-stress and also improves animal energy metabolism and other biological functions.Some study showed that AKG could activate mTOR pathway in Hela cells,but it was not reported that the effects and mechanisms of AKG on protein synthesis and degradation in animals skeletal muscle.This study includes the following five parts.Part 1:Effects of AKG on mice growth development and skeletal muscle protein synthesisTo study the roles of AKG in skeletal muscle protein metabolism,mice were used as objects toobserve that the change of AKG levels in mice serum and skeletal muscle by offering different stimulus,including test,low speed running and high running for mice.The results showed that with the increase of exercise intensity,AKG concentrations were significantly increased in mice serum,soleus muscle and gastrocnemius muscle.Further,to study effects of AKG on mice growth development and skeletal muscle protein synthesis,1 g/kg AKG was injected for 3 h by i.p.and 2%AKG was used to feed mice for 8 weeks by supplementing into drinking water.The results suggested that i.p.AKG significantly increased puromycin?marker for protein synthesis?incorporation and the phosphorylation of S6 and 4E-BP1 of mTOR downstream in mice gastrocnemius muscle.Moreover,AKG significantly inhibited the expression of protein degradation related genes,MuRF1 and MAFbx.2%AKG significantly promoted mice mean body weight gain and cumulative feed intake,increased the weight of gastrocnemius muscle and skeletal muscle fibers areas,enhanced the muscle grip of mice,and extended the time of low speed running and high speed running.AKG also significantly increased total protein,albumin and IGF-1 levels in mice serum,and reduced BUN levels.We also found that AKG significantly promoted phosphorylation of Akt,mTOR,S6,4E-BP1 and FoxO1 in mice gastrocnemius muscle,and inhibited the expression of MuRF1 and MAFbx.The above results suggested that AKG could promote protein synthesis and inhibit protein degradation in mice gastrocnemius muscle.Part 2:Mechanisms of AKG in promoting protein synthesis in skeletal muscle cellsThe above study showed that AKG could promote protein synthesis in mice gastrocnemius muscle both through i.p.and supplementing into drinking water.However,AKG in drinking water and i.p.may be converted into its metabolites to play the roles in vitro.C2C12 cells were used as objects to research whether it was the directly effect of AKG on promoting protein synthesis in skeletal muscle.In this test,2 mM AKG and Glu were supplemented into medium to treat C2C12cells for 48 h to research the effects on protein synthesis.The results showed that AKG significantly increased total protein levels and puromycin incorporation of C2C12 cells,and promoted the phosphorylation of S6 and 4E-BP1 of mTOR downstream and the expression of MHCII.However,there was not significantly effect of Glu on total protein levels of cells and mTOR downstream signaling molecular.The above results suggested that AKG could directly promote protein synthesis in skeletal muscle,but did not converted into its metabolites to play the roles.Part 3:AKG regulated its reporter and the signaling pathway of protein synthesisThe above study already found that AKG could significantly promote protein synthesis in skeletal muscle both in vitro and vivo.Further,in order to reveal its mechanism,C2C12 cells were treated with 2 mM AKG for 1,2 and 4 h to reveal the regulation effects of AKG on Akt/mTOR and Akt/FoxO1 pathway.The results showed that AKG could significantly activated Akt/mTOR and Akt/FoxO1 pathway.At the same time,we also found inhibitors of Akt and mTOR significantly blocked the effects of AKG on activation of Akt and mTOR pathway.It was reported that GPR99 and GPR91 were the recepter of AKG.But in this study,we found that there was only the expression of GPR91 in skeletal muscle,but not GPR99,moreover,GPR91could responded the change of AKG.We also reduced the expression of GPR91 in skeletal muscle cells members by the method of RNA interference.The results suggested that the inhibition effects of AKG on MuRF1 and MAFbx could not be blocked by interference of GPR91,but interference of GPR91 could block the effects of AKG on promoting the phosphorylation of S6 and 4E-BP1,and could not block the effects of AKG on promoting the phosphorylation of mTOR.The above results showed that GPR91 partially mediates the role of AKG in promoting skeletal muscle protein synthesis.In this study,we also found that it could express a kind of depending on AKG oxygen sensor,proline hydroxylase?PHD?.Among them,the expression of PHD3 was the highest,and followed by PHD1 and PHD2.At the same time,we found AKG could inhibit the expression of PHD3 in skeletal muscle.Furthermore,through overexpression of PHD3 in C2C12 cells,we found it could significantly inhibit the effects of AKG on promoting puromycin incorporation and myotubes diameter,the activation effects of mTOR signaling pathway,and the inhibition effects of AKG on FoxO1/MuRF1/MAFbx.The above results suggested that PHD3 played the important roles in AKG-induced protein synthesis of skeletal muscle.Part 4:ADRB2 mediated AKG promoting protein synthesis of skeletal muscleSome papers reported that?_2-adrenergic receptor?ADRB2?play very important roles in skeletal muscle hypertrophy and protein synthesis,and PHD3 could hydroxylate ADRB2 to ubiquitinate ADRB2 and further to degrade ADRB2.To research the effects of PHD3/ADRB2 on AKG-regulating protein synthesis and degradation in skeletal muscle,we found that it had significantly interaction effects between PHD3 and ADRB2 by immunoprecipitation.However,AKG could reduce this interaction effects,thereby reducing the hydroxylation of ADRB2 by PHD3and significantly inhibit ubiquitination of ADRB2.Further,C2C12 cells and mice were co-treated with ADRB2 inhibitor and AKG.The results showed that it could significantly block the effects of AKG on activating of mTOR pathway by blocking ADRB2 and the effects of AKG on inhibition of FoxO1/MuRF1.In addition,we knocked down the expression of ADRB2 by injecting the lentiviral interference vector of ADRB2 into the mice gastrocnemius muscle.Then the mice were fed for 8weeks by supplementing 2%AKG into drinking water.The results showed that it could significantly reduce the muscle grip,high speed running and low speed running of mice,block the effects of AKG on increasing the mice gastrocnemius weight and skeletal muscle fiber areas by knocking down ADRB2.And it could significantly block the effects of AKG on activation of mTOR signaling pathway and inhibition of MuRF1 and MAFbx by knocking down ADRB2.The above results showed that PHD3/ADRB2 could mediate the effects of AKG on regulating protein synthesis in skeletal muscle.Part 5:Effects of AKG on alleviating muscle atrophy and protein degradation Protein turnover includes protein synthesis and degradation.The above study found that AKG could significantly promote protein synthesis in skeletal muscle.The of muscle atrophy?DMD?were used as objects to investigate whether AKG had the effects on regulation of protein degradation.We observed that the effects of AKG on protein degradation of DMD mice by supplementing 2%AKG into drinking water for 8 weeks.The results showed that DMD mice could maintain a normal growth development,but there were serious injury in gastrocnemius muscle.AKG could significantly alleviate gastrocnemius injury,increase gastrocnemius and muscle fiber area,and promote its body weight gain.AKG also significantly increased the muscle grip of DMD mice and endurance excise,significantly downregulated the expression of MuRF1 and MAFbx of DMD mice.Corticosterone was used to induce mice muscle protein degradation by i.p.in this experiment.At the same time,AKG was co-injected to investigate the effects of AKG on relieving protein degradation in skeletal muscle.The results showed that AKG could significantly alleviate the effects of increasing of corticosterone-induced FoxO1,MuRF1 and MAFbx.The above results suggested that AKG could significantly alleviate muscle atrophy of DMD mice and corticosterone-induced muscle protein degradation.In summary,this study results suggested that AKG could promote significantly protein synthesis,inhibit protein degradation in skeletal muscle,and increase skeletal muscle fiber diameter.Its mechanisms were through inhibiting PHD3 expression,reduce hydroxylation of ADRB2,activating ADRB2 and Akt/mTOR and Akt/FoxO1 signaling pathway of ADRB2 downstream,one hand to promote protein synthesis,and the other hand to inhibit protein degradation in skeletal muscle,thereby achieving to increase skeletal muscle fiber diameter and muscle hypertrophy.Moreover,AKG could significantly alleviate muscle atrophy of DMD mice and corticosterone-induced muscle protein degradation by inhibiting the expression of MuRF1 and MAFbx.The above results deeply revealed that nutritional metabolism intermediates,AKG had an important role in regulating protein metabolism in skeletal muscle,enriched a new understanding in the physiological functions of intermediate metabolites.Moreover,it had very important reference value through further to research the effects of AKG on regulating muscle atrophy,alleviating muscle fatigue and muscle weakness and promoting human health.Furthermore,it has also of great reference value for the effective regulation of pig carcass traits and meat production rate in pig production.