The Function Of The Phosphorylation Of AMPKα2 Threonine 172 On The Regulation Of Glucose And Fat Metabolism In Skeletal Muscle Contraction

AMPK is an important cellular energy sensor for maintaining cellular energy homeostasis.AMPK regulates multiple metabolic processes,including glucose metabolism,lipid metabolism,protein metabolism and autophagy/mitochondrial homeostasis,and it’s closely associated with the activation of AMPK.Exercise,nutrient starvation,mitochonsrial poisons（metformin,rotenone and CCCP）,AMPK activator AICAR,A-769662 and 991 are common modifiers to activate AMPK.In order to study the regulated mechanism of AMPK,most studies applied muscle-specific AMPKα1/α2 single or double knock out（DKO）mouse models,the defect of these models are to impair AMPKαstructure and function,so that it leads to the inactivation of AMPK and AMPK downstream effectors and further to worse endurance exercise capacity.Is this due to the lost function of some specific sites or the whole structural impairment of AMPK complex?We don’t know the real resson.Therefore,our study applies point mutation of AMPKα2 Thr172 phospho-site to develop whole body AMPKα2T172A knock in（KI）mice.This model’s biggest advatange is to maintain the integrity of AMPKαstructure and only to inhibit the phosphorylation of AMPK.Thoughα2T172A KI mouse,we could further confirm whether the effects of muscle contraction on glucose and fat metabolism depends on the phosphorylation of AMPKα2 Thr172.Based on this,it’s convenient to provide scientific basis and precise medical targets for exercise improving and treating obesity,type 2 diabetes or other metabolic diseases.OBJECTIVE（1）Based on wild type（WT）mouse model,further confirm the phosphorylated effects of acute exercise and electrical stimulation-induced muscle contraction on the activation of AMPK and its downstream effectors.（2）Based on AMPKα2T172A KI homozygous mouse model,discuss the significance of the phosphorylation of AMPKα2 Thr172 on regulating muscle contraction-induced glucose and fat metabolism.（3）Based on type 2 diabetic mouse model induced by high fat diet and STZ i.p.injection,discuss the association of endurance exercise training improving glucose and fat metabolism in skeletal muscle and AMPK signaling pathway.METHOD1.The effects of acute exercise on the activation of AMPK and its downstream signaling pathway in skeletal muscleIn order to confirm the effetcs of acute exercise on the activation of AMPK,male C57BL/6J mice were selected to be divided into sedentary group（Sed）and exercise group（Ex）.The exercise protocol is 5%incline,90 min including 10 min at 13.4m/min,10 min at 16.1 m/min,50 min at 18.8 m/min,and then 20 min at 21.5 m/min increased load acute treadmill running.Therefore,we could observe the impacts of acute exercise on skeletal muscle AMPK and its downstream effectors.Sedentary group mice were sacrificed on sedentary condition.Exercise group mice were immediately sacrificed or cervical dislocation after 90 min acute exercise.Muscle harvest:plantaris muscle.Western blot method tests the activation of p-AMPKαThr172,p-ACC Ser79,p-Ulk1 Ser555,p-TBC1D1 Ser237,p-TBC1D4Ser318/Ser588/Thr642 and p-Akt Ser473/Thr308 phospho-proteins in skeletal muscle.2.The effects of electrical stimulation on the activation of AMPK and its downstream signaling pathway in skeletal muscleIn order to confirm acute exercise activated AMPK in skeletal muscle is directly because of muscle contraction,our study applied different frequency and different time duration of electrical stimulation to observe the impacts of different electrical stimulation on the activation of AMPK and its downstream signaling pathway.Detailed protocols are shown as following:（1）Adopt high frequency short time（100 Hz,20 min）and low frequency long time（10 Hz,2 h）electrical stimulation separately mimicking acute resistance exercise and acute endurance exercise,western blot method tests the activation of p-AMPKαThr172,p-ACC Ser79,p-TBC1D1 Ser237,p-TBC1D4 Ser318/Ser588/Thr642 and p-Akt Ser473/Thr308 phospho-proteins in skeletal muscle.Meanwhile,observe the effects of 100 Hz 30 min electrical stimulation and 1.5U/ml dose of insulin ip.injection on GLUT4 translocation in skeletal muscle.（2）According to 100 Hz（20 min,1 h and 2 h）and 10 Hz（20 min,1 h and 2 h）different frequency and different time duration of electrical stimulation,western blot method tests the effects of electrical stimulation frequency and time duration on the phosphorylation of p-AMPKαThr172,p-ACC Ser79,p-TBC1D1 Ser237 and p-TBC1D4 Ser318/Ser588/Thr642 phospho-proteins in skeletal muscle.3.The effects of the phosphorylation of AMPKα2 Thr172 on the regulation of glucose and fat metabolism in muscle contractionIn order to confirm whether the activation of acute exercise and electrical stimulation-induced muscle contraction on AMPK and its downstream signaling pathway depends on the phosphorylation of AMPK Thr172,we applied CRISPR-Cas9 gene encoding technology to locate AMPKα2 Thr172 phospho-site and realize point mutation of one DNA nucleotide.Threonine encoded by ACT was mutated to alanine encoded by GCT so that using this technology developed AMPKα2 Thr172 KI homozygous mice.（1）Mouse body composition was tested by Echo MRI machine.Energy metabolism indicators were tested by OXYMAX metabolic cage during exercise and post-exercise recovery inα2（T172）KI homozygous mice and WT littermates.Monitor the comparison of RER（respiratory exchange ratio）,VO₂（oxygen uptake）,according to formula calculate carbohydrate（CHO）utilization and fat utilization betweenα2（T172）KI homozygous mice and WT littermates during 6-day voluntary wheel running and 3-day post-exercise recovery.（2）Harvest gastrocnemius whole cell lysate,cytosolic and mitochondrial fraction ofα2（T172）KI homozygous mice and WT littermates.Observe the effects of acute exercise on the phosphorylation and activation of AMPK and its downstream signaling pathway in skeletal muscle ofα2（T172）KI homozygous mice.Western blot method tests the phosphorylation of p-AMPKαThr172,p-ACC Ser79,p-Ulk1 Ser555,p-TBC1D1 Ser237,p-TBC1D4 Thr642,p-Erk1/2 Thr202/Tyr204 and the protein expression of Cox4,LC3II/I.Thus,we could observe the effects of acute exercise on GLUT4 translocation in skeletal muscle by using cryo-sectioning and immunoflurescent staining.（3）Apply low frequency long time（10 Hz,2 h）electrical stimulation mimicking endurance exercise,and observe the effects of electrical stimulation on the activation of AMPK and the phosphorylation of its downstream signaling pathway inα2（T172）KI homozygous mice.Western blot method tests the phosphorylation of p-AMPKαThr172,p-ACC Ser79,p-TBC1D1 Ser237 and p-TBC1D4 Ser318/Ser588/Thr642phospho-proteins in skeletal muscle.4.The effects of chronic exercise training on blood glucose and skeletal muscle AMPK signaling pathway in type 2 diabetic miceIn order to further confirm whether chronic exercise training improved type 2diabetes is associated with the activation of AMPK and the phosphorylation of its downstream signaling pathway in skeletal muscle,we selected male C57BL/6J mice and performed 4-week high fat diet（45%fat content）and three low dose of 40 mg/kg STZ intraperitoneal injection to build type 2 diabetic mice.The standard of type 2diabetic mice is fasting blood glucose over 11.1 mmol/L.C57BL/6J healthy male mice were randomly divided into control（Con）group and endurance training（ET）group,type 2 diabetic mice were randomly divided into diabetic control（DC）group and diabetic endurance training（DET）group.Exercise training protocol was shown as following:1 h 70%VO₂max exercise training/day,5 days/week,8-week treadmill running.Test all groups of mice body weight,fasting blood glucose during8-week training and fasting blood glucose and serum insulin on week 8.Western blot method tests the phosphorylation of p-AMPKαThr172,p-ACC Ser79,p-TBC1D1Ser237,p-TBC1D4 Thr642 and p-m TOR Ser2448 phospho-proteins and the protein expression of GLUT4 and CPT1 in skeletal muscle.RESULTS1.Acute exercise activated the phosphor-sites of AMPKαThr172 and its downstream effectors ACC Ser79 and Ulk1 Ser555,and upregulated the phosphorylation of TBC1D1 Ser237 in skeletal muscle.High frequency electrical stimulation（100 Hz 20 min and 1 h）significantly activated AMPK/ACC,TBC1D1and Akt signaling pathways in skeletal muscle,which expressed as the phosphorylation of AMPKαThr172,ACC Ser79,TBC1D1 Ser237 and Akt Ser473were significantly increased.Meanwhile,10 Hz 2 h electrical stimulation significantlt activated AMPKαThr172 and Akt Thr308 in skeletal muscle.100 Hz 30 min electrical stimulation promoted skeletal muscle GLUT4 translocation to plasma membrane,which expressed as increased immunoflurescent of GLUT4 on muscular plasma membrane.2.During light cycle of post-exercise recovery period,fat utilization percentage of AMPKα2T172A KI homozygous mice was significantly lower than wild type littermates,fat utilization percentage ofα2（T172A）KI homozygous mice was decreased by 50.4%.But there was no significant difference of oxygen uptake（VO₂）and endurance exercise capacity betweenα2（T172A）KI homozygous mice and wild type littermates.3.The phosphorylation of AMPKα2 Thr172 of skeletal muscle whole cell lysate,cytosolic and mitochondrial fraction were significantly decreased in AMPKα2（T172A）KI homozygous mice.Futhermore,the phosphorylation of ACC Ser79 and Ulk1Ser555 were significantly decreased.Mitochondrial content in skeletal muscle was significantly declined in AMPKα2（T172A）KI homozygous mice.Acute exercise increased muscular autophagy/mitophagy in AMPKα2（T172A）KI homozygous mice.It’s demonstrated that acute exercise upregulated the phosphorylation of TBC1D1Ser237 and promoted GLUT4 translocation to plasma membrane in skeletal muscle of wild type mice.But this effect wasn’t presented in AMPKα2（T172A）KI homozygous mice.4.Low frequency long time electrical stimulation（10 Hz 2 h）significantly activated AMPK in skeletal muscle of AMPKα2（T172A）KI homozygous mice and wild type littermates.At the same time the phosphorylation of AMPKα1 was significantly higher than AMPKα2.However,10 Hz 2 h electrical stimulation didn’t upregulate the phosphorylation of ACC,TBC1D1 and TBC1D4 in skeletal muscle of AMPKα2（T172A）KI homozygous mice and wild type littermates.Under the condition of electrical stimulation,there was no significant difference of the phosphorylation of ACC,TBC1D1 and TBC1D4 in skeletal muscle of AMPKα2（T172A）KI homozygous mice and wild type littermates.5.Eight-week moderate exercise intensity treadmill ruuning significantly decreased body weight in control group mice and improved fasting blood glucose and serum insulin in type 2 diabetic mice.It’s clarified that treadmill running upregulated the phosphorylation of AMPKαThr172 and ACC Ser79 in skeletal muscle of healthy control group mice.But this training didn’t upregulate the phosphorylation of AMPK Thr172,ACC Ser79,TBC1D1 Ser237 and TBC1D4 Thr642 in skeletal muscle in T2D.Moreover,GLUT4 protein expression was significantly decreased in skeletal muscle of type 2 diabetic mice.CONCLUSIONS1.Although acute exercise and electrical stimulation both could upregulate the phosphorylation of AMPKαThr172 in skeletal muscle,the phosphorylation of AMPK’s downstream signaling pathway was not the same.Compared to low frequency electrical stimulation,high frequency electrical stimulation significantly activated the phosphorylation of AMPK’s downstream signaling pathway.Although long time（2 h）low frequency electrical stimulation could increase the phosphorylation of AMPK Thr172,10 Hz 2 h electrical stimulation couldn’t increase the phosphorylation of AMPK’s downstream signaling pathway.This demonstrated that the process of AMPK activating its downstream effectors maybe not last long time,long time electrical stimulation may inhibit the activation of AMPK and its downstream effectors.2.Although the point mutation ofα2（T172A）KI inhibited the phosphorylation of AMPKα2 Thr172 and led to declined mitochondrial content in skeletal muscle,this didn’t significant affect oxygen uptake（VO₂）and exercise endurance capacity inα2（T172A）KI homozygous mice.However,fat utilization ofα2（T172A）KI homozygous mice was significantly decreased at light cycle during post-exercise recovery period.This declared that the phosphorylation of AMPKα2 Thr172 is not necessary for exercise capacity.Under the condition of low frequency long time electrical stimulation,the phosphorylation of AMPKα1was significantly higher than AMPKα2,which indicated that the point mutation ofα2（T172A）KI would lead to the compensation of AMPKα1 for the dysfunction of AMPKα2.3.Acute exercise upregulated the phosphorylation of TBC1D1 Ser237 in skeletal muscle and promoted GLUT4 translocation.This process might rely on the phosphorylation of AMPKα2 Thr172.Although low frequency long time electrical stimulation induced the upregulated phosphorylation of AMPKα1 in skeletal muscle,the phosphorylation of AMPKα2 Thr172 is upstream activator of ACC and TBC1D1.4.Chronic exercise training improved blood glucose and serum insulin level in type 2 diabetic mice,but this wasn’t associated with the activation of AMPK and the phosphorylation of its downstream signaling pathway.Thus,exercise training improved type 2 diabetes independent of the phosphorylation of AMPKαThr172.