Investigation Of The Mechanism Underlying The Decomposition Of Branched-chain Amino Acids By Adiponectin In The Skeletal Muscle Of Type2Diabetic Mice

IntroductionThe morbidity and mortality of diabetes, especially type2diabetes mellitus (T2DM),have rapidly increased in recent years. In2013, the incidence of diabetes was reported tobe114,000,000in China and was estimated to be143,000,000in2035. As the third largestthreat to human health following cancers and cardiovascular diseases, T2MD has becomea global public health problem and a major burden to social and economy developments.Therefore, investigation of the mechanism of diabetes and identification of efficientprevention approaches and effective therapy methods are critically important. A largenumber of studies have demonstrated that obesity, metabolic syndrome, and diabetespatients have increased level of branched-chain amino acids (BCAAs) in the plasma. Inaddition, the plasma level of BCAAs is closely associated with insulin resistance.Newgard et al. have reported that mice with diabetes induced by high-fat diet hadsignificantly increased level of BCAAs in the skeletal muscle. However, the underlying mechanism is still unclear.As essential amino acids of humans, BCAAs include leucine, isoleucine, and valineand foods are the major resources of BCAAs. BCAAs can be decomposed to provideenergy for human body under special condition. They are also resources of the synthesis ofa number of important bioactive substances such as proteins, hormones, and ketones.More importantly, BCAAs are involved in the mTOR pathway to regulation cell growthand metabolism. The homeostasis of BCAAs is largely controlled by highly regulatedcatabolic activities in the skeletal muscle that is the major tissue where BCAAdecomposition occurs. By the rate-limiting enzyme branched-chain α-ketoaciddehydrogenase complex (BCKDC), BCAAs are converted to correspondingbranched-chain-keto acids (BCKAs) that enter the citric acid cycle or are further convertedinto other biologically active substances. The activity of BCKDC is tightly regulated byphosphorylation and dephosphorylation at the Ser293residue of the E1subunit(BCKDE1α). However, whether the elevated BCAA level in the skeletal muscle of T2DManimals is associated with the activity changes of BCKDC and underlying mechanismhave not been reported yet.Adiponectin (APN) is a cytokine of multiple functions secreted by fat cells. Previousstudies have reported that APN is widely involved in regulating the metabolism of glucoseand lipid, inhibiting inflammation, and anti-ischemic myocardial protection. It has beenshown that APN regulated the metabolism of glucose and lipid via peroxisomeproliferator-activated receptor (PPAR), Rab5, and the adenosine monophosphate-activatedprotein kinase (AMPK) pathways. As the most important downstream signaling moleculeof APN, AMPK mediates the insulin sensitizers and cardiac and vascular protective effectsof APN. However, whether APN prompts BCAA decomposition and whether AMPK isinvolved in this process are unknown.Objective1. To establish a T2DM mouse model and investigate the metabolism of BCAAs inthe skeletal muscle. 2. To confirm whether APN regulates the decomposition of BCAAs in the skeletalmuscle of T2DM mice.3. To elucidate the molecular mechanism of the decomposition of BCAAs in theskeletal muscle of T2DM mice.Methods1. Establishment of T2DM mouse model induced by intraperitoneal injection oflow-dose of streptozocin (STZ) combined with a high-fat diet. The blood glucose andweight of animals were monitored daily and intraperitoneal glucose tolerance test (IPGTT)was conducted in the12th week. Elevated blood glucose level and positive IPGTT wereconsider as successful establishment of T2MD model of mice.2. Wild-type (WT), ob/ob, and T2DM mice received intraperitoneal injection ofAPN for four days (2mg/kg/day) and the BCAA level in the skeletal muscle of mice wasmeasured and compared among different groups. Six groups of mice were compared,including the wild-type (WT), ob/ob, T2DM, WT+APN, ob/ob+APN, and T2DM+APNgroups.3. To further investigate the regulatory role of APN in the decomposition of BCAAsin APN knockout (APN-/-) mice, the BCAA and P-BCKDE1α/BCKDE1α levels in theskeletal muscle were measured and compared among five groups of mice including WT,APN-/-, WT+HD (high-fat diet), APN-/-+HD, and APN-/-+HD+APN groups.4. T2DM mice were randomly divided into four groups including the control, theT2DM+APN, T2DM+AICAR (AMPK activator) and T2DM+APN+CC (Compound C, aAMPK inhibitor) groups. The BCAA and P-BCKDE1α/BCKDE1α levels in the skeletalmuscle of mice were measured and compared among these four groups.5. Skeletal myoblast cell line C2C12were cultured with high concentration ofBCAAs and divided into two groups, the control (vehicle) group and the APN group. TheBCAA and P-BCKDE1α/BCKDE1α levels in the culture supernatant at different timepoints (0min,30min,1h,3h, and24h) after APN treatment were measured.6. Skeletal myoblast cell line C2C12were cultured with high concentration of BCAAs and divided into four groups, the control (vehicle), the APN, the AICAR, andAPN+CC groups. The BCAA and P-BCKDE1α/BCKDE1α levels in the culturesupernatant after1h of APN incubation were measured and compared among differentgroups.7. The glucose tolerance was evaluated by IPGTT assay.8. The protein levels of P-AMPK, AMPK, P-BCKDE1α, and BCKDE1α wereevaluated by Western blot.9. The BCAA level in the skeletal muscle was measured by (enzyme-linkedimmunosorbent assay) ELISA.10. The plasma glucose level was measured by ELISA.Results1. The fasting blood glucose and plasma insulin levels were measured and the IPGTTwas conducted in T2DM mice induced by low-dose of STZ combined with a high-fat diet.Elevated blood glucose and plasma insulin, and positive IPGTT were consider to besuccessful establishment of T2MD model of mice.2. The BCAA level in the skeletal muscle of mice in the T2DM and ob/ob groupswere significantly higher than the control group. With APN treatment, the BCAA level inthe skeletal muscle of mice in the T2DM+APN and ob/ob+APN groups decreasedsignificantly, suggesting that APN reduced the BCAA level in the skeletal muscle ofT2DM mice..3. Based on ELISA assay, the BCAA and P-BCKDE1α/BCKDE1α levels in theskeletal muscle of mice in the APN-/-+HD group were significantly higher than the WT,WT+HD, and APN-/-groups, suggesting that inhibition of the activity of BCKDCincreased the level of BCAAs in the skeletal muscle in the APN-/-+HD mice. In addition,the BCAA and P-BCKDE1α/BCKDE1α levels in the skeletal muscle of mice in theAPN-/-+HD+APN group were significantly lower than the APN-/-+HD group, suggestingthat APN reduced the BCAA level in the skeletal muscle of T2DM mice.4. The T2DM mice were treated with APN, AICAR (a AMPK activator), and CC (aAMPK inhibitor) and the BCAA and P-BCKDE1α/BCKDE1α levels in the skeletal muscle were measured and compared among different groups of mice. Compared to theT2DM mice, the BCAA, P-BCKDE1α/BCKDE1α, and P-AMPK/AMPK levels in theskeletal muscle were significantly lower in the AICAR and APN treated groups,suggesting that increased activity of AMPK improved the enzyme activity of BCKD E1αand promoted the decomposition of BCAAs. In addition, the BCAA,P-BCKDE1α/BCKDE1α, and P-AMPK/AMPK levels in the skeletal muscle in theAPN+CC mice were significantly lower than the APN group, suggesting that inhibition ofAMPK increased the BCKD E1α activity in the skeletal muscle of T2DM mice, whichthen reduced the BCAA level.5. Compared to the control group, the P-AMPK/AMPK and P-BCKD E1α/BCKDE1α levels in the supernatant increased and decreased, respectively, after30mins,1h, and3h of APN application, suggesting that APN up-regulated the activity of AMPK andBCKD, which then resulted in the decomposition of BCAAs in the supernatant of C2C12cells. Based on the dynamic changes of BCAA, P-AMPK/AMPK, and P-BCKDE1α/BCKD levels, one hour ofAPN application is the best time for decompose BCAAs.6. The C2C12cells cultured with high concentration of BCAAs were divided intofour groups, the control, APN, AICAR, and APN+CC groups. After1h of treatment ofAPN, AICAR, and CC, respectively, the BCAA and P-BCKD E1α/BCKD E1α levels inthe supernatant decreased in the APN and AICAR groups compared to the control group.Compared to the APN group, the BCAA and P-BCKD E1α/BCKD E1α levels in thesupernatant of the APN+CC group increased. These observations suggest that APNregulates the metabolism of BCAAs by directly activating AMPK in C2C12cells culturedwith high concentration of BCAAs, which is independent to the regulation of glucose andlipid.Conclusions1. T2DM mice have significant higher level of BCAAs in the skeletal muscle than thecontrol of mice. APN up-regulates the activity of BCDKC via the AMPK signalingpathway, leading to the decomposition of BCAAs in the skeletal muscle of T2DM mice. 2. APN promotes the decomposition of BCAAs in the skeletal muscle cell lineC2C12cultured with high concentration of BCAAs by directly up-regulating the activityof BCKDC, which is independent of its regulation of the metabolism of glucose and lipid.