Alterations Of Metabolic Profiles And Mitochondrial Properties Induced By Leucine In Rats At The Different Stages Of Insulin Resistance

Part? Dynamic alterations of serum metabolomic profiles induced by leucine supplemetation in rats at the different stages of insulin resistanceObjective:The mechanisms underlying the different effects of leucine supplementation on insulin resistance and type 2 diabetes is still unknown.BCAAs have been proposed to elicit different or even opposite effects,depending on the prevalence of catabolic and anabolic states.We aimed to explore the metabolic alteration of leucine supplementation in rats fed with high fat diet at the different stage of insulin resistance and provide an insight into the underlying mechanisms.Methods:Male Sprague-Dawley rats were fed with a normal chow(ND),high-fat diet(HFD),HFD with 20%calorie restriction(HFD+CR),or HFD supplemented with 1.5%leucine(HFD+Leu),for 24w or 32w respectively.At the end of experiment,the insulin tolerance test(ITT)and the glucose tolerance test(GTT)were performed.The rats were killed,then the fast serum and tissue samples were collected.Serum levels of glucose,insulin,TC and TG were tested and HOMA-IR was calculated.HPLC-MS/MS-based metabolomics was applied to identify the changes in the serum metabolomic profiles response to leucine treatment at these two time points.Results:(1)Body weight gain and calorie intake:HFD induced more weight gain than rats on ND from the 5th week.Leucine supplementation prevented HFD-induced weight gain from the 13th week.HFD+CR prevented HFD-induced weight gain from the the 5th week.The calorie intake of HFD was significantly more than rats on ND rats from the 3th week.Supplementaion of leucine almost did not alter calorie intake across all time points,except some slightly increase at week 5.Consistent with our experiment design,total calorie intake in calorie restricted rats was decreased 20%throughout the 32-week feeding compared to rats on HFD.(2)Insulin sensitivity:the 24-week HFD feeding did not alter fasting blood glucose concentration while increased fasting serum insulin level significantly.Leucine supplementation for 24 weeks had no obvious effects on fasting serum glucose,insulin levels and the HOMA-IR index,but CR prevented the HFD-induced increase in fasting insulin level,and accompanied with significantly decreased HOMA-IR index.At week 32,both fasting blood glucose and insulin levels elevated in HFD rats compared with ND rats.Serum insulin level were in a tendency of decrease but not statistically significant,while HOMA-IR index was significantly decreased in rats subjected to leucine than corresponding HFD controls at 32 week.CR largely prevented HFD-induced hyperglycemia and hyperinsulinemia,and had lower HOMA-IR index.The results of GTT and ITT confirmed that CR or leucine-supplemented rats were significantly more glucose tolerance and insulin sensitivity than HFD-fed rats at two time points.(3)Fast serum amino acids profile:at week 24,BCAAs(except for isoleucine)concentration were significantly elevated in HFD+Leu rats compared to HFD-fed rats(p<0.05).Other amino acids,such as the aromatic amino acids(phenylalanine and tryptophan),and several gluconeogenic amino acids(asparagine,proline and alanine)were also significantly elevated.However,after 32 weeks of leucine treatment,serum BCAAs and aromatic amino acids were not significantly different,while asparagine and glycine were greatly depressed,accompanied with proline increased.Compared with HFD control,there was a trend toward lower levels of most amino acids at week 24 but differences were not statistically significant(except for leucine,isoleucine,alanine,phenylalanine and lysine),while serum alanine and proline concentrations were significantly increased at 32 weeksin CR group.(4)Metabolic profiles:the integration of results from metabonomic studies revealed that HFD,HFD+Leu and HFD+CR rats had different metabolic signature at both time points.The altered metabolic pathways in leucine-treated rats,included fatty acid metabolism,Krebs cycle,bile acid metabolism and amino acid metabolism,however,the changes were different when compared to their corresponding HFD-fed rats.At 24 weeks,serum levels of TCA intermediates,metabolites related to lipids metabolism and amino acids catabolism were markedly higher in the leucine-treated rats compared to the HFD control values.Whereas at week 32,there were a sharp decline of the TCA intermediates and lipids-related metabolites in the leucine-treated rats compared to the HFD control values.The amino acid catabolites discriminated from HFD control diminished and the terminal catabolites of amino acids greatly decreased.CR feeding resulted in significant elevations for metabolites related to lipid,bile acid metabolism and some amino acids catabolites compared to their corresponding HFD-fed rats at both 24 and 32 weeks.Conclusion:Leucine supplementation triggered dynamic alterations of metabolic signature in the progression of insulin resistance,mainly involved in TCA cycle,fatty acid,bile acid and amino acids metabolism.At the stage of hyperinsulinemia,compared to the HFD rats,the concentration of amino acids and related catabolites,fatty acid metabolites and intermediates of TCA were significantly increased in HFD+Leu rats.However,at the stage of hyperglycemia,the concentration of amino acids and above metabolites were significantly decreased compared to corresponding HFD control.The metabolic profiles of HFD+Leu rats were different from HFD+CR rats.The results suggested that leucine supplementation exerted different effect at the different stage of insulin resistance,and the metabolic signature of leucine supplementation and CR on insulin resistance might be different.Part ? Dynamic alterations of mitochondrial properties induced by leucine supplementation in rats at the different stage of insulin resistanceObjective:To investigate the effect of leucine supplementation on BCAAs catabolism and mitochondrial properties in the progression of insulin resistance.Methods:Animal feeding and treatments were the same as the part ?.At the 24 and 32 weeks,the rats were killed and the fast serum and tissue samples were collected.Serum levels of ATP and branched-chain amino acids derived branched-chain keto acids,C3 and C5 acylcarnitines were determined by LC-MS/MS.The serum concentrations of SOD and MDA were detected by colorimetry method.Mitochondrial structure was visualized by transmission electron microscopy.Protein expression of oxidative phosphorylation complexes,BCAA catabolic enzymes.AKT and phosphorylated AKT(pSer473)were detected by western blot.Quantitative RT-PCR were carried out to assess the mtDNA content and the expression of genes related to mitochondrial biogenesis,such as NRF-1,TFAM,PGC-1?,and SIRT1 in skeletal muscle.Results:(1)Insulin sensitivity of skeletal muscle:insulin-stimulated phosphorylation of AKT was significantly decreased in HFD rats at different stage of insulin resistance compared with ND rats.In leucine-supplemented rats the insulin-stimulated phosphorylation of AKT was only enhanced at 32 weeks compared with HFD control.However,the phosphorylation of AKT in skeletal muscle-was increased in calorie-restricted rats at both 24 and 32 weeks.(2)BCAAs related metabolites:at week 24,the transamination products of BCAAs in HFD rats were not significantly different from ND rats.Compared with HFD-fed controls,the levels of transamination products of leucine(?-Ketoisocaproate,KIC)was significantly increased.In CR rats the level of transamination products of valine(?-Ketoisovalerate,KIV)was significantly decreased compared with HFD-fed controls.At week 32,the concentrations of KIC and KIV were significantly increased in HFD rats compared with ND rats.The isoleucine-derived KMV(?-Keto-?-methylvalerate,KMV)levels were higher in leucine-supplemented rats than HFD control.KIC concentrations in HFD+CR rats were lower than HFD controls.Notably,neither leucine supplementation nor CR had a significant impact on serum BCAA-derived short-chain acylcarnitine(C3 and C5)levels throughout the experiment.(3)Protein expression of key enzyme of BCAAs catabolism:mitochondrial branched-chain aminotransferase(BCATm)protein expression was significantly increased in skeletal muscle and adipose tissue,concurrent with a significant increase in branched-chain?-keto acid dehydrogenase kinase(BCKDK)and no change in branched-chain ?-keto acid dehydrogenase E1 ?(BCKDHE1?)in skeletal muscles,livers and adipose tissues of leucine-supplemented rats compared with those of HFD-fed controls at week 24,while the only changes observed in the levels of BCAA catabolizing enzymes was an increased expression of BCATm in adipose tissue.BCAA-catabolizing enzyme protein expression did not significantly differ between CR rats and HFD-fed controls at week 24.However,the protein expression of BCATm and BCKDK were significantly decreased,whereas BCKDHE1? expression was significantly increased in skeletal muscle,and protein expression of BCATm in adiposity was significantly increased at week 32.(4)Serum levels of oxidative stress:in HFD fed rats the MDA level were higher,while the SOD activitity were lower than that of rats fed with ND.The activity of SOD in leucine-supplementation rats was lower than HFD control at week 24.However,the level of MDA was lower while the SOD activitity was higher in both leucine-supplementation and CR rats at 32 weeks.(5)Mitochondrial properties in skeletal muscle:long-term HFD feeding induced mitochondrial structural damage that was exacerbated by leucine supplementation at 24 weeks.The expression of enzymes involved in oxidative phosphorylation(OXPHOS),serum ATP production and the expression of genes controlling mitochondrial biogenesis,including mitochondrial transcription factor A(TFAM),peroxisome proliferator-activated receptor ?coactivator-1-?(PGC-1?)and sirtuin 1(SIRT1)were greatly decreased in leucine-treated rats compared to HFD control at this time point.In contrast,HFD-induced mitochondrial damage was partially restored by leucine supplementation at 32 weeks,accompanied by increases in the mitochondrial DNA content,the expression of genes involved in mitochondrial biogenesis(TFAM)and ATP production.As expected,CR had a beneficial effect on HFD-induced mitochondrial properties at both time points.including increases in OXPHOS protein levels,ATP production,and mitochondrial biogenesis.Conclusion:Taken together,these results indicated that alterations in the metabolic phenotype were correlated with changes in mitochondrial properties.At the early stage of insulin resistance(24 weeks),abnormal BCAAs metabolism induced by dietary supplementation coupled with HFD intake might lead to the accumulation of BCKAs and incompletely oxidized lipid species.which contributed to mitochondrial dysfunction and insulin resistance in skeletal muscle.In contrast,during the hyperglycaemic stage(32 weeks),when catabolic processes predominate,leucine supplementation enhanced mitochondrial biogenesis,with concomitantly improved lipid oxidation and mitochondrial function,in skeletal muscle.These findings reveal that leucine has opposite effects on metabolic signatures at different stages of insulin resistance and indicate that the overall metabolic status of the patient should be carefully considered to potentiate the health benefits of leucine.