Chronic Supplementation with L-Isoleucine Alone or in Combination with Exercise Reduces Hepatic Cholesterol Levels with No Effect on Serum Glucose, Insulin, or Lipids in Rats Fed a High Fructose Diet

The thought of using branched-chain amino acids (BCAA) in the prevention and treatment of certain disorders is becoming increasingly popular. Individual BCAA use has been associated with improving glucose tolerance and liver disease. Previous studies have cited improvements in glucose metabolism with a single dose of L-isoleucine (ILE). However, it is still unclear whether chronic consumption of ILE has any direct benefit. The objective of this study was to examine the influence of chronic ILE supplementation alone or in combination with exercise on fasting serum glucose, insulin, lipids, and lipoprotein cholesterol levels;glucose tolerance;and hepatic lipids in rats. Male Sprague-Dawley rats (n = 40) were divided into Control (low fructose diet);High Fructose diet (HF);HF plus 1.5% ILE (HF + ILE);HF plus exercise (HF + EX);and HF plus 1.5% ILE and exercise (HF + ILE + EX). The HF diets consisted of 70% kcalories from fructose. After 6 weeks of treatment, no significant differences were observed between groups for changes in fasting serum glucose, insulin, lipids, or lipoprotein cholesterol levels. However, hepatic total cholesterol was significantly lower in the HF + ILE + EX compared to the Control and HF, while, the HF + ILE had significantly lower hepatic free cholesterol compared to the HF. We also found no differences between groups for serum glucose response following an oral glucose tolerance test. In conclusion, our study shows that ILE supplementation in rats does not influence serum glucose and lipid biomarkers but may have an influence on lipid metabolic pathways within the liver.

Surplus dietary isoleucine intake enhanced monounsaturated fatty acid synthesis and fat accumulation in skeletal muscle of finishing pigs

Background: Isoleucine(Ile) has been implicated in the regulation of energy homeostasis and adipogenesis.However,the impact of surplus dietary Ile intake on muscle lipogenesis remains unknown.The present study aimed to investigate the impact of dietary supplementation of extra-Ile on lipogenesis,fatty acid profile and lipid accumulation in skeletal muscle in finishing pigs.Methods: Forty-eight barrows with initial body weight of 77.0 ± 0.1 kg were allotted to one of two groups and fed diets containing 0.39%,0.53% standardized ileal digestible(SID) Ile with six replicates per treatment and four pigs per replicate for 30 d.Results: Dietary Ile intake significantly improved the intramuscular fat(IMF) content and monounsaturated fatty acid(MUFA) concentration in the skeletal muscle(P < 0.05),and decreased the drip loss and shear force(P < 0.05) without influencing the growth performance of pigs(P > 0.05).Moreover,the phosphorylation of adenosine monophosphate activated protein kinase α(AMPKα) and acetyl coenzyme A carboxylase(ACC) proteins that monitor lipid metabolism were decreased in skeletal muscle of pigs offered extra-Ile diet(P < 0.05).The mRNA expression of adipose-specific genes adipocyte determination and differentiation factor 1(ADD1),fatty acid synthase(FAS),and stearoyl-CoA desaturase(SCD) were upregulated and the activity of SCD was increased as well(P < 0.05).Conclusions: Surplus dietary Ile intake could increase IMF accumulation and MUFA synthesis in skeletal muscle through depressing the phosphorylation of AMPKα-ACC and stimulating the expression and activity of SCD,and increasing the capability of lipogenesis in skeletal muscle.

Dietary isoleucine improved flesh quality,muscle antioxidant capacity,and muscle growth associated with AKT/TOR/S6K1 and AKT/FOXO3a signaling in hybrid bagrid catfish(Pelteobagrus vachelli♀×Leiocassis longirostris♂)

Background:Muscle is the complex and heterogeneous tissue,which comprises the primary edible part of the trunk of fish and mammals.Previous studies have shown that dietary isoleucine(Ile)exerts beneficial effects on growth in aquatic animals.However,there were limited studies regarding the benefits of Ile on fish muscle and their effects on flesh quality and muscle growth.Thus,this study was conducted to explore whether dietary Ile had affected flesh quality and muscle growth in hybrid bagrid catfish(Pelteobagrus vachelli♀×Leiocassis longirostris♂).Methods:A total of 630 hybrid fish,with an initial average body weight of 33.11±0.09 g,were randomly allotted into seven experimental groups with three replicates each,and respectively fed seven diets with 5.0,7.5,10.0,12.5,15.0,17.5,and 20.0 g Ile/kg diets for 8 weeks.Results:In the present study,we demonstrated that Ile significantly:(1)increased muscle protein and lipid contents and the frequency distribution of myofibers with≤20μm and≥50μm of diameter;(2)improved pH value,shear force,cathepsin B and L activities,hydroxyproline content,resilience,cohesiveness,and decreased cooking loss,lactate content,hardness,springiness,gumminess,and chewiness;(3)decreased reactive oxygen species(ROS),malondialdehyde(MDA),and protein carbonyl(PC)contents,GCLC and Keap1 mRNA levels,and up-regulated CuZnSOD,CAT,GPX1a,GST,and Nrf2 mRNA levels;(4)up-regulated the insulin-like growth factor 1,2(IGF-1,IGF-2),insulin-like growth factor 1 receptor(IGF-1R),proliferating cell nuclear antigen(PCNA),Myf5,Myod,Myog,Mrf4,and MyHC mRNA levels,and decreased MSTN mRNAlevel;(5)increased muscle protein deposition by activating AKT-TOR-S6K1 and AKT-FOXO3a signaling pathways.Conclusion:These results revealed that dietary Ile improved flesh quality,which might be due to increasing nutritional content,physicochemical,texture parameters,and antioxidant ability;promoting muscle growth by affecting myocytes hyperplasia and hypertrophy,and muscle protein deposition associated with protein synthesis and degradation signaling pathways.Finally,the quadratic regression analysis of chewiness,ROS,and protein contents against dietary Ile levels suggested that the optimal dietary Ile levels for hybrid bagrid catfish was estimated to be 14.19,12.36,and 12.78 g/kg diet,corresponding to 36.59,31.87,and 32.96 g/kg dietary protein,respectively.

Isoleucine, an Essential Amino Acid, Induces the Expression of Human <i>β</i>Defensin 2 through the Activation of the G-Protein Coupled Receptor-ERK Pathway in the Intestinal Epithelia

Anti-microbial peptides are essential for the intestinal innate immunity that protects the intestinal epithelia from attacks by foreign pathogens. Human β-defensin (HBD) is one of the pivotal anti-microbial peptides that are expressed in the colonic epithelia. This study investigated the effect and the signaling mechanism of inducible β-defensin HBD2 by an essential amino acid, isoleucine (Ile) in colonic epithelial cells. Here we examined the expression level of HBD2 on induction of Ile in epithelial cells, and checked this pathway. HBD2 mRNA was induced by co-incubation with IL-1α and Ile in Caco2 cells, but not by Ile alone. An inhibitor of either ERK or Gi, a subunit of G-proteins, reduced the induction of HBD2 mRNA by Ile. The treatment with Ile also increased the intracellular calcium ion concentration, thus suggesting that the GPCR and ERK signaling pathway mediate the effects of Ile. These results indicate that an essential amino acid, Ile, enhances the expression of an inducible β-defensin, namely HBD2, by IL-1α through the activation of GPCRs and ERK signaling pathway. The administration of Ile may therefore represent a possible option to safely treat intestinal inflammation.

Application of quartz crystal nanobalance in conjunction with a net analyte signal based method for simultaneous determination of leucine, isoleucine and valine

The aim of the present investigation was to develop a biosensor for the detection of amino acids, Leucine, Isoleucine and Valine based on a quartz crystal nanobalance. leucine (Leu), isoleucine (Ile), and valine (Val) were selectively determined by quartz crystal nanobalance (QCN) sensor in conjunction with net analyte signal (NAS)-based method called HLA/GO. An orthogonal design was applied for the formation of calibration and prediction sets including Leu, Ile and Val compounds. The selection of the optimal time range involved the calculation of the net analyte sig-nal regression plot in any considered time window for each test sample. The searching of a region with maximum linearity of NAS regression plot (minimum error indicator) and minimum of PRESS value was carried out by applying a moving window strategy. On the base of obtained results, the differences on the adsorption profiles in the time range between 1 and 300 s were used to determine mixtures of compounds by HLA/GO method. The results showed that the method was successfully applied for the determina-tion of Leu, Ile and Val.

Behaviors of Leucine and Isoleucine in Ion Mobility- Quadrupole Time of Flight Mass Spectrometry

In this study, ion mobility separation coupled with tandem mass spectrometry （IM-tandem MS） was utilized to investigate the ionization behaviors of two amino acids including leucine and isoleucine. Under the electrospray ionization （ESI） mode, two protonation sites in each molecular sturcture caused two forms of protomer. One arose from the amino being protonated （amino-protomer） and the other from the carboxyl being protonated （car- boxyl-protomer）. In the two-dimensional （drift time, m/z） spectrum, the protomers had the same mass, but the dis- tinguishable drift times and fragmentation patterns. For the characterization purpose, the theoretical collision cross section （CCS） values of the protomers were calculated and proven to be consistent with the experimental. Moreover, the quantified relationship between the amino acids and their protomers was evaluated. It showed that the abun- dance of the carboxyl-protomer was proportional to the concentration of the amino acid, whereas that of the amino- protomer did not have the same trend. Under the atmospheric pressure chemical ionization （APCI） mode, only the carboxyl-protomer was observed. In addition, the amino-protomer and the cluster ions observed under ESI were absent completely. The results demonstrate that the ionization mode impacts heavily on the ionization behaviors of leucine and isoleucine not only on the form of therir protomers but also On the quantified relationship.

Dietary isoleucine affects muscle fatty acid and amino acid profiles through regulating lipid metabolism and autophagy in hybrid catfish(Pelteobagrus vachelli ♀×Leiocassis longirostris ♂)

The present study explored the impacts of Ile on muscle fatty acid and amino acid profiles,lipid metabolism,and autophagy in hybrid catfish.Seven isonitrogenous(387.8 g/kg protein)semi-purified diets were formulated to contain 5.0(control),7.5,10.0,12.5,15.0,17.5,and 20.0 g Ile/kg diet respectively.The fish(initial weight of 33.11±0.09 g)were randomly assigned to 7 groups for a 56-day trial.Each group has 3 replicates with 30 fish per replicate,fed at 08:00 and 18:00 each day.Results showed that muscle protein and lipid,C14:0,C18:0,C22:0,C14:1,C18:1n-9,polyunsaturated fatty acid(PUFA),Arg,Ile,Ala,Cys,Gly,Tyr,essential amino acid(EAA),and total amino acid(TAA)contents and flavor amino acid(FAA)/TAA in muscle had positive linear and/or quadratic responses to dietary Ile levels(P<0.05).Fatty acid synthase(FAS),stearoyl-Co A desaturase(SCD),acetyl-Co A carboxylase(ACC),and lipoprotein lipase(LPL)activities had positive linear and/or quadratic responses,but carnitine palmitoyl transferase 1(CPT1)activity had a negative response with increasing dietary Ile levels(P<0.05).The m RNA expressions of FAS,SCD,ACC,LPL,fatty acid binding protein 4(FABP4),FATP1,sterol response element-binding protein 1c(SREBP-1c),sequestosome 1(SQSTM1),and adenosine 5’-monophosphateactivated protein kinase(AMPK)had positive linear and/or quadratic responses to dietary Ile levels(P<0.05).The m RNA expressions of hormone-sensitive lipase(HSL),CPT1,peroxisome proliferatoractivated receptor a(PPARa),PPARγ,uncoordinated 51-like kinase 1(ULK1),beclin1(Becn1),autophagy-related protein 9a(Atg9a),Atg4b,Atg7,autophagy marker light chain 3 B(LC3B),and SQSTM1in muscle had negative linear and/or quadratic responses to dietary Ile levels(P<0.05).The p-AMPK and ULK1 protein levels,and p-AMPK/AMPK were decreased by 12.5 g Ile/kg in the diet(P<0.05).Finally,SQSTM1 protein level had the opposite effect(P<0.05).The above results indicate that dietary Ile improves fish muscle fatty acid and amino acid profiles potentially via respectively regulating lipid metabolism and autophagy.The Ile requirement of hybrid catfish(33 to 72 g)were estimated to be 12.63,13.77,13.75,11.45,10.50,12.53 and 12.21 g/kg diet based on the regression analysis of protein,lipid,SFA,PUFA,FAA,EAA,and TAA muscle contents,respectively.

An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand

Glucocorticoid-induced tumor-necrosis factor receptor(GITR)and its ligand,GITRL,play significant roles in regulating immune responses.It is clear that human soluble GITRL(hsGITRL)transduces signal activity through multiple oligomerization states.To develop human soluble trimeric GITRL protein as a potential therapeutic target,we explored the link of the isoleucine-zipper(ILZ)motif to the N-terminus of the human soluble GITRL with two leucine sequences.hsGITRL,with the ILZ motif(ILZ-hsGITRL),was firstly expressed in Escherichia coli,which exhibited a predominant trimer when identified by Sephadex G-100 filtration and non-reducing SDS–polyacrylamide gel electrophoresis(SDS-PAGE).The significantly higher biological activity of the ILZ-hsGITRL compared with hsGITRL was confirmed by CD41 T proliferation,interferon-c(IFN-c)secretion and binding activity assay.To reveal and compare the underlying mechanisms,the level of extracellular signal-regulated kinase-1/2(ERK1/2)phosphorylation was examined,indicating that ILZ-hsGITRL induced more persistent and stronger ERK1/2 activation than hsGITRL.In conclusion,the incorporation of an ILZ motif could markedly improve the costimulation of hsGITRL.

Effect of different dietary protein levels and amino acids supplementation patterns on growth performance, carcass characteristics and nitrogen excretion in growing-finishing pigs

Background:This study was conducted to determine the effects of different dietary protein levels and amino acids supplementation patterns in low protein diets on the growth performance,carcass characteristics and nitrogen excretion in growing-finishing pigs.Forty-two barrows(25.00±0.39 kg)were randomly assigned to 7 diets.Diet 1:the high crude protein diet with balanced for 10 essential amino acids(EAAs).Diet 2:the medium crude protein diet with 2%(approx)decreased protein level of Diet 1 and balanced 10 EAAs.Diet 3:the low crude protein diet with 4%decreased protein level of Diet 1 and balanced 10 EAAs.The protein levels of Diet 4,5,6 and 7 were the same as that of Diet 3.Diet 4 was only balanced for lysine(Lys),methionine(Met),threonine(Thr)and tryptophan(Trp);Diet 5 and 6 were further supplemented with extra isoleucine(Ile)or valine(Val),respectively;Diet 7 was further supplemented with extra Ile+Val.Results:Over the 112 days trial,the reduction of dietary protein by 2%or 4%with balanced10 EAAs significantly decreased nitrogen excretion(P<0.05),but had no effects on growth performance and carcass characteristics(P>0.05).In low protein diet,Val supplementation significantly increased body weight gain at 25–50 kg phase(P<0.05),while Ile supplementation at 75–100 kg phase and 100–125 kg phase significantly reduced the ratio of feed to gain(P<0.05).No effect of different dietary protein levels and amino acids supplementation patterns in low protein diets on carcass characteristics was observed(P>0.05).The total N excretion of pigs supplemented with only Lys,Met,Thr and Trp was numerically higher than that of pigs fed with extra Ile,or Val,or Ile+Val diets.Conclusion:In low protein diet,Val is more required than Ile in the early growing phage(25–50 kg),while Ile becomes more required in the late growing and finishing phage(75–125 kg).

Dietary branched-chain amino acid assessment in broilers from 22 to 35 days of age

Background:Valine and isoleucine are similar in chemical structure and their limitation in broiler chicken diets.To evaluate their limitation and interactive effects,multivariate assessment nutrition studies for the branched-chain amino acids(BCAA)are needed.A three level(−1,0,+1),three-factor Box-Behnken design study was conducted to assess dietary BCAA ratios to lysine of 65,75,and 85 for valine,58,66,and 74 for isoleucine,and 110,130,and 150 for leucine in male and female Lohman Indian River broilers from 22 to 35 d of age.Results:Live performance of male broilers was not affected by BCAA level.However,male broilers fed increasing isoleucine had improved(P=0.07)carcass yield as leucine and valine were reduced.Female broilers had improved body weight gain(P=0.05)and feed conversion(P=0.003)when leucine and isoleucine were at their lowest levels,independent of valine,but increasing leucine impaired live performance and warranted concomitant increases in isoleucine to restore responses.Increasing dietary isoleucine and valine in female broilers increased breast meat yield(P=0.05),but increasing leucine tended to diminish the response.Conclusion:The female Lohman Indian River broiler is more sensitive to BCAA diet manipulation than males.Specifically,as dietary leucine is increased in female broilers,dietary isoleucine increases were needed to offset the negative effects.Both increases in dietary valine and isoleucine improved breast meat yield in female broilers,but only when birds were fed the lowest dietary leucine.

Refractory very early-onset inflammatory bowel disease associated with cytosolic isoleucyl-tRNA synthetase deficiency:A case report

BACKGROUND Aminoacyl tRNA synthetases/ligases(ARSs)are highly conserved enzymes involved in attaching amino acids to tRNA promoting protein synthesis.Although deficiencies of ARSs localized to the mitochondria classically present with neuropathology,the clinical features of cytosolic ARS deficiencies are more variable.They have previously been associated with neonatal hepatitis,but never with early-onset inflammatory bowel disease.CASE SUMMARY A nine-year-old Bangladeshi boy presented with neonatal liver failure and deranged clotting,transaminitis and cholestasis.His parents were first cousins.Two older brothers and a sister were well.The patient suffered from loose stools from early infancy which became more troublesome and persistent from five years old with ten bloody motions a day.Repeated endoscopies showed persistent pancolitis,which was refractory to mesalazine,corticosteroids,azathioprine,sirolimus and anti-TNF(adalimumab)therapy,but has improved recently with subcutaneous methotrexate.Whole Genome Sequencing revealed a novel pathogenic missense variant(c.290A>G)in the cytosolic isoleucyl-tRNA synthetase gene,leading to an amino acid substitution(p.Asp97Gly).Pathogenic variants in other genes associated with inflammatory bowel disease(IBD)(ADAM17,EGFR,FOXP3,IL10RA,IL10RB,IL21R,NCF4,STAT3)were excluded.Cytokine assays demonstrated markedly elevated IL-2,IL-5,IL-13,IL-9 and IL-10 by the patient’s CD4+T-cells,while IL-17A,IL-17F,IFNβwere lower,and TNFαnot significantly different when compared to healthy controls.CONCLUSION This case report provides evidence that recessive mutations in cytosolic isoleucyltRNA synthetase are a novel monogenic cause of IBD,which should be considered,particularly in infants and children with a history of neonatal hepatitis and very early-onset IBD poorly responsive to treatment.

Systematic screening and structural characterization of dipeptides using offline 2D LC-LTQ-Orbitrap MS:A case study of Cordyceps sinensis

Cordyceps sinensis(C.sinensis)is a widely used and highly valuable traditional Chinese medicine.Several dipeptides have been detected in C.sinensis,but current scientific knowledge of its chemical makeup remains limited.In this study,an improved approach that integrates offline two-dimensional liquid chromatography(2D LC)separation,precursor ion list,library screening,and diagnostic ion filtering was established to systematically screen and characterize dipeptides in C.sinensis.Offline 2D LC integrating hydrophilic interaction LC and reverse phase separations was established to eliminate interference and identify the target dipeptides.A library containing the potential 400 dipeptides was created,and a precursor ion list with all theoretical precursor ions was adopted to trigger the MS/MS scan with high sensitivity.To identify dipeptides,the type and connection sequence of amino acids were determined according to the product ions.Ile and Leu residues were differentiated for the first time according to the characteristic ion at m/z 69.07.Ultimately,170 dipeptides were identified or tentatively characterized from C.sinensis,and most are reported for the first time in this species herein.In addition,the identified dipeptides were also applied for discrimination among the three Cordyceps species,and 11 markers were identified.The obtained results provide a deeper understanding of the chemical basis of C.sinensis.

Insulin resistance and the metabolism of branched-chain amino acids

Insulin resistance(IR)is a key pathological feature of metabolic syndrome and subsequently causes serious health problems with an increased risk of several common metabolic disorders.IR related metabolic disturbance is not restricted to carbohydrates but impacts global metabolic network.Branched-chain amino acids(BCAAs),namely valine,leucine and isoleucine,are among the nine essential amino acids,accounting for 35%of the essential amino acids in muscle proteins and 40%of the preformed amino acids required by mammals.The BCAAs are particularly responsive to the inhibitory insulin action on amino acid release by skeletal muscle and their metabolism is profoundly altered in insulin resistant conditions and/or insulin deficiency.Although increased circulating BCAA concentration in insulin resistant conditions has been noted for many years and BCAAs have been reported to be involved in the regulation of glucose homeostasis and body weight,it is only recently that BCAAs are found to be closely associated with IR.This review will focus on the recent findings on BCAAs from both epidemic and mechanistic studies.

Global analysis of a reaction-diffusion blood-stage malaria model with immune response

Malaria is one of the most dangerous diseases that threatens people's lives around the world.In this paper,we study a reaction-difusion model for the within-host dynamics of malaria infection with an antibody immune response.The model is given by a system of partial differential equations(PDEs)to describe the blood-stage of malaria life cycle.It addresses the interactions between uninfected red blood cells,antibodies,and three types of infected red blood cells,namely ringinfected red blood cells,trophozoite-infected red blood cells and schizont-infected red blood cells.Moreover,the model contains a parameter to measure the efficacy of isoleucinc starvation and its effect on the growth of malaria parasites.We show the basic properties of the model.We compute all equilibria and derive the thresholds from the conditions of existence of malaria equilibrium points.We prove the global stability of all equilibrium points based on choosing suitable Lyapunov functionals.We use the characteristic equations to verify the local instability of equilibrium points.We finally execute numerical simulations to validate the theoretical results and highlight some important observations.The results indicate that isoleucine starvation can have a critical impact on the stability of equilibrium points.When the efficacy of isoleucine starvation is high,it switchcs the system from the infection state to the malaria-free state.The presence of an antibody immune response does not lead to the elimination of malaria infection,but it suppresses the growth of malaria parasites and increases the amount of healthy red blood cells.