This study tested the hypothesis that elevated L-leucine concentrations in plasma reduce nitric oxide (NO) synthesis by endothelial cells (ECs) and affect adiposity in obese rats. Beginning at four weeks of age, male Sprague-Dawley rats were fed a casein-based low-fat (LF) or high-fat (HF) diet for 15 weeks. Thereafter, rats in the LF and HF groups were assigned randomly into one of two subgroups (n = 8/subgroup) and received drinking water containing either 1.02% L-alanine (isonitrogenous control) or 1.5% L-leucine for 12 weeks. The energy expenditure of the rats was determined at weeks 0, 6, and 11 of the supplementation period. At the end of the study, an oral glucose tolerance test was performed on all the rats immediately before being euthanized for the collection of tissues. HF feeding reduced (P < 0.001) NO synthesis in ECs by 21% and whole-body insulin sensitivity by 19% but increased (P < 0.001) glutamine:fructose-6-phosphate transaminase (GFAT) activity in ECs by 42%. Oral administration of L-leucine decreased (P < 0.05) NO synthesis in ECs by 14%, increased (P < 0.05) GFAT activity in ECs by 35%, and reduced (P < 0.05) whole-body insulin sensitivity by 14% in rats fed the LF diet but had no effect (P > 0.05) on these variables in rats fed the HF diet. L-Leucine supplementation did not affect (P > 0.05) weight gain, tissue masses (including white adipose tissue, brown adipose tissue, and skeletal muscle), or antioxidative capacity (indicated by ratios of glutathione/glutathione disulfide) in LF- or HF-fed rats and did not worsen (P > 0.05) adiposity, whole-body insulin sensitivity, or metabolic profiles in the plasma of obese rats. These results indicate that high concentrations of L-leucine promote glucosamine synthesis and impair NO production by ECs, possibly contributing to an increased risk of cardiovascular disease in diet-induced obese rats.
WidlanskyMEGokceNKeaneyJFVitaJA. The clinical implications of endothelial dysfunction.J Am Coll Cardiol2003;42:1149–60
2.
Van GaalLFMertensILDe BlockCE. Mechanisms linking obesity with cardiovascular disease.Nature2006;444:875–80
3.
FeligPMarlissECahillGFJr.Plasma amino acid levels and insulin secretion in obesity.N Engl J Med1969;281:811–6
4.
MelsCMSchutteAESchutteRHuismanHWSmithWFourieCMKrugerRVan RooyenJMMalanNTMalanL. The link between vascular deterioration and branched chain amino acids in a population with high glycated haemoglobin: the SABPA study.Amino Acids2013;45:1405–13
5.
ChevalierSBurgessSCMalloyCRGougeonRMarlissEBMoraisJA. The greater contribution of gluconeogenesis to glucose production in obesity is related to increased whole-body protein catabolism.Diabetes2006;55:675–81
6.
ChevalierSBurgosSAMoraisJAGougeonRBassilMLamarcheMMarlissEM. Protein and glucose metabolic responses to hyperinsulinemia, hyperglycemia, and hyperaminoacidemia in obese men.Obesity2015;23:351–8
7.
ShePVan HornCReidTHutsonSMCooneyRNLynchCJ. Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism.Am J Physiol2007;293:E1552–63
8.
HolečekM. Branched-chain amino acids in health and disease: metabolism, alterations in blood plasma, and as supplements.Nutr Metab2018;15:33
9.
AdevaMMCalviñoJSoutoGDonapetryC. Insulin resistance and the metabolism of branched-chain amino acids in humans.Amino Acids2012;43:171–81
10.
YangYWuZLMeiningerCJWuG. L-Leucine and NO-mediated cardiovascular function.Amino Acids2015;47:435–47
11.
VanweertFSchrauwenPPhielixE. Role of branched-chain amino acid metabolism in the pathogenesis of obesity and type 2 diabetes-related metabolic disturbances BCAA metabolism in type 2 diabetes.Nutr Diabetes2022;12:35
12.
BinderEBermúdez-SilvaFJElieMLeste-LasserreTBelluomoIClarkSDuchamptAMithieuxGCotaD. Leucine supplementation modulates fuel substrates utilization and glucose metabolism in previously obese mice.Obesity2014;22:713–20
13.
ZhangYGuoKLeBlancRELohDSchwartzGJYuYH. Increasing dietary leucine intake reduces diet-induced obesity and improves glucose and cholesterol metabolism in mice via multimechanisms.Diabetes2007;56:1647–54
14.
BinderEBermúdez-SilvaFJAndréCElieMRomero-ZerboSYLeste-LasserreTBelluomoIDuchamptAClarkSAubertAMezzulloMFanelliFPagottoULayéSMithieuxGCotaD. Leucine supplementation protects from insulin resistance by regulating adiposity levels.PLoS One2013;8:e74705
15.
LiHXuMLeeJHeCXieZ. Leucine supplementation increases SIRT1 expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice.Am J Physiol2012;303:E1234–44
16.
BaumJIWashingtonTAShouseSABottjeWDridiSDavisGSmithD. Leucine supplementation at the onset of high-fat feeding does not prevent weight gain or improve glycemic regulation in male Sprague-Dawley rats.J Physiol Biochem2016;72:781–9
17.
CeglarekVMCoelhoMLCoelhoRLAlmeidaDLde Souza RodriguesWNCamargoRLBarellaLFde Freitas MathiasPCGrassiolliS. Chronic leucine supplementation does not prevent the obesity and metabolic abnormalities induced by monosodium glutamate.Clin Nutr Exp2020;29:62–75
18.
ZampieriTTTorres-LealFLCampañaABLimaFBDonatoLJr.l-Leucine supplementation worsens the adiposity of already obese rats by promoting a hypothalamic pattern of gene expression that favors fat accumulation.Nutrients2014;6:1364–73
19.
BaronADSteinbergHOChakerHLeamingRJohnsonABrechtelG. Insulin-mediated skeletal muscle vasodilation contributes to both insulin sensitivity and responsiveness in lean humans.J Clin Invest1995;96:786–92
20.
VincentMABarrettEJLindnerJRClarkMGRattiganS. Inhibiting NOS blocks microvascular recruitment and blunts muscle glucose uptake in response to insulin.Am J Physiol2003;285:E123–9
21.
JobgenWSFriedSKFuWJMeiningerCJWuG. Regulatory role for the arginine-nitric oxide pathway in metabolism of energy substrates.J Nutr Biochem2006;17:571–88
22.
CayatteAJPalacinoJJHortenKRohenRA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits.Arterioscler Thromb1994;14:753–9
23.
KuhlencordtPJGyurkoRHanFScherrer-CrosbieMAretzTHHajjarRPicardMHHuangPL. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein endothelial nitric oxide synthase double-knockout mice.Circulation2001;104:448–54
24.
JobgenWSMeiningerCJJobgenSCLiPLeeMJSmithSBSpencerTEFriedSKWuG. Dietary L-arginine supplementation reduces white fat gain and enhances skeletal muscle and brown fat masses in diet-induced obese rats.J Nutr2009;139:230–7
25.
TekweCDYaoKLeiJLiXGuptaALuanYMeiningerCJBazerFWWuG. Oral administration of α-ketoglutarate enhances nitric oxide synthesis by endothelial cells and whole-body insulin sensitivity in diet-induced obese rats.Exp Biol Med2019;244:1081–8
26.
NewgardCBAnJBainJRMuehlbauerMJStevensRDLienLFHaqqAMShahSHArlottoMSlentzCARochonJGallupDIlkayevaOWennerBRYancyWSJrEisensonHMusanteGSurwitRSMillingtonDSButlerMDSvetkeyLP. A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance.Cell Metab2009;9:311–26
27.
TekweCDLeiJYaoKRezaeiRLiXLDahanayakaSCarrollRJMeiningerCJBazerFWWuG. Oral administration of interferon tau enhances oxidation of energy substrates and reduces adiposity in Zucker diabetic fatty rats.Biofactors2013;39:552–63
28.
MeiningerCJWuG. Regulation of endothelial cell proliferation by nitric oxide.Methods Enzymol2002;352:280–95
JobgenWSWuG. L-Arginine increases AMPK phosphorylation and the oxidation of energy substrates in hepatocytes, skeletal muscle cells, and adipocytes.Amino Acids2022;54:1553–68
32.
JobgenWSLeeMJFriedSKWuG. L-Arginine supplementation regulates energy-substrate metabolism in skeletal muscle and adipose tissue of diet-induced obese rats.Exp Biol Med2023;248:209–16.
33.
WuGHaynesTELiHYanWMeiningerCJ. Glutamine metabolism to glucosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis.Biochem J2001;353:245–52
34.
HouYLiXDaiZWuZBazerFWWuG. Analysis of glutathione in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde.Methods Mol Biol2018;1694:105–15
35.
LowryOHRosebroughNJFarrALRandallRJ. Protein measurement with the Folin phenol reagent.J Biol Chem1953;193:265–75
36.
FolchJLeesMSloane-StanleyGH. A simple method for the isolation and purification of total lipids from animal tissues.J Biol Chem1957;226:497–509
37.
ShapiroSSWilkMB. An analysis of variance test for normality (complete samples).Biometrika1965;52:591–611
AnthonyJCYoshizawaFAnthonyTGVaryTCJeffersonLSKimballSR. Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway.J Nutr2000;130:2413–9
40.
BrunettaHSde CamargoCQNunesEA. Does L-leucine supplementation cause any effect on glucose homeostasis in rodent models of glucose intolerance? A systematic review.Amino Acids2018;50:1663–78
41.
PikoskyMCifelliCAgarwalSFulgoniVIII. Association of protein/leucine intake and grip strength among adults aged 19+ years: analysis of NHANES 2011–2014.Curr Dev Nutr2020;4:65
42.
ElangoRRasmussenBMaddenK. Safety and tolerability of leucine supplementation in elderly men.J Nutr2016;146:2630S–4S
43.
KrebsMKrssakMBernroiderEAnderwaldCBrehmAMeyerspeerMNowotnyPRothEWaldhäuslWRodenM. Mechanism of amino acid-induced skeletal muscle insulin resistance in humans.Diabetes2002;51:599–605
44.
SunHOlsonKCGaoCProsdocimoDAZhouMWangZJeyarajDYounJYRenSLiuYRauCDShahSIlkayevaOGuiWJWilliamNSWynnRMNewgardCBCaiHXiaoXChuangDTSchulzePCLynchCJainMKWangY. Catabolic defect of branched-chain amino acids promotes heart failure.Circulation2016;133:2038–49
45.
KapurSBedardSMarcotteBCoteCHMaretteA. Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action.Diabetes1997;46:1691–700
46.
YoungMELeightonB. Fuel oxidation in skeletal muscle is increased by nitric oxide/cGMP—evidence for involvement of cGMP-dependent protein kinase.FEBS Lett1998;424:79–83
47.
WuGMeiningerCJ. Nitric oxide and vascular insulin resistance.Biofactors2009;35:21–7
48.
WooSLYangJHsuMYangAZhangLLeeRPGilbuenaIThamesGHuangJRasmussenACarpenterCLHenningSMHeberDWangYLiZ. Effects of branched-chain amino acids on glucose metabolism in obese, prediabetic men and women: a randomized, crossover study.Am J Clin Nutr2019;109:1569–77
49.
LeeJVijayakumarAWhitePJXuYIlkayevaOLynchCJNewgardCBKahnBB. BCAA supplementation in mice with diet-induced obesity alters the metabolome without impairing glucose homeostasis.Endocrinology2021;162:bqab062
50.
LynchCJAdamsSH. Branched-chain amino acids in metabolic signalling and insulin resistance.Nat Rev Endocrinol2014;10:723–36
51.
NairKSSchwartzRGWelleS. Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans.Am J Physiol1992;263:E928–34
52.
EscobarJFrankJWSuryawanANguyenHVDavisTA. Amino acid availability and age affect the leucine stimulation of protein synthesis and eIF4F formation in muscle.Am J Physiol2007;293:E1615–21
53.
MaQZhouXHuLChenJZhuJShanA. Leucine and isoleucine have similar effects on reducing lipid accumulation, improving insulin sensitivity and increasing the browning of WAT in high-fat diet-induced obese mice.Food Funct11:2279–90
54.
WangFXiaoFDuLNiuYYinHZhouZJiangXJiangHYuanFLiuKChenSDuanSGuoF. Activation of GCN2 in macrophages promotes white adipose tissue browning and lipolysis under leucine deprivation.FASEB J2021;35:e21652
55.
RezaeiRWuG. Branched-chain amino acids regulate intracellular protein turnover in porcine mammary epithelial cells.Amino Acids2022;54:1491–504
56.
YoneshiroTWangQTajimaKMatsushitaMMakiHIgarashiKDaiZWhitePJMcGarrahRWIlkayevaORDeleyeYOguriYKurodaMIkedaKLiHUenoAOhishiMIshikawaTKimKChenYSpontonCHPradhanRNMajdHGreinerVJYoneshiroMBrownZChondronikolaMTakahashiHGotoTKawadaTSidossisLSzokaFCMcManusMTSaitoMSogaTKajimuraS. BCAA catabolism in brown fat controls energy homeostasis through SLC25A44.Nature2019;572:614–9
57.
BishopCAMachateTHenningTHenkelJPüschelGWeberDGruneTKlausSWeitkunatK. Detrimental effects of branched-chain amino acids in glucose tolerance can be attributed to valine induced glucotoxicity in skeletal muscle.Nutr Diabetes2022;12:20
ChenXXiangLJiaGLiuGZhaoHHuangZ. Effects of dietary leucine on antioxidant activity and expression of antioxidant and mitochondrial-related genes in longissimus dorsi muscle and liver of piglets.Anim Sci J2019;90:990–8
60.
CojocaruEFilipNUngureanuCFilipCDanciuM. Effects of valine and leucine on some antioxidant enzymes in hypercholesterolemic rats.Health2014;6:50368
61.
ZhenyukhOCivantosERuiz-OrtegaMSánchezMSVázquezCPeiróCEgidoJMasS. High concentration of branched-chain amino acids promotes oxidative stress, inflammation and migration of human peripheral blood mononuclear cells via mTORC1 activation.Free Radic Biol Med2017;104:165–77
62.
WuGFangYZYangSLuptonJRTurnerND. Glutathione metabolism and its implications for health.J Nutr2004;134:489–92
63.
ShePReidTMBronsonSKVaryTCHajnalALynchCJHutsonSM. Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle.Cell Metab2007;6:181–94
64.
EllerLKSahaDCShearerJReimerRA. Dietary leucine improves whole-body insulin sensitivity independent of body fat in diet-induced obese Sprague-Dawley rats.J Nutr Biochem2013;24:1285–94
65.
SunKJWuZLJiYWuG. Glycine regulates protein turnover by activating Akt/mTOR and inhibiting expression of genes involved in protein degradation in C2C12 myoblasts.J Nutr2016;146:2461–7
66.
El HafidiMPérezIZamoraJSotoVCarvajal-SandovalGBañosG. Glycine intake decreases plasma free fatty acids, adipose cell size, and blood pressure in sucrose-fed rats.Am J Physiol2004;287:R1387–93
67.
NairiziAShePVaryTCLynchCJ. Leucine supplementation of drinking water does not alter susceptibility to diet-induced obesity in mice.J Nutr2009;139:715–9
68.
LiJHuXSelvakumarPRussellRRIIICushmanSWHolmanGDYoungLH. Role of the nitric oxide pathway in AMPK-mediated glucose uptake and GLUT4 translocation in heart muscle.Am J Physiol2004;287:E834–41
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
