Branched chain amino acids—friend or foe in the control of energy substrate turnover and insulin sensitivity?

Figures & data

Figure 1. Associations between glucose, fatty acid and amino acid metabolism. BCAA are metabolized within mitochondria through branched chain amino acid aminotransferase (Bcat) that catalyzes the transamination of BCAA by the transfer of amino group to α-ketoglutarate with a simultaneous generation of glutamate. Glutamate provides amino group to generate alanine from pyruvate or for condensation with ammonia to form glutamine. Branched chain keto acids (BCKA) are oxidatively decarboxylated by branched chain keto acid dehydrogenase (Bckdh). Finally, acyl-CoA metabolism diverges into separate pathways to CO₂, and succinyl-CoA (valine, glucogenic) or acetyl-CoA (leucine, ketogenic), or acetyl-CoA and succinyl-CoA (isoleucine, glucogenic and ketogenic) production. Acetyl-CoA may also derive from lipolysis or glycolysis, and its conversion to CO₂ and H₂O depends on tricarboxylic acid cycle capacity. Among valine intermediates, β-hydroxyisobutyrate (3-HIB) and β-aminoisobutyric acid (BAIBA), can be secreted outside the cell and exert paracrine/endocrine functions. Abbreviations: Abat, 4-aminobutyrate aminotransferase; Acat, acetyl-CoA acetyltransferase; Acc, acetyl-CoA carboxylase; Acl, ATP citrate lyase; Acs, fatty acyl-CoA synthetase; Agpat, 1-acylglycerol-3-phosphate-O-acyltransferase; Aldh6a1/Mmsdh, aldehyde dehydrogenase 6 family member A1/methylmalonic semialdehyde dehydrogenase; Alt, alanine transaminase; Ast, aspartate transaminase; Atgl, adipose triglyceride lipase; BCFA, branched chain fatty acids; Bckdk, branched chain keto acid dehydrogenase kinase; Cpt1/2, carnitine palmitoyltransferase 1/2; Cs, citrate synthase; Dgat, diacylglycerol acyltransferase; Fasn, fatty acid synthase; Gldh, glutamate dehydrogenase; Gpat, glycerol-3-phosphate acyltransferase; Gs, glutamine synthetase; Hadha, hydroxyacyl-CoA dehydrogenase subunit α; Hbdh, 3-hydroxybutyrate dehydrogenase; Hibadh, 3-hydroxyisobutyric acid dehydrogenase; Hibch, 3-hydroxy-isobutyryl-CoA hydrolase; HMG lyase, 3-hydroxy-3-methylglutaryl-CoA lyase; Hsl, hormone-sensitive lipase; Ibdh, isobutyryl-CoA dehydrogenase; Ivd, isovaleryl-CoA dehydrogenase; Kicd, α-ketoisocaproate dioxygenase; Ldh, lactate dehydrogenase; Mcd, malonyl-CoA decarboxylase; Me, malic enzyme; Mgat, acyl CoA:monoacylglycerol acyltransferase; Mgl, monoacylglycerol lipase; Mhbd, α-methyl-β-hydroxybutyryl-CoA dehydrogenase; MUFA, monounsaturated fatty acids; Mut, methylmalonyl-CoA mutase; Oxct1, 3-oxoacid CoA transferase; Pc, pyruvate carboxylase; Pcc, propionyl-CoA carboxylase; Pdh, pyruvate dehydrogenase; Pepck, phosphoenolpyruvate carboxykinase; Pk, pyruvate kinase; PP2Cm, protein phosphatase 2 Cm; Sbcad, methylbutyryl CoA dehydrogenase; Scd1, stearoyl-CoA desaturase; Sdh, serine dehydratase; Shmt, serine hydroxymethyltransferase.

Figure 2. The network of intracellular signals transduction that modulate mammalian target of rapamycin (mTOR) activity. BCAA within the cell are sensed by leucyl tRNA synthetase and Sestrin, what precludes related GTP binding (Rag) complex activation that in turn stimulates GTP binding to Ras homolog enriched in brain (Rheb) protein and its activation, ultimately leading to mTORC1 upregulation. BCAA also suppress mTORC1 inhibitors, such as general control nonderepressible 2 (Gcn2) and 5’AMP-activated protein kinase (AMPK). The activity of mTORC1 is positively regulated by oxygen through protein regulated in DNA damage and development (Redd1), hormones or growth factors through phosphoinositide-3-kinase (PI3K)/Akt or mitogen-activated protein kinase kinase 1/2 (Mek1/2)/extracellular signal-regulated kinase 1/2 (Erk1/2) signaling cascades, which inactivate tuberous sclerosis complex 2 (TSC2) subunit of the TSC complex that stimulates Rheb activity. Upon activation, mTORC1 regulates various proteins, including unc-51 like autophagy activating kinase 1 (Ulk1), p70 S6 kinase (p70 S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). Abbreviations: Deptor, DEP domain-containing mTOR-interacting protein; Gator, GTP-ase activating protein activity toward Rag; HIF-1α, hypoxia-inducible factor 1α; IRS1/2, insulin receptor substrate 1/2; mLST8, mammalian lethal with SEC13 protein 8; mSin1, mammalian stress-activated protein kinase-interacting 1; PDK1, 3-phosphoinositide-dependent kinase 1; Pras40, proline-rich Akt substrate of 40 kDa; Protor 1/2, protein observed with Rictor 1/2; Raptor, regulated associated protein of mTOR; Rictor, rapamycin insensitive companion of mTOR.

Table 1. Selected factors affecting BCAA metabolism in the blood, skeletal muscle, liver and adipose tissue.

Factor	Serum/plasma	Skeletal muscle	Liver	Adipose tissue
Fasting/short-term starvation	↑ BCAA (human) (Fryburg et al. 1990); ↑ BCKA (ob/ob mice) (Holeček 2018); ↓ BCAA (healthy individuals), ↔ BCAA (type 2 diabetes) (Tobias et al. 2021)	↑ Bcat activity (rats) (Holeček 2018)	↓ Bckdh activity (rats) (Holeček 2018); ↑ Bckdk (rats) (Harris et al. 2001)	↑ BCAA and BCKA, ↑ BCAA oxidation (mice) (Valerio, D’Antona, and Nisoli 2011)
Low protein diet	↓ BCAA and BCKA (human, rats, mice) (Zhou et al. 2019; Horiuchi et al. 2017; Maida et al. 2016; Fontana et al. 2016; Maida et al. 2017)	↓ Bckdh activity (rats) (Holeček 2018)	↓ Bckdh activity (rats) (Holeček 2018); ↑ Bckdk (rats) (Harris et al. 2001)	↓ p70 S6K (T389) (mice) (Maida et al. 2017)
Adiponectin	↓ BCAA and BCKA (diabetic mice) (Lian et al. 2015)	↑ Bckdh activity (diabetic mice) (Lian et al. 2015)	↑ PP2Cm, ↓ Bckdk, ↑ Bckdh activity (diabetic mice) (Lian et al. 2015)	↑ PP2Cm, ↓ Bckdk, ↑ Bckdh activity (diabetic mice) (Lian et al. 2015)
Insulin	↓ BCAA (mice, rats) (Neinast, Murashige, et al. 2019; Shin et al. 2014)	↑ BCAA oxidation (mice) (Neinast, Murashige, et al. 2019)	↑ Bckdh activity and BCAA oxidation (rats) (Shin et al. 2014; Gannaban et al. 2021)	↑ Bckdh activity (rats) (Holeček 2020); ↑ Bckdh (rats) (Shin et al. 2014)
Type 1 diabetes	↑ BCAA (human) (Borghi et al. 1985; Karusheva et al. 2021); ↑ 3-HIB (human) (Andersson-Hall et al. 2018)	↑ BCAA (human) (Borghi et al. 1985)	↓ Bckdh activity (rats) (Gibson et al. 1993)	↔ alanine oxidation (rats) (Szkudelska, Nogowski, and Szkudelski 2014)
High fructose diet	↑ BCAA (rats) (David et al. 2019)	↓ Bcat activity, ↑ pBckdha-E1α (rats) (David et al. 2019)	↔ pBckdha-E1α (rats) (David et al. 2019)	↔ Bcat activity, pBckdha-E1α (rats) (David et al. 2019)
High fat diet	↔ BCAA and BCKA (mice) (Lee et al. 2021; Maida et al. 2017); ↑ BCAA and BCKA (rats, mice) (Zhang et al. 2016; She, Reid, et al. 2007)	↔ BCAA and BCKA, ↔ BCAA oxidation (mice) (Lee et al. 2021); ↔ Bcat2, Bckdha-E1α, pBckdha-E1α and Bckdk (rats) (She, Reid, et al. 2007); ↓ Bckdhb, Mut and Ivd, ↓ BCKA (mice) (Lerin et al. 2016); ↔ Bckdh activity (mice) (Zhang et al. 2016)	↔ BCAA and BCKA (mice) (Lee et al. 2021); ↓ Bckdh activity (mice) (Zhang et al. 2016); ↓ Bckdk, ↑ Bckdh activity (rats) (Kadota et al. 2013)	↔ BCAA and BCKA, ↓ BCAA oxidation (mice) (Lee et al. 2021); ↓ Bckdh, ↑ pBckdh/Bckdh (mice) (Lackey et al. 2013); ↓ Bcat2 and Bckdha-E1α, ↑ Bckdk content, ↔ pBckdha-E1α (rats) (She, Reid, et al. 2007); ↓ Bckdh activity (mice) (Zhang et al. 2016)
Ob/ob mice	↑ BCAA and BCKA (Lian et al. 2015; She, Reid, et al. 2007; Hernández-Alvarez et al. 2017); ↔ BCAA, ↑ BCKA (Zhou et al. 2019)	↔ BCATm, Bckdh-E1α, pBckdh-E1α, Bckdk (She, Reid, et al. 2007); ↓ PP2Cm and ↑ Bckdk (Lian et al. 2015); ↔ Bcat2 and Bckdha (Hernández-Alvarez et al. 2017) ↔ BCAA catabolic genes (Zhou et al. 2019);	↓ Bckdh activity (She, Reid, et al. 2007); ↓ PP2Cm and ↑ Bckdk (Lian et al. 2015); ↑ pBckdh/Bckdh (Hernández-Alvarez et al. 2017); ↓ BCAA catabolic genes, ↑ BCAA and BCKA (Zhou et al. 2019)	↓ BCATm and Bckdh-E1α, ↑ Bckdk (She, Reid, et al. 2007); ↓ PP2Cm and ↑ Bckdk (Lian et al. 2015); ↓ Bcat2 and Bckdha (Hernández-Alvarez et al. 2017); ↓ BCAA catabolic genes, ↑ BCAA (Zhou et al. 2019)
Zucker fatty rats	↑ BCAA and BCKA, ↑ systemic leucine oxidation (She et al. 2013)	↑ BCAA and BCKA, ↓ Bckdh activity (She et al. 2013)	↔ Bckdha-E1α, ↑ Bckdk and pBckdha-E1α expression, ↓ Bckdh activity (White et al. 2016); ↑ Bckdk (9 week), ↓ Bckdk (18 week) activity (rats) (Doisaki et al. 2010); ↑ BCKA, ↔ Bckdh activity (She et al. 2013)	↓ Bckdh activity (White et al. 2016)
Obesity (human)	↔ BCAA (Biswas, Dao, et al. 2020); ↑ BCAA (Newgard et al. 2009; Cifarelli et al. 2020)	↓ BCAA metabolism (Lynch and Adams 2014)	↓ Bckdh (men), ↔ Bckdh (women) (Shin et al. 2014)	↓ Bckdha and Bckdhb (Lackey et al. 2013); ↓ BCAA catabolic genes (Biswas, Dao, et al. 2020)
Type 2 diabetes	↑ BCAA and BCKA (human, mice) (Hernández-Alvarez et al. 2017; Lian et al. 2015; Xu, Tavintharan, et al. 2013; Kujala et al. 2016); ↑ 3-HIB (human) (Andersson-Hall et al. 2018)	↑ pBckdh/Bckdh (mice) (Lian et al. 2015); ↓ BCAA degradation genes (human) (Lerin et al. 2016); ↓ Bcat2 and Bckdhb, ↑ methylation of Bckdhb, ↔ Bckdha, ↔ Bcat2, Bckdha, pBckdha and Bckdhb (human) (Hernández-Alvarez et al. 2017)	↑ pBckdh/Bckdh (mice) (Hernández-Alvarez et al. 2017); ↓ PP2Cm, ↑ Bckdk and pBckdh/Bckdh (mice) (Lian et al. 2015)	↓ Bcat2 and Bckdha (mice) (Hernández-Alvarez et al. 2017); ↓ PP2Cm, ↑ Bckdk and pBckdh/Bckdh (mice) (Lian et al. 2015)
Bariatric surgery	↓ BCAA (human, mice, rats) (She, Reid, et al. 2007; Laferrère et al. 2011; Bozadjieva Kramer et al. 2020; Seyfried et al. 2021)	↔ Bcat2 and Bckdh (mice) (Bozadjieva Kramer et al. 2020)	↔ Bcat2 and Bckdh (mice) (Bozadjieva Kramer et al. 2020)	↔ pBckdh/Bckdh ratio and Bckdk level, ↑ BCATm and total Bckdh-E1α (human) (She, Reid, et al. 2007); ↑ Bcat2 and Bckdh (mice) (Bozadjieva Kramer et al. 2020)
Exercise	↓ BCAA (human) (Gawedzka et al. 2020; Liu et al. 2020)	↑ Bckdh activity (human) (Wagenmakers 1998); ↑ Bckdk free form (rats) (Xu et al. 2001); ↑ BCAA uptake and Bckdh activity (human) (Gawedzka et al. 2020)	↑ Bckdk free form (rats) (Xu et al. 2001)	↑ BCAA oxidation (mice) (Valerio, D’Antona, and Nisoli 2011)
Thiazolidynedione	↓ BCAA (human) (Iwasa et al. 2015; Kalavalapalli et al. 2018)	↑ Bckdha and Bcat2 (mice) (Kalavalapalli et al. 2018)	↑ Bckdha and Bcat2 (mice) (Kalavalapalli et al. 2018)	↑ Bckdh (mice) (Lackey et al. 2013); ↑ Bckdha and Bcat2 (mice) (Kalavalapalli et al. 2018)

Abbreviations: 3-HIB, 3-hydroxyisobutyrate; BCAA, branched chain amino acids; Bcat, branched chain aminotransferase; BCKA, branched chain keto acids; Bckdh, branched chain keto acid dehydrogenase; Bckdk, branched chain keto acid dehydrogenase kinase; Ivd, isovaleryl-CoA dehydrogenase; Mut, methylmalonyl-CoA mutase; p70 S6K, p70 S6 kinase; PP2Cm, protein phosphatase 2 Cm.

Table 2. Metabolic characteristic of subjects undergone dietary BCAA deprivation or supplementation.

Experimental model	Diet	Body weight	Insulin sensitivity		Metabolic features	Reference
Dietary BCAA restriction
Lean C57BL/6J mice (male and female)	Isocaloric diet; one-fifth of the standard content of BCAA for 6 weeks	↓ fat mass	↔ basal blood glucose level and glucose tolerance ↓ fasting insulin level		↔ plasma TAG ↓ hepatic lipogenesis (↓Acl, Scd1, Fasn and Acc) ↑ hepatic gluconeogenesis (↑ Pepck) ↑ oxidation of carbohydrates ↑ glucagon content within pancreatic islets	(Solon-Biet et al. 2019)
Lean C57BL/6J mice (male and female)	Isocaloric leucine-deficient diet for either 7 or 17 days	↓ food intake and body weight ↓ abdominal adipose mass (−50% after 7 days, −97% after 17 days)	↓ plasma insulin ↔ serum glucose level and glucose clearance		↓ serum TAG and FFA levels Liver: ↓ Srebp-1c, PPARγ, Fasn, Acl, Scd, G6pd, and Me expression ↔ cholesterol level ↔ β-oxidation ↔ PPARα, Aco, Lcad, and Mcad content ↔ fatty acid transport proteins (i.e., Fabp, CD36/SR-B2, Fatp, Lpl)	(Guo and Cavener 2007)
Male C57BL/6J mice	7 days of leucine-deficient diet	↓ food intake ↓ abdominal fat and body weight	ND		↑ energy expenditure ↔ physical activity ↑ serum noradrenaline and T3 levels ↓ adipocyte volume (-42%) ↑ pHSL, PPARα, Cpt1 and Aco expression, and glycerol release in WAT ↓ Srebp-1c, Fasn, Acc1, Scd1 and Me expression in WAT ↑ β-oxidation- and fatty acid transport-related genes in BAT and skeletal muscle ↓ serum FFA level ↑ thermogenesis and oxygen consumption in BAT	(Cheng et al. 2010)
Male C57BL/6J mice fed the Western diet (leucine 25.4 g/kg, isoleucine 7.8 g/kg, valine 8.4 g/kg) for 12 weeks	Switched to normal calorie, BCAA-restricted diet (leucine 5.9 g/kg, isoleucine 1.81 g/kg, valine 1.95 g/kg) for additional ∼15 weeks	↔ food intake Rapid weight loss and stabilization ↓ adiposity	↑ glucose tolerance		Liver: ↓ lipid droplet size ↓ expression of many lipogenic genes	(Cummings et al. 2018)
	Switched to Western diet with low BCAA amount (-67%) for additional ∼14 weeks	↔ food intake ↓ body weight ↓ adiposity ↑ lean fraction	↑ glucose tolerance ↓ fasting blood glucose and insulin levels		↓ hepatic droplet size ↑ TAG content in the liver ↑ RER ↑ energy expenditure ↑ circulating FGF21 after 12 days, but ↔ after 12 weeks ↓ adipocyte size
Genetically obese (ob/ob) male mice	Low protein diet (6%) supplemented with BCAA in drinking water (3 mg/mL) for 2 weeks versus Low protein diet	↔ weight loss	↓ systemic insulin response ↓ insulin response in liver, WAT, and, slightly, in skeletal muscle ↔ fasting plasma glucose level		ND	(Zhou et al. 2019)
Male C57BL/ksJ-db (db/db) mice	Standard chow with intermittent leucine deprivation every other day for 8 weeks	↔ body weight ↑ fat mass ↓ lean mass	↓ fasting blood glucose level ↓ insulin resistance		↑ O2 consumption and CO2 production ↓ physical activity ↑ blood levels of valine, isoleucine, threonine, alanine, proline, lysine, and tyrosine ↑ β cells number in the pancreatic islets ↑ hepatic steatosis ↑ blood Alt and Ast levels ↑ Bacteroidetes and Actinobacteria, ↓ Firmicutes species within gut microbiota	(Wei et al. 2018)
Male Zucker fatty rats	Low fat and low BCAA diet (45% less BCAA than the normal BCAA diet) for up to 15 weeks versus low fat diet	↓ calorie consumption (-10%) ↓ weight gain (-15%) ↓ final body weight (-10%)	↔ systemic glucose tolerance ↑ skeletal muscle insulin sensitivity and glucose disposal		↓ eWAT mass ↑ circulating glycine ↓RER (↑ reliance on fatty acid oxidation) ↔ circulating TAG, glycerol, NEFA, ketones and lactate ↓ even chain acyl-CoA species in skeletal muscle ↑ muscle glycogen, pyruvate and lactate levels ↔ liver metabolites	(White et al. 2016)
Male Zucker fatty rats fed low fat diet	Low fat diet with reduced BCAA amount (-45%) for 15 weeks	↓ body weight	↔ circulating glucose and insulin concentrations ↔ pAkt and total Akt protein levels in the heart		↔ circulating TAG and NEFA Heart: ↑ palmitic acid oxidation to acetyl-CoA ↓ glucose uptake and catabolism ↑ long and even-chain acylcarnitines ↓ TAG level ↓ Glut4 and hexokinase II expression	(McGarrah et al. 2020)
Cobb 500 female chickens fed standard diet containing 77% Val/Lys and 69% Ile/Lys	Low BCAA diet containing 72% Val/Lys and 63% Ile/Lys for 2 weeks	↔ body weight	↔ plasma glucose		↓ plasma TAG Liver: ↑ Srebp-1, Srebp-2 and PPARα levels ↑ pAMPKα (Thr172), Ucp, Nrf-1 levels ↓ ACCα and Scd1 expression ↓ phosphorylation of mTOR (Ser2448) and p70 S6 kinase (Thr389)	(Bai et al. 2015)
8 healthy males and 4 females	BCAA-restricted diet (isoleucine 1.1%; leucine 1.8% and valine 1.3%) for 7 days	↔ body weight and BMI	↓ HOMA-IR		↓ circulating BCAA content ↔ circulating non-essential amino acids and essential amino acids minus the three BCAA amount	(Ramzan et al. 2020)
Lean male C57BL/6J mice	Isocaloric diet with all BCAA or isoleucine reduced by 67% for 3 weeks	↑ food intake ↓ weight gain ↓ fat and lean mass	↑ glucose tolerance		Low isoleucine: ↔ hepatic S6K1, 4E-BP1 and eIF2α phosphorylation ↑ Foxa2, Fasn and glycolytic gene expression in the liver ↑ FGF21 level in the plasma, WAT, liver and skeletal muscle ↑ adipocyte beigeing ↑ Acc, Fasn, Atgl and Hsl in WAT ↑ energy expenditure ↑ RER (↑ carbohydrate utilization)	(Yu et al. 2021)
Male C57BL/6J mice fed Western diet for 12 weeks	Switched to Western diet with 67% lower BCAA or isoleucine/valine level for up to 12 weeks	↑ food intake ↓ adiposity and weight gain	↑ glucose tolerance ↑ insulin sensitivity		↑ energy expenditure ↑ peripheral glucose uptake ↓ hepatic gluconeogenesis ↓ lipid droplets size in the liver	(Yu et al. 2021)
Type 2 diabetic patients (men/women, BMI 28-35 kg/m^2) with near-normal glycemic control	Isocaloric diet (55% carbohydrates, 30% fat and 15% protein) during weeks 1 and 3; 60% reduction of dietary BCAA in weeks 2nd and 4th	↔ body weight	↔ pAkt (Ser473), pAkt (Thr308) and p70 S6K (Ser2481) in skeletal muscle and adipose tissue ↑ postprandial insulin sensitivity		↔ circulating FFA in meal tolerance test ↔ skeletal muscle oxidative capacity ↑ mitochondrial efficiency in adipose tissue ↓ Firmicutes and ↑ Bacteroidetes in gut microbiota	(Karusheva et al. 2019)
Dietary BCAA supplementation
Lean C57BL/6J mice (male and female)	Isocaloric diet; doubled BCAA content for 6 weeks	↑ food intake (+20%; hyperphagia) ↑ body weight and fat mass	↔ basal blood glucose levels ↔ glucose tolerance	↔ hypothalamic gene expression of Lepr and Socs3 ↓ median lifespan (-10%) ↓ central Trp uptake and serotonin synthesis ↑ hypothalamic expression of genes promoting appetite, fat storage and inflammation ↑ hepatic TAG content and hepatosteatosis ↑ plasma Alt and Ast levels ↔ plasma TAG		(Solon-Biet et al. 2019)
Middle-aged (9 months old) male mice (sedentary and exercise-trained)	BCAA supplementation (1.5 mg/g body weight/day in drinking water) for 3 months	↔ food intake ↔ body weight and adiposity	↔ plasma insulin	↑ mitochondrial mass and biogenesis (↑ PGC-1α, Nrf1, Tfam, Sirt1) in the cardiac and skeletal muscle ↑ ROS defence system (↑ SOD1/2, catalase, GPx) in cardiac and skeletal muscles, but not in WAT and liver ↑ endurance capacity ↑ average lifespan		(D’Antona et al. 2010)
Male BALB/c mice	BCAA supplementation (in drinking water; 1.5 mg/g body weight/day) for 4 months	↔ body weight	ND	↑ gut microbial diversity ↑ Firmicutes/Bacteroides ratio ↑ Akkermansia and Bifidobacterium content in the gut (species protecting against diet-induced obesity)		(Yang et al. 2016)
C57BL/6J mice fed high fat (male and female) or high-fat high-sucrose diet (female) for up to 40 weeks	BCAA (0.75% leucine, 0.48% isoleucine, 0.53% valine) additionally supplemented in drinking water	↔ food intake ↔ weight gain	↔ insulin resistance	↑ C5-OH/C3-DC acylcarnitine content in the skeletal muscle and liver ↔ metabolome in the skeletal muscle and liver		(Lee et al. 2021)
Male C57BL/6J mice fed high fat (60%) diet	High fat (60%) diet supplemented with four times higher BCAA level (versus only high fat diet) for 8 weeks	↓ food intake ↔ weight gain and adiposity ↔ lean mass	↔ fasting blood insulin and glucose levels ↓ glucose tolerance	ND		(Bozadjieva Kramer et al. 2020)
Obese male C57BL/6J mice fed Western diet (leucine 25.4 g/kg, isoleucine 7.8 g/kg, valine 8.4 g/kg) for 12 weeks	Western diet with high BCAA content (leucine 16.6 g/kg, isoleucine 8.8 g/kg, valine 10.7 g/kg) for additional 15 weeks	↔ weight gain	↓ glucose tolerance	Hepatic steatosis with large lipid droplets ↓ expression of many lipogenic genes in the liver		(Cummings et al. 2018)
	Western diet with doubled BCAA content (leucine 50.8 g/kg, isoleucine 15.6 g/kg, valine 16.8 g/kg) for additional ∼14 weeks	↔ weight gain	Fasting hyperglycemia and hyperinsulinaemia ↑ HOMA-IR	Liver: ↓ TAG level ↓ lipid droplet size ↓ expression of lipogenic genes (Fasn, Scd1)
Wild-type male C57BI/6NCrl mice fed amino acid deficient diet	Diet deficient in all amino acids except BCAA for up to 14 weeks	↔ caloric intake ↑ feed efficiency ↓ fat loss ↔ lean mass loss	↓ improvement in intraperitoneal glucose tolerance and insulin tolerance ↑ fasting plasma insulin ↑ hepatic and peripheral mTORC1 activity	↓ serum FGF21		(Maida et al. 2017)
Hyperphagic New Zealand obese male mice fed amino acid deficient diet	Diet deficient in all amino acids except BCAA for up to 7 weeks	↔ caloric intake ↔ body mass accrual ↔ feed efficiency	↔ decrease in fasting plasma glucose ↔ fasting plasma insulin ↔ insulin sensitivity ↑ hepatic and peripheral mTORC1 activity	↔ hepatic FGF21
Male C57BL6J mice fed high fat (60%) diet	High fat diet supplemented with BCAA (L-leucine 25 g, L-isoleucine 12.5 g, and L-valine 12.5 g) in drinking water for 12 weeks	↓ weight gain and fat mass	↑ insulin resistance ↑ hepatic phospho-mTOR and ↓ pAKT	↑ plasma Ast and Alt ↑ circulating TAG and FFA Liver: ↓ amount of lipid droplets and liver TAG content ↑ FFA content ↓ Srebp-1c, Acc1, Fasn, Scd1, Elovl6 and Dgat1 expression ↑ proinflammatory cytokine levels (TNF-α, IL-6, IL-1β, and MCP-1) ↑ lipid peroxidation (↑ MDA and 4-HNE) ↓ inhibitory effects of insulin upon CL-316243-induced lipolysis		(Zhang et al. 2016)
Male C57BL/6J mice fed high fat (45%) diet for 6 weeks	20 mg BCAA/ml (2%) co-supplemented in drinking water for the last 2 weeks	↔ food intake ↔ body weight and adiposity	↓ serum insulin ↔ serum glucose	↔ serum TAG and FFA ↓ TAG in the liver and skeletal muscle ↑ PPARα and Acox1 expression in WAT, liver and skeletal muscle ↓ CD36/SR-B2 in the liver, but ↑ in skeletal muscle		(Arakawa et al. 2011)
Male C57BL/6J mice fed standard or high fat (60%) diet for 8 weeks	Standard or high fat diet supplemented with leucine (+1.5%) in drinking water for additional 14 weeks	Standard diet: ↔ food intake ↔ body weight or composition High fat diet: ↓ weight gain (-32%) and fat mass (-25%)	High fat diet: ↓ hyperglycemia ↓ basal plasma insulin and glucagon concentration ↑ insulin sensitivity	High fat diet: ↓ locomotor activity ↑ total energy expenditure ↑ fat oxidation (↓RER) ↑ Ucp3 expression in skeletal muscle, BAT and WAT ↓ hepatic G6P, but ↔ PGC-1α and Pepck expression ↓ total and LDL-C in plasma ↔ HDL-C, total TAG and FFA level ↓ proteolysis		(Zhang et al. 2007)
Male C57BL/6J mice fed high-fat/cholesterol (46%) diet	High-fat/cholesterol diet supplemented with 1.5% or 3% leucine for 24 weeks	↔ kcal/day intake ↓ weight gain ↔ ratios of fat and liver weights to body weight	↓ fasting insulin level and HOMA index	↓ serum and liver cholesterol level ↓ serum leptin, IL-6 and TNF-α level ↓ adipocyte size ↓ hepatic lipid accumulation ↓ hepatic Srebp-1, LXRα, Acc and Fasn expression ↑ pHSL (Ser563 and Ser660) and Atgl expression in eWAT ↑ expression of proteins involved in thermogenic program (Ucp1, β3AR, PGC-1 and FGF21) in WAT and BAT		(Jiao et al. 2016)
Male C57BL/6J mice initially fed high fat diet (8 weeks)	Standard chow supplemented with 1.5% leucine in water for 21 weeks	↔ food intake ↔ weight loss and fat mass	↓ fasting glucose level ↔ fasting insulin level and HOMA index ↑ glucose tolerance	↔ plasma levels of FFA, HDL-C, and LDL-C as well as hepatic TAG content ↓ RQ value ↑ fatty acid oxidation (Cox III, Ucp1, Ucp3, and Cpt1) and synthesis (Scd1, Acc, Fasn) in WAT ↑ fatty acid oxidation (Cox III, Ucp3, Cpt1 and PPARα) in BAT ↔ fatty acid metabolizing gene expression in skeletal muscle		(Binder et al. 2014)
C57BL/6J mice fed high-fat (60%) diet	High-fat high-BCAA (4%) diet for 16 weeks	Slower weight gain	↑ fasting blood glucose and insulin levels ↑ systemic insulin resistance ↓ hepatic pAkt1 and pAkt2 ↑ Akt2 degradation in hepatocytes ↑ hepatic mTORC1 activity	↑ plasma FFA, LDL and TAG concentration ↓ hepatomegaly ↓ hepatic TAG content and steatosis ↑ gluconeogenesis and glycogen content in the liver ↔ hepatic fatty acid β-oxidation ↓ expression of Srebp-1, Fasn and Scd1 in the liver ↑ muscular and kidney lipid content		(Zhao et al. 2020)
Male C57BL/6 mice with high fat (60%) diet-induced obesity	High fat diet with the addition of leucine (24 g/kg diet) for 6 weeks	ND	↑ glucose tolerance	↑ Sirt1 activity, pAMPK/AMPK ratio and pAcc/Acc ratio in adipocytes when combined with metformin		(Fu et al. 2015)
Male Wistar rats fed high fat (45%) diet	High fat (43%) diet supplemented with BCAA (↑ by 150% compared to high fat diet alone) for 12-16 weeks	↓ food intake ↓ weight gain	↔ insulin resistance ↑ phospho-mTOR (Ser2448), pS6K1 (Thr389), and pIRS1 (Ser302) in the skeletal muscle	↑ plasma C3 and C5 acylcarnitines ↔ total energy expenditure (VO2) and RER (RQ)		(Newgard et al. 2009)
Male Sprague-Dawley rats fed high fructose (60%) diet with streptozotocin-induced diabetes	50 mg/kg of 4-hydroxyisoleucine supplemented for 2 weeks	↔ body weight	↓ fasting glucose level ↑ insulin sensitivity ↑ Akt, pAkt and Glut4 expression in muscle	↓ plasma TAG, cholesterol, LDL-cholesterol, ↑ plasma HDL-C ↑ pAMPK/AMPK, ↓ PPARγ and PPARα expression (liver and muscle)		(Rawat et al. 2014)
Chinese 63 men and 69 women with BMI 25-36 kg/m^2 (nondiabetic overweight/obese)	BCAA-supplemented (0.1 g/kg body mass) hypocaloric diet with standard protein content (14%) for 16 weeks compared to placebo-supplemented diet	↔ weight loss and fat/lean mass loss	↔ fasting plasma glucose and insulin ↔ insulin sensitivity improvement (↔ HOMA-IR)	↔ plasma cholesterol, TAG, HDL-C and LDL-C		(Ooi et al. 2021)

Abbreviations: 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1; 4-HNE, 4-hydroxynonenal; β3AR, β3 adrenergic receptor; Acc, acetyl-CoA carboxylase; Aco, fatty acyl-CoA oxidase; Acl, ATP citrate lyase; Acox1, peroxisomal acyl-CoA oxidase 1; Alt, alanine transaminase; AMPK, 5’AMP-activated protein kinase; Ast, aspartate transaminase; Atgl, adipose triacylglycerol lipase; BAT, brown adipose tissue; BMI, body mass index; CD36/SR-B2, cluster of differentiation 36/scavenger receptor class B protein; Cox III, cyclooxygenase III; Cpt1, carnitine palmitoyltransferase 1; Dgat1, diacylglycerol acyltransferase 1; eIF2α, eukaryotic initiation factor 2α, Elovl6, elongation of very long chain fatty acids protein 6; eWAT, epididymal white adipose tissue; FABP, fatty acid binding protein; Fasn, fatty acid synthase; FATP, fatty acid transport protein; FFA, free fatty acids; FGF21, fibroblast growth factor 21; Foxa2, forkhead box protein A2; G6P, glucose-6-phosphatase; G6PD, glucose-6-phosphate dehydrogenase; Glut4, glucose transporter type 4; GPx, glutathione peroxidase; HDL-C, high-density lipoprotein cholesterol; HOMA, homeostatic model assessment; HSL, hormone sensitive lipase; IL-6, interleukin 6; IRS1, insulin receptor substrate 1; Lcad, long-chain acyl-CoA dehydrogenase; LDL, low-density lipoproteins; Lepr, leptin receptor; Lpl, lipoprotein lipase; LXRα, liver X receptor α; Mcad, medium-chain acyl-CoA dehydrogenase; MCP-1, monocyte chemoattractant protein 1; MDA, malondialdehyde; Me, malic enzyme; mTORC1, mammalian target of rapamycin complex 1; ND, not determined; NEFA, non-esterified fatty acids; Nrf1, nuclear respiratory factor 1; Pepck, phosphoenolpyruvate carboxykinase; PGC-1, peroxisome proliferator-activated receptor γ co-activator 1; PPARα, peroxisome proliferator-activated receptor α; RER, respiratory exchange ratio; ROS, reactive oxygen species; RQ, respiratory quotient; Scd1, stearoyl-CoA desaturase 1; Sirt1, sirtuin 1; Socs3, suppressor of cytokine signaling; SOD, superoxide dismutase; Srebp-1c, sterol regulatory element-binding protein 1; TAG, triacylglycerol; Tfam, mitochondrial transcription factor A; TNF-α, tumor necrosis factor α; Trp, tryptophan; Ucp1, uncoupling protein 1.

Table 3. Metabolic characteristic of subjects undergone genetic or pharmacological modulation of enzymes responsible for Bckdh phosphorylation/dephosphorylation.

Experimental model	Diet	Body weight	Insulin sensitivity	Metabolic features	Reference
PP2Cm inactivation
C57BL/6 mice with PP2Cm knockout	Standard diet	ND	ND	Heart: ↑ BCAA and BCKA levels ↓ systolic function ↑ superoxide production ↑ glucose, glycolytic intermediates and glucose-derived carbohydrate content	(Sun et al. 2016)
C57BL/6 mice with PP2Cm knockout	Standard diet (18% protein)	ND	ND	↑ plasma BCAA concentration ↑ hepatic Bckdh-E1α phosphorylation ↑ ROS production in isolated fibroblasts ↑ incidence of premature death in PP2Cm^–/– offspring compared with PP2Cm^+/– littermates	(Lu et al. 2009)
Lean C57BL/6 male mice with PP2Cm knockout	Standard diet; measurements at age of 10-14 weeks	↔ food intake ↔ energy expenditure and physical activity ↓ body weight	↑ glucose clearance and insulin sensitivity ↔ fasting plasma insulin level	↑ plasma BCAA and BCKA concentrations ↑ RER (preference toward carbohydrate usage) ↓ downstream BCKA metabolites in the skeletal muscle, liver and WAT ↑ hepatic glycogenolysis and glycolysis ↓ hepatic respiratory complex II ↔ glucose metabolism in the skeletal muscle and WAT	(Wang et al. 2019)
C57BL/6 mice with PP2Cm knockout	Standard diet	↔ body weight	↔ blood glucose and insulin levels ↔ glucose tolerance ↔ insulin sensitivity (↔ mTOR, p-mTOR, Akt, p-Akt, PI3K and p-PI3K levels) in the heart	Heart: ↓ glucose and ↑ fatty acid oxidation ↓ glucose uptake, glycogen content, mitochondrial pyruvate utilization and Pdh activity ↔ genes involved in glucose uptake, glycolysis, glycogenesis, and fatty acid oxidation ↑ susceptibility to ischemia/reperfusion injury	(Li et al. 2017)
C57BL/6 mice with PP2Cm knockout	Standard diet or high fat diet (60%) for 8 weeks	Standard diet: ↔ food intake, body weight and fat mass High fat diet: ↔ food intake and weight gain High fat diet + bariatric surgery: ↔ weight loss	Standard diet: ↔ glucose tolerance at 6-, 9-, 12- and 30-week High fat diet + bariatric surgery: ↔ blood glucose ↔ insulin sensitivity improvement	↑ plasma BCAA to all amino acids ratio	(Bozadjieva Kramer et al. 2020)
Obese insulin-resistant Zucker fatty rats	Low fat diet, adenoviral transfection of human PPM1K, metabolic features assessed 7 days later	↔ body, liver, adipose, or skeletal muscle weight	↑ insulin sensitivity	↓ plasma BCAA and BCKA concentrations ↓ circulating lactate level ↔ circulating TAG, cholesterol, glycerol, NEFA and ketones ↑ hepatic, but not cardiac, Bckdh activity ↓ hepatic TAG content ↑ acylcarnitines amount in the liver ↓ Acl (Ser455) phosphorylation	(White et al. 2018)
Bckdk inactivation
Lean, male C57BL/6 mice	Standard diet, metabolic parameters assessed 30 min after a single intravenous injection of BT2 (40 mg/kg)	ND	ND	↓ circulating BCAA and BCKA levels ↑ circulating FFA and acyl-carnitines ↔ proteolysis	(Neinast, Jang, et al. 2019)
Lean C57BL/6 mice with Bckdk knockout	Standard diet	ND	↔ glucose clearance	↑ systemic BCAA oxidation ↓ circulating BCAA and BCKA levels ↑ circulating 3-HIB content ↓ circulating ratios of α-ketoglutarate to glutamate and pyruvate to alanine ↔ blood FFA content
Obese and insulin-resistant Zucker fatty rats	Low fat diet, BT2 (20 mg/kg/day) injected intraperitoneally for 1 week	↔ body weight ↔ liver, adipose, or skeletal muscle weight	↑ insulin sensitivity	↔ circulating TAG, cholesterol, glycerol, NEFA and ketones ↔ O2 consumption or heat production ↓ RER (↑ preference for fatty acids over glucose) ↓ hepatic Acl expression and phosphorylation (Ser454) ↓ TAG level in the liver ↑ hepatic even-chain acylcarnitines content	(White et al. 2018)
Ob/ob mice or mice with diet-induced obesity	High fat diet with daily oral doses of BT2 40 (mg/kg/day) for 8-10 weeks	↔ food intake ↔ body weight	↑ glucose tolerance and insulin sensitivity ↓ hyperinsulinemia	↓ basal and insulin-stimulated mTORC1 activity indicated by ↓ p70 S6K phosphorylation (Thr389)	(Zhou et al. 2019)

Abbreviations: Acl, ATP citrate lyase; BT2, 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid; FFA, free fatty acids; mTORC1, mammalian target of rapamycin complex 1; ND, not determined; NEFA, non-esterified fatty acids; Pdh, pyruvate dehydrogenase; PI3K, phosphoinositide-3-kinase; RER, respiratory exchange ratio; ROS, reactive oxygen species; TAG, triacylglycerol; WAT, white adipose tissue.

Figure 3. Systemic representation of BCAA-regulated processes.

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