Brown fat thermogenesis and branched-chain amino acids in metabolic disease

Abstract

Since the 1960s, researchers have recognized an association between elevated plasma branched chain amino acids (BCAA) and metabolic disease, including type 2 diabetes mellitus and obesity, but the cause for it remained poorly understood. Recent advances in metabolomics, advanced imaging techniques, and genetic analyses over the past decade have enabled newfound insights into the mechanism of BCAA metabolic dysregulation across a variety of peripheral tissues and its impact on metabolic disease, suggesting a key role for brown adipose tissue (BAT) in determining BCAA metabolic homeostasis. Previous investigations into BAT have emphasized fatty acids and glucose as substrates for BAT thermogenesis. Here, we address the importance of BAT in systemic BCAA metabolism, driven via the newly identified mitochondrial BCAA carrier (MBC), as well as the impact of BAT-driven BCAA clearance on glucose homeostasis and metabolic disease. The newly identified MBC offers new therapeutic avenues by which BAT activity may be enhanced to improve metabolic and cardiovascular health, as well as other diseases in which increases of circulating BCAA may play a role in pathogenicity.

Introduction

Brown adipose tissue (BAT) is a metabolically dynamic organ that has attracted much clinical attention in recent years for its unique ability to dissipate vast amounts of chemical energy into heat. BAT activity can be stimulated in mammals in a variety of ways, including environmental cues such as cold exposure and sympathomimetics, both of which elicit sympathetic nervous system (SNS) activity [1]. In turn, BAT responds with rapid uptake of circulating metabolites, including glucose, triglyceride-rich lipoproteins, and fatty acids (FA), all of which are utilized to fuel the energy-demanding process of thermogenesis [2-4]. In mammalian neonates, interscapular depots of thermogenic BAT serve as the major site of heat production as they have insufficient musculature to shiver. It was long assumed that BAT as well as its physiological relevance degenerates in healthy adult humans. However, in 2009, a series of groundbreaking reports using newly available advanced imaging techniques, namely, positron emission tomography/computed tomography (PET/CT), challenged this assumption, indicating BAT could in fact be detected in adult humans [5-8]. PET/CT imaging in healthy individuals with radioactive glucose (¹⁸F-fluorodeoxyglucose, ¹⁸F-FDG) and radioactive FA tracers indicate that human BAT indeed behaves as a metabolic sink for glucose and FA [4]. Other studies suggest that BAT contributes to glucose and FA clearance in the circulation of humans [9-11]. Researchers have further demonstrated that BAT activity is inversely correlated with obesity and insulin resistance in adult humans [5, 6, 8, 12], supporting its function as a metabolic sink and suggesting a potential role in treating metabolic disease [10, 13].

It is well-known that FA and glucose are major substrates for BAT thermogenesis by fueling the tricarboxylic acid (TCA) cycle. However, until recently, the contributions of other substrates to BAT thermogenesis have largely remained poorly understood. Now, the apparent fact that BAT recruits circulating metabolites beyond the historically recognized substrates for thermogenesis is increasingly being recognized. For example, recent reports posit a role for succinate, a TCA intermediate, wherein uptake or accumulation of succinate drives thermogenesis through its oxidation and generation of reactive oxygen species [14, 15]. Moreover, several studies have now also shown that SNS stimulation strongly activates BAT thermogenesis and decreases the levels of circulating branched-chain amino acids (BCAAs), leucine (Leu), isoleucine (Ile), and valine (Val) [16-18]. It was also recently revealed that BAT actively oxidizes BCAAs through a series of anaplerotic reactions that drive thermogenesis, via the recently identified mitochondrial BCAA carrier (MBC) in brown adipocytes [16]. Owing to the established role of BCAAs in the pathogenesis of cardiovascular and metabolic disease [19-21], this recent recognition of the role of BAT as a metabolic sink for BCAAs indicates that MBC may be a powerful new target for the treatment of obesity and related metabolic diseases.

In this review, we examine the role of BAT in systemic BCAA metabolism, driven via the newly identified mitochondrial BCAA transporter MBC, its impact on metabolic disease, including obesity and type 2 diabetes (T2DM), and its potential therapeutic applications.

Association of BCAA Metabolism with Systemic Glucose Homeostasis

The role of BCAAs in diseases has long been speculated, since at least the 1960s, when Felig et al. first reported that elevated serum BCAA levels in humans are correlated with the eventual development of insulin resistance, T2DM, and cardiometabolic disease [22]. Since then, advances in metabolomics have enabled investigators to corroborate their findings, demonstrating the utility of circulating BCAAs as a powerful prognostic marker for the development of metabolic disease [23-27].

The relationship between BCAAs and glucose homeostasis is directly tied to cellular utilization of BCAAs in the anabolic processes of protein synthesis and gluconeogenesis, and the catabolic process of anaplerosis. BCAA uptake and oxidation occur variably across multiple tissues, especially in skeletal muscle, liver, heart, brain, and adipose tissue [27-29]. Transport across the plasma membrane occurs predominantly via a heterodimeric complex involving proteins LAT1 and CD98 [27, 30]. The first step of BCAA catabolism, common to all three dietary BCAAs, is an initial, reversible transamination reaction mediated via BCAA transaminase (BCAT) producing their cognate branched-chain alpha-ketoacids (BCKAs) (Fig. 1) [30-32].

Fig. 1

Subcellular metabolic fat of BCAA and its impact on insulin sensitivity

Cellular uptake of BCAA via LAT1 can lead to catabolism, predominantly in the mitochondria, by entering the TCA cycle. In this way, BCAA contributes to TCA anaplerosis, in turn contributing to thermogenesis and whole-body energy metabolism. Conversely, cytosolic BCAA can lead to activation of mTORC1 activity. In the short-term, this leads to enhanced autophagy and increased protein synthesis and cell differentiation. In the long-term, mTORC1-mediated phosphorylation of IRS1 and IRS2 targets these proteins for degradation, and in turn increases the burden on insulin.

Notably, there are two isoforms of the BCAT enzyme: a cytosolic isoform (BCAT1 or BCATc) and a mitochondrial isoform restricted to the cristae (BCAT2 or BCATm). In the majority of tissues, BCAT2 is the dominant isoform, necessitating transport of BCAAs into mitochondria via a transporter, MBC, which has only recently been identified [16]. Conversely, BCAT1 is expressed in select tissues including the brain, lungs, and endocrine organs such as the thyroid and pancreas [33-35]. In order to enter the TCA cycle, after transamination a fraction of the resultant BCKAs undergo a subsequent, rate-limiting step of irreversible oxidative decarboxylation, carried out by the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex within the mitochondria [32, 34, 35]. BCAA-derived metabolites are subsequently ligated with coenzyme A (CoA) to form either succinyl-CoA (derived from Leu and Ile) or acetyl-CoA (derived from Val and Leu) for participation in the TCA cycle [36, 37]. Alternatively, Val, in addition to its partially metabolized derivatives ketoisovalerate (KIV) and 3-hydroxyisobutyrate (3-HIB), can serve as anabolic substrates for gluconeogenesis [38-40].

BCAA Catabolic Activity in Various Tissues

To understand how BCAA metabolism impacts glucose homeostasis and why elevated serum BCAA levels should correspond with metabolic disease, Neinast et al. performed in vivo isotope tracing to quantify the contributions of various peripheral tissues to systemic BCAA clearance [41]. BCAAs are not catabolized uniformly across tissues. Metabolomic studies and tissue-specific gene expression analysis revealed that the key enzymes responsible for BCAA catabolism are variably expressed across different organs [41] (Table 1). As a result, different organs make variable contributions to BCAA clearance, and consequently whole-body energy homeostasis. Analysis of oxidative capacity for BCAA of different tissues using radioactive BCKA isotope tracers in mice demonstrated the heterogenous contributions of peripheral tissues to BCAA breakdown in mice, with combined skeletal muscle and BAT contributions comprising almost 80% of total BCAA oxidation across the entire body [41].

Table 1

Expression of rate-limiting enzymes of BCAA oxidation in various tissues and their roles in BCAA clearance

	Enzyme expression			Role in BCAA clearance
	BCAT1	BCAT2	BCKDHc	BCAA catabolic activity per mass	Mass	Contribution to BCAA clearance	Effect of HFD on catabolic activity
Brain	High	Low	Low	Low	Low	Low	Unclear
Skeletal muscle	Low	High	High	High	High	Very high	Downregulated
Liver	Low	Low	High	Low	High	High	Downregulated
Heart	Low	High	High	Very high	Low	Modest	Downregulated
WAT	Low	Modest	Modest	Low	Variable	Modest	Downregulated
BAT	Low	High	High	Very high	Low	Very high	Unclear

The enzymes responsible for BCAA catabolism are differentially expressed across different organs in both humans and mice [25-27, 39, 41, 42, 46, 56, 61]. Likewise, the estimated rates of BCAA clearance also vary across tissues, with skeletal muscle, BAT, and liver making the greatest contributions [39, 46]. Supplementing mice with HFD reportedly attenuates the catabolism of BCAA in select tissues [14]. BAT = brown adipose tissue; HFD = high fat diet; WAT = white adipose tissue.

It has long been well established that skeletal muscle makes a major contribution to whole-body BCAA clearance [42]. The myocytes that compose skeletal muscle predominantly express the mitochondrial isoform of transaminase, BCAT2, and also express heightened levels of genes responsible for enzymes involved in BCAA oxidation [42]. Defective BCAA catabolism in myocytes has a posited role in insulin resistance, with several reports suggesting that BCAA metabolic dysregulation in myocytes specifically contributes to metabolic dysfunction [43-45]. In a study of humans with insulin resistance, gene expression analysis of skeletal muscle revealed depressed expression of enzymes required for BCAA oxidation, including BCKDH [43]. Additionally, in rodent models of obesity, chronic high-fructose diet induces decreased skeletal muscle BCAT activity and resulted in increased fasting plasma BCAA levels, which impedes whole-body BCAA clearance [46]. Most strikingly, a recent randomized controlled trial evaluating the efficacy of pharmacological activation of BCAA catabolism in tissues including skeletal muscle showed a remarkable 27% improvement in insulin sensitivity in humans [47].

Cardiac muscle also exhibits BCAA catabolic activity [41]. BCAT2 and BCKDH are expressed at high levels in cardiomyocytes, indicating that the tissue exhibits robust BCAA oxidative capacity. However, a recent study by Walejko et al. observed that actual rates of BCAA oxidation in cardiomyocytes are low [48]. Conversely, BCAA anabolism predominates with BCAA intermediates, primarily BCKAs, preferentially undergoing reamination to form BCAAs. This phenomenon is likely due to low expression of the mitochondrial BCAA transporter in cardiomyocytes, and can be associated with programs of pathologic protein synthesis, leading to cardiac hypertrophy in mice [48]. Researchers have also established that BCAAs reduce cardiomyocyte glucose oxidation directly by suppressing pyruvate dehydrogenase (PDH) activity [49]. However, given the low prevalence of cardiac muscle in the body by mass, relative to other tissues, its effect on whole-body BCAA clearance as well as systemic glucose homeostasis remains insubstantial.

Although the liver is the site of catabolism for most amino acids, it performs relatively little BCAA oxidation. This is due in part to the fact that hepatocytes express both isoforms of the transaminase BCAT at hardly detectable levels, whereas BCAT expression is upregulated in certain forms of hepatoma [50, 51]. In turn, BCAAs primarily undergo transamination in extrahepatic tissues, and BCKAs may be recirculated back to the liver for metabolic breakdown at the point of BCKDH [52, 53]. Studies of diet-induced obese mice indicate that excess dietary BCAA intake results in increased lipogenesis and lipid accumulation in the hepatocytes, leading to hepatic insulin resistance and systemic glucose intolerance [54-56]. Furthermore, genetic modulation of hepatic BCAA catabolism indicates a clear role in systemic BCAA clearance. Liver-specific over-expression of protein phosphatase 1K (Ppm1k), a positive regulator of BCAA oxidation, leads to reduced hepatic steatosis, lower circulating BCAAs, and improved systemic glucose tolerance in Zucker fatty rats [36]. Additionally, review of Hybrid Mouse Diversity Panel data revealed variable contribution of hepatic BCAA catabolic gene expression to insulin resistance and fasting glucose, indicating no clear role of the liver in driving the pathogenesis of metabolic disease associated with elevated circulating BCAA [57].

Metabolism of BCAA in the brain is unique from that of many other tissues, in that the dominant transaminase expressed is the cytosolic isoform BCAT1 [58]. Consequently, BCAAs are not transported directly to the mitochondria of astrocytes, the most metabolically active cell in the brain, but rather undergo conversion into their conjugate BCKAs within the cytosol prior to mitochondrial transport. The sequestration of BCAAs to the cytosol of astrocytes corresponds with the observed low rates of catabolic turnover of BCAAs, as this tissue is not thought to make a major contribution to whole-body BCAA clearance [59]. However, a recent report challenging this assumption demonstrated that BCAA oxidation does occur in cultured human astrocytes, and suggested that the upregulation of astrocyte metabolism of Leu may in fact be associated with the pathogenesis of Alzheimer’s disease [58].

Finally, adipose tissue also plays a crucial role in systemic BCAA metabolism, and consequently glucose homeostasis [19]. Studies in mice and humans revealed that white adipose tissue (WAT) transcribes genes responsible for mitochondrial BCAA oxidation, such as those composing the BCKDH complex, and that expression of these enzymes is markedly decreased in obese and diabetic states [60-63]. In addition, transplantation of WAT in rodents with impaired BCAA metabolism significantly reduces circulating BCAAs [63, 64]. Given its competence for BCAA oxidation and relatively high mass, WAT may exert an important role in regulating systemic BCAA levels. However, despite its clear potential as a powerful organ in BCAA clearance, whole-body metabolomics data suggest that the relative contribution of WAT to BCAA clearance is quite minimal due to low tissue oxidative flux [41]. In stark contrast to WAT, current estimates indicate that BAT may contribute to up to 19% of systemic BCAA catabolism [41].

BAT Utilizes BCAA as a Key Fuel for Thermogenesis

BAT is composed of mitochondria-enriched brown adipocytes, which possess multilocular lipid droplets primed for rapid mobilization of fatty acid stores. BAT accomplishes thermogenesis through rapid catabolism of fuels such as FA and glucose, and subsequent uncoupling the mitochondrial proton gradient from mitochondrial ATP synthesis by uncoupling protein 1 (UCP1) which dissipates chemical energy in the form of heat. Accordingly, enzymes required for mitochondrial transport and metabolism of FA and glucose, including carnitine palmitoyltransferase 1 (CPT1b), CPT2, plasma membrane glucose transporter 1 (GLUT1), GLUT4, hexokinase (HK2), mitochondrial pyruvate carrier (MPC), and PDH are present at elevated levels in both murine and human brown adipocytes [65-67]. BAT thus serves as a metabolic sink of glucose and FA for clearance in the circulation in humans [68, 69]. However, the utilization of other possible substrates beyond FA and glucose has not historically been fully addressed, especially in humans. Notably, BAT also expresses heightened levels of enzymes responsible for BCAA catabolism, including BCAT2 and BCKDHA [16]. Furthermore, analysis of the adipose tissue of mice shows that cold exposure robustly induces a program of mitochondrial BCAA oxidation [70, 71].

In 2019, by employing ¹⁸F-FDG-PET/CT and serum metabolite analysis before and after cold exposure, Yoneshiro et al. discovered that humans with high BAT activity exhibit significantly decreased BCAA levels in the circulation following cold exposure, whereas no change was observed in individuals with low BAT activity [16]. Consistent with this finding, plasma BCAA was decreased by acute cold exposure in wild type mice whereas it remained unchanged in BAT-ablated mice [16]. Gene and protein expressions of BCAA catabolic enzymes (i.e., BCAT2, BCKDHA, BCKDHB) in BAT are increased by cold exposure both in mice and humans. Cold-induced utilization of BCAA in BAT has also been verified in a mouse model by visualizing Leu uptake in murine BAT via PET-CT using the radioactive Leu-analogue tracer, ¹⁸F-fluciclovine (Fig. 2A). Additionally, pharmacological activation of β3 adrenergic receptor has also been shown to decrease circulating BCAAs and increase BCAA-derived metabolites in humans [72].

Fig. 2

BCAA catabolism via MBC fuels BAT thermogenesis

A. Typical images of PET/CT using ¹⁸F-fluciclovine, Leu analog. Mice were acclimated to either thermoneutral temperature at 30°C or cold temperature at 8°C for 2 weeks. ¹⁸F-fluciclovine uptake into interscapular BAT depots (pink arrowheads) was significantly enhanced by cold acclimation.

B. A model of BCAA oxidation in brown adipocytes. Cold exposure or psychological stress stimulates mitochondrial BCAA oxidation in brown adipocytes. MBC transports BCAA from cytosol into mitochondria, thereby serving as a gatekeeper for BCAA catabolism.

The importance of BCAA clearance to BAT function is further corroborated by in vitro investigations. Metabolic tracing using ¹³C-labeled stable isotope of Leu revealed that norepinephrine (NE)-induced Leu catabolism results in an accumulation of TCA cycle intermediates [16], including succinate, in brown adipocytes, suggesting that BCAA is an important substrate for BAT thermogenesis [14]. In fact, BAT thermogenic function as well as cold-induced BCAA clearance is significantly blunted in mice with a brown adipocyte-specific knock-out of Bckdha, which is driven by Ucp1-Cre (BCKDHA^UCP1-KO) [16]. Moreover, BCKDHA^UCP1-KO mice are unable to maintain core body temperature in a cold environment. Conversely, disrupting BAT thermogenic capacity by Ucp1 knockout results in accumulation of BCAAs in BAT [73]. BCAA-associated thermogenesis is regulated cell-autonomously because NE-induced oxygen consumption in cultured brown adipocytes is largely diminished in BCKDHA-KO cells, whereas it is restored by supplementation of succinate which bypasses the synthesis of BCAA-derived TCA intermediates and anaplerosis [16].

Collectively, these data indicate that BAT is responsible for BCAA clearance following cold exposure, and that BCAA catabolism is indispensable for BAT thermogenesis and thermoregulation in cold environment (Fig. 2B).

BCAA: Good or Bad for Glucose Homeostasis?

It can be extrapolated from the above data that BCAA supplementation might enhance BAT thermogenesis, and possibly improve energy homeostasis in humans. Consistent with this assumption, BCAA supplementation is a frequent tactic employed by individuals seeking to promote anabolic uptake of BCAAs, while researchers have also demonstrated that BCAA supplementation often improves insulin sensitivity [64]. Pharmacological activation of BCAA oxidation via sodium phenylbutyrate or peroxisome proliferator-activated receptor-gamma (PPARγ) agonists in humans also leads to enhanced insulin sensitivity [45, 74]. Paradoxically, however, it has also been reported that when paired with high-fat diet (HFD), BCAA supplementation leads to diabetes, insulin resistance, and obesity [64]. Additionally, a mouse model in which BCAA oxidation is genetically impaired by a whole-body knock-out of Ppm1K exhibits impaired insulin sensitivity [36, 49]. These somewhat contradictory findings indicate BCAA can have disparate effects in different settings; that is, BCAAs can improve metabolic health by enhancing their oxidation and energy expenditure, but in the context of impaired catabolic activity due to obesity, a concomitant accumulation of BCAA may be deleterious for metabolic health and promote insulin resistance and T2DM [64, 75, 76].

Mechanistically, there are several pathways, both direct and indirect, by which incomplete oxidation of BCAAs may exert their pathogenic effects on whole-body metabolism. Historically, the best studied direct mechanism implicates Leu as an activator of mammalian target of rapamycin complex 1 (mTORC1), a potent anabolic signaling factor which controls protein synthesis, cell proliferation, and differentiation (Fig. 1). Leu achieves this effect by directly binding and inhibiting Sestrin2, a negative regulator of mTORC1 function [77-80]. Sestrin2, in the absence of Leu, binds and inhibits GATOR2, a positive regulator mTORC1. Consequently, mTORC1 is active in the context of an abundance of intracellular Leu, leading to excess phosphorylation of insulin receptor substrate-1 (IRS1) and IRS2, and subsequent inhibition of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway. In the setting of chronically elevated plasma BCAAs, continued phosphorylation of IRS1 targets the receptor for degradation via protease, and leads to reduced expression of IRS1 and IRS2 on the cellular membrane, which increases the signaling burden on insulin. Long-term, IRS1 and IRS2 degradation leads to reduced insulin sensitivity, and eventually T2DM [81]. Of note, a recent study purports a role for Val, in addition to Leu, in the activation of mTORC1 [82].

Furthermore, BCAA-associated modulation of endocrine signaling in the brain or secretion of satiety-related hormones across a variety of peripheral tissues is another branch whereby dietary BCAAs may influence insulin sensitivity through separate, parallel mechanisms [83-93]. One powerful indirect mechanism is through satiety signaling, where, in the hypothalamus, BCAAs are thought to influence satiety [87]. Experiments in mice fed BCAA-rich diet showed that patterns of gene expression in the hypothalamus changed upon excess BCAA intake to promote hunger, inflammation, and obesity [87]. Additionally, dietary supplementation of Leu triggers the release of satiety hormones, such as cholecystokinin (CCK), glucagon-like peptide 1 (GLP1), gastric inhibitory polypeptide (GIP), and leptin [27, 91, 92]. BCAA metabolism is also tightly coupled with the function of pancreatic islet cells. BCAA deprivation reduces insulin secretion in pancreatic β cells [88, 89]. In contrast, disordered BCAA catabolism promotes glucagon secretion in diabetic mice [90].

As previously mentioned, BCAA-derived 3-HIB plays an important role in regulating insulin sensitivity [38]. This may be due in part to the observation that 3-HIB promotes lipid droplet accumulation and fatty acid uptake in adipocytes, as well as insulin-mediated glucose transport. 3-HIB has also been shown to play divergent roles in brown and white adipocytes, stimulating TCA-cycle activity in the former while inhibiting it in the latter [38]. In addition, monomethyl branched-chain fatty acids (mmBCFAs) serve as another BCAA-derived metabolite that promotes de novo lipogenesis in BAT [70, 71, 93]. In humans, mmBCFAs levels are inversely correlated with obesity and insulin resistance [94]. The precise mechanism by which mmBCFAs may contribute to metabolic health, however, remains unestablished. In contrast to the beneficial effect of their metabolites, accumulation of BCAA-derived Acetyl-CoA due to incomplete oxidation in TCA cycle induces acetylation of PR domain containing 16 (PRDM16), the master regulator of beiging of WAT, thereby preventing beige fat development [95]. Researchers have shown that accumulation of BCAAs is coupled with disrupting PDH activity [49] and insulin resistance. In this regard, it is worth noting that BCKDHA^UCP1-KO mice fed HFD exhibited insulin resistance, partly due to impaired phosphorylation of PDH and reduced glucose oxidation in BAT [16].

With the caveat that so long as BCAAs are actively being catabolized, they apparently perform a protective function for metabolic health, defects in BCAA oxidation lead to impaired insulin resistance, and consequently BCAA accumulation is frequently observed in instances of metabolic disease. Taken together, the beneficial effects of BCAAs on energy metabolism and insulin sensitivity seem to demand high catabolic potential toward BCAA. It is thus possible that BAT’s function as a BCAA metabolic sink prevents excess accumulation of BCAAs in the circulation, thereby preventing insulin resistance.

Identification of Mitochondrial BCAA Transporter

BCAA metabolic fate is regulated by cellular BCAA uptake and catabolism. As previously addressed, the first step of BCAA catabolism is transamination mediated via BCAT1 or BCAT2. The mitochondrial isoform, BCAT2, is selectively expressed in the majority of metabolically active tissues, including adipose, indicating that mitochondrial uptake of BCAA is a crucial gatekeeper for BCAA metabolic fate. Although LAT1 has long been identified as a plasma membrane transporter for BCAA and other large neutral amino acids [96, 97], the mitochondrial BCAA transporter MBC has only recently been identified [16].

Given that BAT actively oxidizes BCAAs in the mitochondria, it was reasoned that BAT would likely express this previously uncharacterized mitochondrial BCAA transporter. Researchers had previously performed amino acid sequence similarity analysis which suggested solute carrier family 25 (SLC25A) proteins included candidates for a putative mitochondrial amino acid transporter [98]. Transcriptome analysis of murine and human thermogenic adipocytes indicated that several transcripts from this SLC25A family subset were expressed at heightened levels, while a further refined cohort with previously undetermined function was additionally upregulated in response to cold exposure [16]. Among those transcripts upregulated by cold exposure, only one was subsequently confirmed to play a functional role in BAT thermogenic function: SLC25A44, named as mitochondrial BCAA carrier (MBC) [16]. Cell-free proteoliposome assay using radioactive Val and Leu isotopes confirmed that MBC mediates the transport of BCAAs, but not that of other amino acids [16] (Fig. 2B). Murine whole-body MBC depletion by CRISPR inference resulted in thermogenic dysfunction of BAT, impairment of BCAA oxidation, and loss of thermoregulatory ability during cold exposure. Similarly, adeno-associated virus (AAV)-mediated BAT-specific depletion of MBC significantly blunted thermogenic function of BAT in mice. These findings clearly demonstrated that mitochondrial BCAA transport via MBC is required for BAT function and thermoregulation (Fig. 2B).

Pathophysiological Roles of MBC

Since MBC is crucial for BCAA catabolism and thermogenesis in BAT, it is highly probable that MBC is involved in the control of systemic energy balance and glucose tolerance, although it remains to be tested. Moreover, the anti-obesity and anti-diabetic roles of MBC and BAT may be mediated through mechanisms beyond thermogenesis. Since MBC may be a gatekeeper of the subcellular fate of BCAA, i.e., mitochondrial catabolism versus cytosolic accumulation, it is thus possible that MBC may control BCAA-mediated intracellular signaling in the context of obesity, as, for instance, a constitutive activation of mTORC1, impaired autophagy, and impaired insulin sensitivity. Further studies are needed to test the roles of MBC in the cellular metabolic flexibility of BCAAs and in systemic energy and glucose homeostasis.

On the other hand, multiple studies have implied that mitochondrial BCAA transport contributes to a wide range of pathophysiological processes. For instance, MBC has been shown to play a role in the febrile response to psychological stress [99]. Psychological stress, such as social defeat stress, induces BAT thermogenesis and hyperthermia in rats and probably in humans [100-102]. Interestingly, social defeat stress indeed increases BCAA oxidation in BAT of rats [99]. However, this febrile thermogenic response is diminished in brown adipocyte-specific MBC or BCKDHA knock-out mice, suggesting that active BCAA catabolism in BAT might be involved in stress-induced hyperthermia and defensive stress responses. Consistent with this finding, clinical studies have shown that circulating BCAA levels are elevated in patients with depression or chronic fatigue syndrome, who often show low-grade fever [103-105]. BCAAs are also posited to play a complex role in mood regulation, through competition for uptake at the hypothalamus with dopamine and 5-hydroxytryptamine precursors [84-87]. In this model, BCAAs may contribute to prevalence of mood disorders in individuals with metabolic disease [106]. These findings may offer an effective intervention by targeting MBC to treat stress-associated disorders. In this regard, BCAA supplementation reportedly increases resilience to social defeat stress in mice [107].

Although BCAA is rarely catabolized in the heart because of the undetectable levels of MBC, Walejko et al. demonstrated the function of MBC in a different experimental model, where targeted overexpression of MBC in the heart dramatically altered the metabolic fate of BCKA and BCAA [48]. Another research group, Lee et al., performed skeletal muscle biopsies on males to demonstrate upregulation of MBC transcription in this tissue after a period of exercise [44]. These findings point to the potential role of MBC in BCAA processing in multiple peripheral tissues.

Perspectives

The association of elevated circulating BCAAs with metabolic pathogenicity has long been an established yet poorly understood phenomenon for much of the past half-century. Moreover, the apparently paradoxical finding of BCAA supplementation improving metabolic health in the context of exercise further shrouded any putative mechanism in mystery. Recent advances in diagnostic imaging, metabolomics, and tissue-dependent gene expression have enabled a greater understanding of the process of BCAA catabolism [5-8, 16, 41], yielding breakthrough revelations indicating the crucial role of BAT in systemic energy homeostasis and BCAA regulation, including the discovery of MBC (Fig. 3).

Fig. 3

Relationship of BAT activity to BCAA metabolism, thermogenesis, and energy metabolism

BAT activity is a significant determinant of adaptive thermogenesis in humans, and is thereby involved in the control of energy expenditure and body fat content. BAT selectively utilizes BCAAs as a key substrate for thermogenesis, thereby serving as a metabolic sink for BCAAs. Age-related attenuation of BAT’s function as a metabolic sink for BCAAs is correlated with impaired insulin sensitivity.

It is worth noting that BAT activity is significantly attenuated with aging [108]. The age-related attenuation of BAT activity is coupled with reduced BCAA oxidation and is attributed to reduced protein lipoylation in the E2 subunit of the BCKDH complex, reducing the complex activity to catabolize BCKA [109]. Because dietary supplementation of α-lipoic acid restores the protein lipoylation and thermogenic function of BAT in aged mice [109], enhancing BCAA catabolism by α-lipoic acid supplementation may represent an effective means to alleviate age-associated decrease in BAT activity and development of obesity. Beige adipocytes, the brown-like thermogenic adipocytes induced in WAT deposits in response to chronic cold exposure, also display functional decline with aging through an age-dependent upregulation of ubiquitin E3 ligase complex that mediates ubiquitination and degradation of PRDM16 [110]. Depletion of the ubiquitin E3 ligase complex stabilizes PRDM16 protein, inducing beige adipocyte differentiation and upregulating expression of BCAA-catabolic enzymes [110]. Thus, controlling BCAA mitochondrial transport via MBC affords the therapeutic potential for the treatment of age-associated obesity and insulin resistance developed in the context of chronic elevated circulating levels of the partial metabolites of BCAA and BCAA.

In light of the role of BCAAs in the pathogenesis of cardiovascular disease [111], the implications of MBC activity modification are immense. BCAA dysregulation is implicated in cardiac hypertrophy and lacunar stroke, as well as neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease [48, 58, 112, 113]. It should be emphasized, however, that in most cases the exact mechanism of BCAA dysmetabolism remains uncertain and some findings may be a secondary phenotype to the fundamental cause of disease. The same logic applies for the early identification of malignant neoplasms associated with irregular BCAA metabolism. For instance, defects in BCAA metabolism are consistently associated with certain malignancies including non-small cell lung carcinoma, oral cancer, and pancreatic ductal adenocarcinoma [114-116]. The recent finding that increased BAT activity, induced by cold acclimatization, suppresses tumor proliferation further suggests a possible role of BCAA metabolism in BAT for attenuation of cancer burden, in addition to prognostic utility [117].

Acknowledgments

This research was supported in part by grants to TY from the Japan Society for the Promotion of Science (21K08548, 20K22647), the Japan Science and Technology Agency (JPMJFR2014), and the Naito Foundation (Grant-in-Aid for Raising Next Generation).

Conflict of Interests

The authors declare no conflict of interest.

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