Introduction
Type 2 diabetes is a metabolic disease characterised by chronic hyperglycaemia and insulin resistance [
1]. These metabolic derangements severely affect pathways controlling the appropriate sensing and handling of nutrients, thereby leading to a positive energy balance and metabolic inflexibility, which further compromises whole-body glucose homeostasis [
2]. Overnutrition and type 2 diabetes-related disturbances also affect non-glucose metabolites such as branched-chain amino acids (BCAAs; leucine, isoleucine and valine) [
3]; the utilisation and metabolism of these essential amino acids are exquisitely regulated in healthy individuals. While BCAAs and related metabolites play a role in protein synthesis, they also modulate liver gluconeogenesis and lipogenesis rates, cell growth and nutrient signalling, and can enter the tricarboxylic acid cycle to produce energy [
4]. Under pathological conditions, BCAAs, especially in a context of overnutrition, disrupt insulin sensitivity and secretion [
5].
Circulating levels of BCAAs are elevated in individuals with obesity, insulin resistance and/or type 2 diabetes [
6]. Metabolomic profiling of blood metabolites has revealed a signature of altered BCAA catabolism in obese individuals, strongly associated with insulin resistance [
7]. BCAA-related metabolites are predictive of type 2 diabetes pathogenesis [
8] as well as being prognostic markers for intervention outcomes [
9,
10]. Mendelian randomisation analysis suggested a causal link between genetic variants associated with impaired BCAA catabolism and higher risk of type 2 diabetes [
11]. Therefore, there is growing evidence that high levels of BCAAs and related intermediate metabolites are not only type 2 diabetes biomarkers but also pathophysiological factors. However, many of these studies were conducted in individuals after an overnight fast, under conditions in which protein degradation in skeletal muscle and a concomitant release of amino acids to support gluconeogenesis [
12] could mask deeper alterations in BCAA homeostasis. Moreover, these studies do not provide insight into the dynamic shift in metabolism that occurs in response to nutritional challenges. An analysis of metabolomic signatures in fasted individuals with glucose tolerance or type 2 diabetes before and after a glucose challenge may provide insight into dynamic changes in BCAAs and related metabolites and the consequences of insulin resistance.
Skeletal muscle is the largest contributor to systemic BCAA oxidation [
13] and therefore impairments in glucose and BCAA metabolism in myocytes have an impact on whole-body metabolic homeostasis. While BCAA-related gene expression and oxidation rates are reduced in vastus lateralis muscle from individuals with insulin resistance [
14], studies of BCAA metabolism in human skeletal muscle are scarce. BCAA catabolism occurs in the mitochondrial matrix, implying that alterations in mitochondrial proteins may influence BCAA metabolism. In transgenic mice overexpressing the mitochondrial biogenesis inducer peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α), BCAA levels are reduced in gastrocnemius muscle [
15]. Conversely, administration of the PPARγ agonist thiazolidinedione rosiglitazone improves glycaemic control and increases circulating levels of BCAAs in individuals with type 2 diabetes [
16]. Nevertheless, mechanisms underlying the direct role of PGC-1α in the regulation of BCAA catabolism are unclear.
The aim of this study was to assess whether an OGTT can further reveal an impairment in BCAA metabolism in individuals with type 2 diabetes. We also aimed to identify a PGC-1α transcriptional partner in the regulation of the BCAA gene program by using cell and mouse models in which PGC-1α expression was altered.
Discussion
A link between increased plasma BCAAs and insulin resistance was established as early as 1969 [
3] but this association has remained relatively unexplored until the last decade. Here, we elucidate the mechanisms underpinning BCAA metabolism in type 2 diabetes. While we corroborate the association between BCAA metabolites and type 2 diabetes [
9,
26‐
28] and the role of PGC-1α in BCAA catabolism [
15,
24,
29], we provide new evidence that an OGTT unmasks impairments in BCAA catabolism in individuals with type 2 diabetes. Moreover, we reveal that PGC-1α-mediated regulation of genes important for BCAA catabolism is dependent on ERRα, a canonical PGC-1α-interacting protein.
Circulating plasma BCAA levels are altered in individuals with type 2 diabetes; however, metabolomic profiles of peripheral tissues involved in glucose homeostasis are scarce and the transcriptomic regulation of genes involved in BCAA catabolism is unknown. Studies focusing on skeletal muscle BCAA content are limited to an analysis of adults with insulin resistance [
14], rather than type 2 diabetes. We performed untargeted metabolomic analysis on both plasma and vastus lateralis biopsies obtained before and after an OGTT from men with either NGT or type 2 diabetes. Corroborating an earlier study of plasma samples from the Framingham Heart Study Offspring cohort [
30], circulating BCAAs were decreased after an OGTT and this reduction was attenuated in type 2 diabetes. Concomitantly, we found that this blunted response also affected leucine- and isoleucine-derived BCKAs. In skeletal muscle, insulin inhibits proteolysis [
31] and increases the preference for BCAA oxidation [
13], which may explain the blunted excursion of BCAA and BCKA metabolites in insulin-resistant individuals with type 2 diabetes as compared with NGT. Consistent with this hypothesis, BCAAs, BCKAs and 3-HIB levels after the glucose challenge were significantly less decreased in the skeletal muscle from individuals with type 2 diabetes. Although we did not detect changes in BCKDHA phosphorylation state, BCAA catabolic flux cannot be predicted by BCKDH phosphorylation status [
13], thus, we cannot exclude the possibility that BCKDH activity per se is impaired. Moreover, insulin modulates BCAA transamination in type 2 diabetes [
32] and BCAT2 activity [
33], suggesting that insulin resistance could lead to an accumulation of BCKAs due to defects in both BCAA transamination and oxidation.
Generally, plasma metabolites mirrored the skeletal muscle BCAA profile. However, the intramuscular accumulation of isovalerylcarnitine, isobutyrylcarnitine and 3-HIB was not reflected in plasma, suggesting defects in processes controlling the export of metabolites from myocytes in type 2 diabetes. Accordingly, the accumulation of 3-HIB after an OGTT is notable since this valine-derived metabolite promotes insulin resistance through increased fatty acid uptake in skeletal muscle [
24]. This could lead to a vicious cycle in which secretion of 3-HIB from skeletal muscle may decrease insulin sensitivity and further impair insulin signalling. Although elevated levels of circulating C3 and C5 acylcarnitines have been also detected in individuals with obesity [
7], whether accumulation of short-chain acylcarnitines mediates in insulin resistance remains to be elucidated [
34]. These results suggest that the degradation of BCAA-derived metabolites is incomplete. Additionally, we found a strong correlation of post-OGTT BCAAs and derived BCKA circulating levels with blood glucose and HbA
1c. Since plasma fasting BCAA levels exhibited a much weaker relationship with clinical biomarkers of type 2 diabetes, this finding underscores a tight connection between impaired glucose homeostasis and whole-body BCAA catabolism.
The accumulation of BCAA metabolites in individuals with type 2 diabetes was accompanied by reduced expression of genes involved in BCAA catabolism, suggesting that alterations in the transcriptional regulation of these genes could attenuate BCAA catabolism in skeletal muscle. Contrasting with previous findings [
35], we did not find a correlation between BCAA genes and related metabolites, suggesting that post-transcriptional modifications play a key role in the impairment of BCAA catabolism. Conversely, the BCAA gene set was inversely correlated with blood glucose levels, suggesting a connection between glucose homeostasis and alterations in the transcriptional regulation of genes involved in BCAA catabolism.
PGC-1α mediates the expression of genes involved in BCAA catabolism [
15,
24,
29]. Concordantly, expression of
PPARGC1A was positively correlated with BCAA genes. PGC-1α also coordinates metabolic and transcriptomic programs linked to cellular energy homeostasis [
36‐
38], and reduced PGC-1α mRNA and protein levels are linked to insulin resistance in type 2 diabetes [
39,
40]. In the present study, primary HSMCs and mouse skeletal muscle with reduced
PPARGC1A expression exhibited a mild reduction in expression of several BCAA genes, whereas
PPARGC1A overexpression was associated with a consistent upregulation. Thus, while PGC-1α was not essential for basal BCAA gene transcription, a role in the adaptive response to increased BCAA catabolic demands, such as during exercise or nutritional changes, cannot be excluded [
41]. We next hypothesised that alterations in the BCAA gene network would impact BCAA metabolism. We found that mice overexpressing
Ppargc1a in skeletal muscle exhibited an upregulation of BCAA genes and reduced intramuscular accumulation of valine, suggestive of increased BCAA catabolic flux. Similarly, adenovirus-mediated overexpression of
Ppargc1a in C2C12 myotubes increased leucine oxidation rates. However, intramuscular levels of BCAA metabolites were unaltered in mKO mice. Thus, other organs such as adipose tissue may compensate for a putative impairment in BCAA catabolism [
42]. Furthermore, a metabolic challenge may be necessary to reveal functional alterations in BCAA metabolism in mKO and mTG mice.
PGC-1α is a transcriptional coactivator and does not directly bind DNA in a sequence-specific manner. The nuclear orphan receptor ERRα is one of the main transcriptional partners of PGC-1α [
25], and through this physical interaction, elicits the transcription of genes involved in mitochondrial biogenesis and oxidative phosphorylation [
43], lipid metabolism [
44] and ketone body homeostasis [
45]. We found that
ESRRA silencing, as well as inhibition of ERRα with an inverse agonist, downregulated BCAA genes and abrogated the PGC-1α-induced responses, indicating that ERRα is necessary for the PGC-1α-mediated transcription of BCAA genes. Moreover,
ESRRA silencing was associated with a modest decrease in BCKDH-stimulated leucine oxidation, suggesting a functional impact of the transcriptional downregulation of BCAA genes under energy-demanding conditions. Thus, we next hypothesised that alterations in ERRα may explain the defects in BCAA catabolism in the individuals with type 2 diabetes. However, expression of
ESRRA was unaltered in skeletal muscle of individuals with NGT or type 2 diabetes, suggesting that reduction in PGC-1α is sufficient to impair BCAA expression or, alternatively, the PGC-1α–ERRα interaction is compromised in type 2 diabetes.
Some limitations of our study warrant discussion. Our analysis was confined to male participants and therefore we cannot exclude sex-specific differences in the analysed outcomes. Most of the individuals with type 2 diabetes included in this study were treated with metformin. Considering that mitochondria are the major target of metformin, this may affect BCAA metabolism. Due to limitations in the LC/MS methodology, quantification of BCAA metabolites with CoA moieties was not possible. To overcome this, we used specific derived carnitines from these metabolites as a proxy. Alterations in PGC-1α expression may influence fibre type distribution [
19,
46], which could impact the results. Nevertheless, similar results were obtained in transgenic mice, in vitro primary HSMCs and C2C12 myotubes, suggesting that alterations in BCAA metabolism are unrelated to fibre type.
In conclusion, altered expression of PPARGC1A is associated with disturbances in BCAA metabolism in skeletal muscle of individuals with type 2 diabetes. Experimental approaches to reduce PPARGC1A levels partially recapitulates the BCAA gene set profile identified in skeletal muscle from individuals with type 2 diabetes, without impacting circulating and intramuscular BCAA levels. Our results indicate that glucose loading exacerbates disturbances in the BCAA profile, revealing that the metabolic inflexibility that characterises type 2 diabetes encompasses BCAA catabolism. Additionally, our data demonstrate that ERRα is essential for PGC-1α-mediated transcriptional regulation of genes involved in BCAA metabolism in primary human myotubes, thereby unravelling a new role for this orphan nuclear receptor.
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