Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

Abstract

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability.

MiR‐330‐5p negatively regulates ovine preadipocyte differentiation by targeting branched‐chain aminotransferase 2

“…Our results showed that BCAT2 could promote the differentiation of ovine preadipocytes, which was supported by previous research on mice. In the present study, BCAT2 exhibits a low expression level at the early stage and a high level at the late stage of differentiation, the BCAT2 expression is in accordance with the result of experiments in 3T3‐L1 preadipocytes (Estrada‐Alcalde et al., ; Kitsy et al., ). Lower BCAT2 expression at an early differentiation stage can be explained as possibly preserving BCAAs for later protein synthesis.…”

“…Some research displayed that BCAT2 can promote the differentiation of 3T3‐L1 preadipocytes. BCAT2 is the upstream gene of BCKDHA and BCKDHB , and may regulate the expression of them during preadipocyte differentiation (Kitsy et al., ). The sequences of the three genes are known and have been cloned (Lu, Xie, Jia, & Jia, ).…”

Administration of valine, leucine, and isoleucine improved plasma cholesterol and mitigated the preatherosclerotic lesions in rats fed with hypercholesterolemic diet

“…Xiao et al found that a diet deficient in amino acids used for a period of 7 days quickly reduces abdominal fat mass in mice [17]. Recent scientific evidence from studies conducted on animals and humans have reported a link between levels of branchedchain essential amino acids and obesity [18]. Moreover, recent research suggests that leucine is involved in systemic cholesterol metabolism.…”

Mitochondrial function/dysfunction in white adipose tissue

“…The catabolism of BCAAs proceeds through transamination to the respective 2-oxo acids, followed by an intramitochondrial oxidative decarboxylation catalysed by the branched-chain 2-oxo acid dehydrogenase. It is not surprising, therefore, that during adipogenesis, when mitochondrial mass is elevated, leucine catabolism and the expression of enzymes involved in BCAA catabolism increased dramatically (Frerman et al 1983;Kitsy et al 2014). Furthermore, mice lacking the mitochondrial branched-chain aminotransferase enzyme at the whole-body level exhibited reduced adiposity and enhanced energy expenditure despite higher food intake and increased circulating levels of BCAAs (She et al 2007).…”

“…; Kitsy et al . ). Furthermore, mice lacking the mitochondrial branched‐chain aminotransferase enzyme at the whole‐body level exhibited reduced adiposity and enhanced energy expenditure despite higher food intake and increased circulating levels of BCAAs (She et al .…”