Branched‐chain amino acids at supraphysiological but not physiological levels reduce myotube insulin sensitivity

Abstract

Aims Branched‐chain amino acids (BCAA) are often emphasized in the diets of avid exercisers, yet population data demonstrates a correlation between circulating BCAA and insulin resistance. However, it is unclear if BCAA independently promote insulin resistance in otherwise healthy cells. The purpose of this study is to examine the effect of a BCAA mixture on muscle insulin signaling in vitro in both insulin resistant and sensitive cells. Materials and Methods C2C12 myotubes were treated with a BCAA mixture containing leucine:isoleucine:valine at a ratio of 2:1:1 at 0.2, 2, or 20 mM (based on leucine content) for either 30 min, 1 day, or 6 days. Western blot was used to assess insulin sensitivity of cells treated with BCAA both with and without concurrent insulin resistance, and, with and without insulin stimulation. Results BCAA treatment for 1 day significantly reduced basal, but not insulin‐stimulated pAkt expression. BCAA treatment for 6 days resulted in significantly reduced basal insulin signaling in healthy cells and insulin‐stimulated insulin signaling in insulin resistant (but not insulin sensitive) cells. Conclusion Similar to previous observations demonstrating BCAA may correlate with insulin resistance during metabolically stressed conditions, we demonstrate excessively high BCAA exposure can negatively influence basal insulin signaling, as well as insulin sensitivity in insulin resistant myotubes. However, given the intentionally high concentrations of BCAA used in this study, the extent to which these observations translate to in vivo models is unclear and warrants further investigation.

5‐Aza‐2′‐deoxycytidine‐mediated DNA hypomethylation with and without concurrent insulin resistance suppresses myotube mitochondrial capacity

“…Senesi et al demonstrated treatment with myotubes with 5‐Aza for 24 h was sufficient to reduce total DNA methylations measured via elisa 29 . Insulin resistance was accomplished by the addition of insulin at 100 nM for the final 3 days of differentiation (and during the treatment period) similar to previous experiments 30–32 . Insulin resistance was confirmed during the present experiments via pAkt expression following insulin stimulation (Figure S1).…”

“…29 Insulin resistance was accomplished by the addition of insulin at 100 nM for the final 3 days of differentiation (and during the treatment period) similar to previous experiments. [30][31][32] Insulin resistance was confirmed during the present experiments via pAkt expression following insulin stimulation (Figure S1). DNA methylation was assessed using dot blot which revealed reduced DNA methylation in 5-Aza-treated cells (Figure S2).…”

“…Cells were treated as described above and collected in RIPA buffer. For insulin stimulation experiments (to validate hyperinsulinemia as a model of insulin resistance), cells were subjected to acute insulin stimulation in serum‐free media at 100 nM for 30 min with and without insulin resistance as previously performed 32 . Whole cell lysates were prepared by harvesting the cells on ice in RIPA buffer supplemented with protease inhibitor mix (0.1%), followed by incubation on ice for 60 min.…”

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

“…BCAA-induced activation of the mTOR/p70S6K pathway has been shown by multiple rodent studies [ 3 , 133 , 146 , 181 , 182 , 185 , 186 ] and cell culture experiments [ 187 – 189 ]. In addition, in vivo and in vitro BCAA deprivation in mice reduced the activation of the mTOR pathway and increased pAkt in liver and muscle, resulting in improved insulin sensitivity [ 190 – 192 ]. Interestingly, Newgard et al reported that dietary BCAA-induced mTOR activation only occurred in the presence of a high fat load [ 3 , 104 ].…”

Branching-out

“…BCAA-induced activation of the mTOR/p70S6K pathway has been shown by multiple rodent studies (3,133,146,181,182,185,186) and cell culture experiments (187)(188)(189). In addition, in vivo and in vitro BCAA deprivation in mice reduced the activation of the mTOR pathway and increased pAkt in liver and muscle, resulting in improved insulin sensitivity (190)(191)(192). Interestingly, Newgard et al reported that dietary BCAA-induced mTOR activation only occurred in the presence of a high fat load (3,104).…”