Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease

Abstract

Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD.

Keywords: branched chain amino acids; insulin resistance; mitochondrial metabolism; nonalcoholic fatty liver disease.

Fig. 1.

Targeted metabolomics coupled to hyperinsulinemic euglycemic clamps in human subjects. A: correlation between indices of insulin sensitivity and metabolites profiled after an overnight fast (basal) and during insulin stimulation (euglycemic hyperinsulinemic clamp). Only correlations that are significant at P < 0.05 are represented with circles in the matrix. Areas and the color intensity of the circles in the correlation plot indicate the absolute value of the corresponding correlation coefficients. B: impaired suppression of BCAA during insulin stimulation in human subjects with increasing severity of muscle insulin resistance [glucose disposal (R_d)]. C–E: relationships between leucine (C), isovalerylcarnitine myristoylcarnitine (D), and palmitoylcarnitine (E) during euglycemic hyperinsulinemic clamp. Values in the bar graph are represented as means ± SE. *P < 0.05 between “R_d ≥ 10” and “R_d (5–10)”; **P < 0.05 between R_d ≥ 10 and “R_d ≤ 5”; #P < 0.05 between R_d (5–10) and R_d ≤ 5. Ala, Alanine; Gly, Glycine; Pro, Proline; Ser, Serine; Asx, aspartate/asparagine; Glx, glutamate/glutamine; Val, valine; Leu, leucine; Ile, isoleucine; Met, methionine; Thr, threonine; Phe, phenylalanine; Tyr, tyrosine; C2, acetylcarnitine; C3, propionylcarnitine; C4, butyrylcarnitine; C5, isovalerylcarnitine; C6, hexanoylcarnitine; C8, octanoylcarnitine; C14, myristoylcarnitine; C16, palmitoylcarnitine.

Fig. 2.

Hepatic metabolism failed to respond optimally to an acute branched-chain amino acid (BCAA) challenge in mice with nonalcoholic fatty liver disease (NAFLD). A: hepatic short-chain acylcarnitines (C3, propionyl; C5, isovaleryl) responded similarly to BCAA challenge in both control (C) and high-trans-fat diet (TFD) groups. B: incorporation of ¹³C from intracellular [¹³C₆]leucine into its degradation product [¹³C₅]isovalerylcarnitine was impaired in TFD-fed mice, suggesting an impairment in BCAA degradation. C: BCAA induced phosphorylation of p70S6K and S6, both indices of mammalian target of rapamycin complex 1 activity in control mice, but this induction was blunted in mice with NAFLD. D: BCAA challenge increased plasma ketones in control mice, but this response was blunted in mice with NAFLD. E: the response of long-chain acylcarnitines (C14, myristoyl; C16, palmitoyl) to BCAA challenge was impaired in mice with NAFLD. Values in the bar graph (n = 6–7) are represented as means ± SE. *P < 0.05 between C + saline and C + BCAA; **P < 0.05 between C + saline and TFD + saline; #P < 0.05 between C and TFD.

Fig. 3.

BCAA failed to regulate tricarboxylic acid (TCA) cycle activity in mice with NAFLD. A–D: endogenous glucose production remained similar in both the C and high-TFD groups following BCAA challenge. Acute 4-h BCAA challenge resulted in the induction of TCA cycle metabolism in control mice (A); however, mice with NAFLD failed to respond to BCAA, as evidenced by impaired mitochondrial TCA cycle fluxes of gluconeogenesis (B), anaplerosis (C), and pyruvate cycling (D) determined relative to citrate synthase. E: absolute TCA cycle flux was suppressed by BCAA in control mice, whereas TCA cycle failed to respond to BCAA infusion in mice with NAFLD. Values in the bar graph (n = 6–7) are represented as means ± SE. *P < 0.05 between C + saline and C + BCAA; **P < 0.1 between C + saline and C + BCAA.