Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity

Abstract

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

Keywords: bariatric; diabetes; hyperinsulinemia; mammalian target of rapamycin; protein.

Fig. 1.

Branched-chain α-ketoacid dehydrogenase (BCKD) (E1α) protein abundance and phosphorylation (p) in retroperitoneal (RP) white adipose tissue (WAT) of female Zucker lean (Fa/Fa, n = 10) and obese (fa/fa, n = 9) rats (A–C), male C57BL/6J mice fed diets varying in fat energy for 12 wk (D–F), and in lean male C57BL/6J and obese db/db mice (G–I). Effects of treatment with rosiglitizone (RSG) are also shown for lean vs. db/db mice. BCKD protein abundance was normalized to β-actin or GAPDH as shown. Phosphorylated BCKD was normalized to total BCKD. All values are expressed as fold relative to the lean group. Ctl, control. Values are means ± SE; means without a common letter differ significantly, P < 0.001. **P < 0.01, ***P < 0.001, and ****P < 0.0001, statistically different from lean by Student's t-test.

Fig. 2.

mRNA expression profile of BCKD complex components during 3T3-L1 preadipocyte-to-adipocyte differentiation with or without acute (18 h) 10 μg/ml GW-1929 peroxisome proliferator-activated receptor (PPAR)-γ agonist treatment. Transcript levels for each gene are expressed relative to the mean expression for that gene on day 14 postdifferentiation and expressed as the means ± SE of n = 3 independent samples/day. *P < 0.05, **P < 0.01, and #P < 0.0001 indicates significantly different from control at the same time point by 2-way ANOVA followed by Bonferonni's post hoc test. All mRNA expression differs significantly by the time factor; treatment factor differs significantly in B–D, and there is a significant time × treatment interaction in B–D by 2-way ANOVA.

Fig. 3.

mRNA expression profile of branched-chain amino acid (BCAA)-associated genes during 3T3-L1 preadipocyte-to-adipocyte differentiation with or without acute (18 h) 10 μg/ml GW-1929 PPARγ agonist treatment. Transcript levels for each gene are expressed relative to the mean expression for that gene on day 14 postdifferentiation and expressed as means ± SE of n = 3 independent samples/day. #P < 0.0001 indicates significantly different from control at the same time point by 2-way ANOVA followed by Bonferonni's post hoc test. All mRNA expression differs significantly by the time factor.

Fig. 4.

mRNA expression profile of BCKD complex components in mature 3T3-L1 adipocytes treated with rosiglitazone in the presence or absence of 10 μM PPARγ antagonist T0070907 for 24 h. Transcript levels for each gene are expressed relative to the mean expression for that gene treated without rosiglitazone or antagonist and expressed as means ± SE of n = 15 (untreated) or n = 8 (all other treatments) combined from 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, significantly different from unstimulated condition.

Fig. 5.

mRNA expression profile of BCKD complex components (A–D) and associated genes (E–H) in mature 3T3-L1 adipocytes treated with the glucose oxidation inhibitor 2-deoxy-d-glucose (2-DG) in the presence or absence of insulin for 48 h. Transcript levels for each gene are expressed relative to the mean expression for that gene treated without insulin or 2-DG and expressed as means ± SE of n = 8 combined from 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicates significantly different from control within insulin treatment status. mRNA expression differs significantly in B–E by insulin treatment. There is a significant 2-DG treatment × insulin treatment interaction in E by 2-way ANOVA.

Fig. 6.

BCKDHA (A), BCKDHB (B), and DLD (C) mRNA abundances in subcutaneous adipocytes isolated from nonobese and obese male and female Pima Indians (n = 10/group). Values are means ± SE. *P < 0.05, **P < 0.01, and ****P < 0.0001 by Student's t-test.

Fig. 7.

BCKD complex components (A and B) and BCAA-associated (C and D) gene transcript abundances are altered in omental adipose tissue (but not subcutaneous WAT) of obese adult female subjects with metabolic syndrome relative to healthy weight-matched obese women. All values are expressed relative to healthy subject omental adipose tissue values. Values are means ± SE. *P < 0.05 and **P < 0.01, metabolic syndrome obese group differs statistically from healthy obese group by Student's t-test. There was a significant effect of WAT depot in BCKDHA (P < 0.05) and BCKDHB (P < 0.01) expression (2-way ANOVA).