Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels

Abstract

Whereas the role of adipose tissue in glucose and lipid homeostasis is widely recognized, its role in systemic protein and amino acid metabolism is less well-appreciated. In vitro and ex vivo experiments suggest that adipose tissue can metabolize substantial amounts of branched chain amino acids (BCAAs). However, the role of adipose tissue in regulating BCAA metabolism in vivo is controversial. Interest in the contribution of adipose tissue to BCAA metabolism has been renewed with recent observations demonstrating down-regulation of BCAA oxidation enzymes in adipose tissue in obese and insulin-resistant humans. Using gene set enrichment analysis, we observe alterations in adipose-tissue BCAA enzyme expression caused by adipose-selective genetic alterations in the GLUT4 glucose-transporter expression. We show that the rate of adipose tissue BCAA oxidation per mg of tissue from normal mice is higher than in skeletal muscle. In mice overexpressing GLUT4 specifically in adipose tissue, we observe coordinate down-regulation of BCAA metabolizing enzymes selectively in adipose tissue. This decreases BCAA oxidation rates in adipose tissue, but not in muscle, in association with increased circulating BCAA levels. To confirm the capacity of adipose tissue to modulate circulating BCAA levels in vivo, we demonstrate that transplantation of normal adipose tissue into mice that are globally defective in peripheral BCAA metabolism reduces circulating BCAA levels by 30% (fasting)-50% (fed state). These results demonstrate for the first time the capacity of adipose tissue to catabolize circulating BCAAs in vivo and that coordinate regulation of adipose-tissue BCAA enzymes may modulate circulating BCAA levels.

FIGURE 1.

Expression of BCAA-oxidizing enzymes in adipose tissue. A, microarray results from adipose tissue from AG4OX versus control and AGKO versus control female mice at 5 weeks of age that were sacrificed in the fed state (n = 3 per group). RE, relative expression (the expression in the experimental group relative to its control group). B, diagram of the BCAA oxidation pathway indicating genes included in the analysis using the numbering in A. αKIC, α-ketoisocaproic acid; αKIV, α-ketoisovaleric acid; αKMV, α-keto-β-methylvaleric acid. C and D, Q-PCR results for selected enzymes of the BCAA oxidation pathway in perigonadal fat (PG fat), gastrocnemius muscle (Gastroc), and liver from fed, 5-week-old female mice. (*, p < 0.05 versus WT).

FIGURE 2.

Valine oxidation and E1α phosphorylation in skeletal muscle and adipose tissue. A, valine oxidation and αKIV accumulation were measured with and without addition of α-chloroisocaproic acid (αIC) in adipose tissue (n = 10 per group), soleus (n = 6 per group), and EDL (n = 6 per group) explants from fed, 7-month-old female AG4OX and wild-type control mice. Comparisons within tissues performed by 2-way ANOVA using Tukey's test for post-hoc analysis between groups; *, p < 0.05 comparing effect of genotype within αIC; #, p < 0.05 comparing effect of αIC within genotype; Comparison across tissues in wild-type animals without αIC performed by one-way ANOVA, ‡, p < 0.05. B and C, quantitation of Western blots for total BKDH E1α, phospho-BCKDH, and the ratio of total to phospho-BCKDH E1α for (B) perigonadal fat with representative blot, and for (C) soleus and (D) EDL muscle from tissues harvested in experiment described in A. (n = 5–6 per group, *, p < 0.05 versus WT by t test).

FIGURE 3.

Changes in glycemia, protein metabolism, and circulating amino acid levels following food removal. A, serial glucose and insulin levels were measured in 4-month-old, female AG4OX and wild-type controls (n = 10 per group). Tail vein bleeds were performed at baseline (8 a.m.) and 2 h and 6 h following food removal (*, p < 0.05 for WT versus AG4OX at each time). B–D, serial body composition measurements were made by DEXA in 7-month-old female AG4OX and wild-type control mice at baseline and after 24 h and 48 h of fasting (n = 9 per group). Comparisons were made for changes in (B) total body weight, lipid weight, lean weight (C) total cumulative weight loss, and (D) % lean mass and % lipid mass (○, WT, ■, AG4OX). Body weight, lipid weight, and lean weight differed between genotypes (*, p < 0.05). Body weight, lipid weight, and lean weight differed within genotypes at 24 and 48 h of fasting compared with time 0 (p < 0.05, paired Student's t test). % lean mass and % lipid mass differed in AG4OX mice after 24 and 48 h of fasting compared with time 0 (p < 0.05, paired Student's t test), but remained unchanged in wild-type mice. E and F, serial plasma amino acid levels were measured under the conditions described in A (*, p < 0.05 for WT versus AG4OX at each time).

FIGURE 4.

Changes in mTOR signaling. A, p70 S6 kinase activity in perigonadal adipose tissue, liver, gastrocnemius muscle, and tibialis anterior muscle of 2-month-old female, WT, and AG4OX mice. Awake mice were injected with saline or insulin (10 units/kg ip) and sacrificed 5 min later (n = 7–9 per group). Tissues were frozen for assays. Statistical comparisons in performed by 2-way ANOVA with Tukey's post-hoc testing; *, p < 0.001 for insulin effect within genotype; †, p < 0.001 compared with WT insulin group; &, p = 0.057 compared with WT-saline group. B, representative blot demonstrating p70 S6 kinase gel mobility shift measured by Western blotting in perigonadal fat and gastrocnemius muscle in animals described in A. Mice were pretreated with rapamycin (10 mg/kg ip) or vehicle 1 h before saline or insulin injection.

FIGURE 5.

BCAT2 fat transplantation. A and B, body weight, body composition, and plasma BCAAs in the fed state and following 6 h of food removal were measured in male BCAT2^−/− mice transplanted with 750 mg of wild-type fat versus sham operated controls (n = 6–8 per group; *, p < 0.05). C, plasma alanine and glutamine were measured in the fed mice described above (n = 4–7 per group; *, p < 0.05). Mice had free access to a choice of normal chow (NC) or BCAA-free diet and food intake was measured for 1 week at ∼10 weeks of age, following the 2 week recovery period. D, food intake is presented as average daily values. E, fed plasma BCAA levels for individual mice versus average daily intake of normal chow.