A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance

Abstract

Metabolomic profiling of obese versus lean humans reveals a branched-chain amino acid (BCAA)-related metabolite signature that is suggestive of increased catabolism of BCAA and correlated with insulin resistance. To test its impact on metabolic homeostasis, we fed rats on high-fat (HF), HF with supplemented BCAA (HF/BCAA), or standard chow (SC) diets. Despite having reduced food intake and a low rate of weight gain equivalent to the SC group, HF/BCAA rats were as insulin resistant as HF rats. Pair-feeding of HF diet to match the HF/BCAA animals or BCAA addition to SC diet did not cause insulin resistance. Insulin resistance induced by HF/BCAA feeding was accompanied by chronic phosphorylation of mTOR, JNK, and IRS1Ser307 and by accumulation of multiple acylcarnitines in muscle, and it was reversed by the mTOR inhibitor, rapamycin. Our findings show that in the context of a dietary pattern that includes high fat consumption, BCAA contributes to development of obesity-associated insulin resistance.

Figure 1. A banched-chain amino acid-related metabolic “signature” correlates with insulin sensitivity

The figure shows the relationship between insulin sensitivity (HOMA) and a principal component comprised of BCAA-related metabolites including the branched-chain amino acids valine, leucine, and isoleucine, Glx (glutamate + glutamine), the aromatic amino acids phenylalanine and tyrosine, and C3 and C5 acylcarnitines (See Methods and Results for further description). Diamonds, lean subjects (N=67); Squares, obese subjects (N=74).

Figure 2. Effects of BCAA supplementation on body weight, food intake, and insulin signaling

Rats were fed on standard chow (SC), high fat (HF) or HF supplemented with BCAA (HF/BCAA) diets for 12-16 weeks. A) Body weight expressed as % of initial weight; B) Food intake expressed as Kcal/rat/week. Data for panels A and B represent 5-9 animals per group. In panel A, HF was different from both the SC and HF/BCAA groups at all time points, with p < 0.036. In panel B, HF/BCAA was different from both the SC and HF groups at all time points, with p < 0.006. C) Intraperitoneal glucose tolerance test; n = 5-9 rats/group. (*) the SC group was different from the other two groups, with p < 0.036; D) Representative immunoblot of p-AKT levels in skeletal muscle 30 minutes after an acute insulin bolus in overnight fasted rats; E) Quantitative summary of muscle p-AKT studies, expressed as ratio of insulin stimulated: non-insulin stimulated conditions and normalized to total AKT protein; F) Representative immunoblot of p-AKT levels in liver, 30 minutes after an acute insulin bolus in overnight fasted rats; G) Quantitative summary of liver p-AKT studies, expressed as ratio of insulin stimulated: non insulin-stimulated conditions and normalized to total AKT protein levels. For panels E and G, (*) indicates that SC-fed animals had higher levels of p-AKT than the other two groups, with p < 0.033; H) p-mTOR₂₄₄₈, representative immunoblot; I) p-P70S6K1₃₈₉, representative immunoblot; J) p-IRS-1_ser302, representative immunoblot; K) p-IRS1_ser307, representative immunoblot; L) Quantitative summary of phosphoproteins normalized to total levels of mTOR, p70S6K1, or IRS-1, respectively. Data in H-L are for skeletal muscle samples from 3-5 animals/group fed on the three diets, fasted for 48 h and then refed on the same diets for 4 h. (*) in panel L indicates higher levels of phosphoproteins in the HF/BCAA group compared to the other two groups, with p < 0.05; M) Rapamycin treatment reverses insulin resistance in HF/BCAA-fed but not HF-fed rats. Data shown are for 5-7 animals per group. (*) p < 0.05, blood glucose significantly different between the HF-fed, rapamycin treated or HF/BCAA-fed, non treated groups versus the SC, rapamycin treated and HF/BCAA, rapamycin treated groups.

Figure 3. Indirect calorimetry analysis

A) RER (RQ); C) VO₂ measured continuously over a 34 h period that includes a typical dark/light cycle. Panels B, D represent the average RER (RQ) and VO₂, respectively. (*) p<0.05, SC group compared to the other two groups.

Figure 4. BCAA supplementation of HF diet causes accumulation of acylcarnitines in skeletal muscle and chronic activation of mTOR and JNK

A) Skeletal muscle samples were collected from animals fed on the indicated diets and used for acylcarnitine analysis by MS/MS. Data represent the mean ± SEM for 6 animals per group. (*) p < 0.05 for comparison of SC and SC/BCAA to HF and HF/BCAA groups. B) Representative p-mTOR₂₄₄₈, immunoblot in muscle from overnight fasted SC, SC/BCAA, HF, HF/AA, and HF/BCAA-fed rats; C) Quantitative summary of p-mTOR₂₄₄₈ analyses; D) Representative p-cJUN_ser63 immunoblot in muscle from the same sets of animals studied in panel B; E) Quantitative summary of p-cJUN analyses. (*) p < 0.05 for comparison of HF/BCAA to the other groups. (#) p<0.05 for comparison of SC and SC/BCAA to the other groups. In panels C and E, n = 6-9 animals/group.

Figure 5. Schematic Summary of BCAA overload hypothesis

In the physiological context of overnutrition and low IGF-1 levels, as found in our obese subjects, circulating branched-chain amino acids (BCAA) rise, leading to increased flux of these amino acids through their catabolic pathways. We detected changes in several of the intermediary metabolites of the BCAA catabolic pathway in obese subjects, as indicated by the symbol *. A consequence of increased BCAA levels is the activation of the mTOR/S6K1 kinase pathway and phosphorylation of IRS-1 on multiple serines, contributing to insulin resistance. In addition, increased BCAA catabolic flux may contribute to increased gluconeogenesis and glucose intolerance via glutamate transamination to alanine.