The relationship between protein intake and metabolic health is complex, often referred to as the "protein paradox." While high protein diets show positive anabolic effects, epidemiological studies often link low protein diets to improved health outcomes. BCAAs (leucine, valine, isoleucine) are essential amino acids with conflicting roles in metabolic health, depending on context. Elevated circulating BCAAs are associated with obesity and type 2 diabetes, and impaired BCAA metabolism is linked to diabetes development. Studies using high BCAA mixtures have shown negative effects on metabolic health, but high protein diets, particularly those rich in leucine and isoleucine, have shown beneficial effects on weight loss and metabolic parameters in various studies. This study aimed to clarify the long-term effects of individual BCAA supplementation (specifically leucine and valine) under high-fat diet conditions, investigating their roles in glucose homeostasis and metabolic health.

Existing literature presents conflicting findings regarding the effects of BCAA intake on metabolic health. Increased circulating BCAAs are frequently observed in obese and type 2 diabetic patients, suggesting a potential causal link. However, studies have also shown that leucine and isoleucine supplementation can positively impact metabolic health, improving insulin sensitivity and protecting against weight gain in high-fat diet models. Conversely, studies indicate negative metabolic consequences of high BCAA intake, including increased obesity and reduced lifespan. The role of valine, in particular, remained unclear, necessitating this research to determine the long-term metabolic effects of individual BCAA supplementation, focusing on valine’s contribution under high-fat conditions.

Male C57BL/6JRj mice were fed low-fat (LF), high-fat (HF), or high-fat diets supplemented with casein (HFMP), leucine (HFL), or valine (HFV) for 4 or 20 weeks. Body weight, body composition (using NMR), energy expenditure, food intake, and locomotor activity were monitored. Glucose tolerance tests (OGTTs) and insulin tolerance tests (ITTS) were performed at weeks 6 and 16. Plasma and tissue samples were collected for analysis of BCAAs, 3-HIB, ketone bodies, lipids (TAG and DAG), and glycogen. Gene expression (qRT-PCR) and protein expression (Western blotting) were analyzed in liver and skeletal muscle. In vitro studies using C2C12 myocytes were performed to assess the effects of valine and 3-HIB on glucose uptake and insulin signaling pathways. Statistical analyses included one-way ANOVA with Bonferroni’s post hoc test or Kruskal–Wallis tests, depending on data distribution.

Valine supplementation significantly worsened high-fat diet-induced adiposity, increasing subcutaneous and epididymal white adipose tissue. While leucine supplementation and casein supplementation led to reduced body weight and fat mass gains compared to the HF group, valine supplementation resulted in a significant increase in fat mass. Moreover, long-term valine supplementation caused severe glucose intolerance, with impaired glucose clearance and significantly elevated plasma 3-HIB levels. These elevated 3-HIB levels correlated with increased fat mass and impaired glucose tolerance. Valine feeding appeared to shunt BCAA catabolism toward skeletal muscle, with increased expression of BCAA catabolic genes and increased basal muscle glycogen and lactate. In skeletal muscle, valine feeding led to increased Glut1 expression, increased basal glucose uptake, and upregulation of Trib3, a known inhibitor of AKT signaling, ultimately resulting in decreased AKT phosphorylation and impaired insulin signaling. In vitro studies using C2C12 myotubes confirmed that both valine and 3-HIB increased Glut1 expression, basal glucose uptake, and Trib3 expression, while reducing insulin-stimulated AKT phosphorylation. Inhibition of GLUT1 reversed the effect of valine and 3-HIB on Trib3 expression and AKT phosphorylation.

This study reveals the detrimental impact of valine supplementation in a high-fat diet context, contrasting with the beneficial effects of leucine. The increased circulating 3-HIB, resulting from valine metabolism, plays a crucial role in this detrimental effect. 3-HIB increases basal glucose uptake in skeletal muscle, leading to glucotoxicity and the subsequent downregulation of AKT signaling via TRIB3 induction. These findings highlight the importance of considering individual BCAA effects rather than relying on total BCAA levels, emphasizing the need for a nuanced understanding of the dietary protein quality on metabolic health. The distinct roles of leucine and valine in glucose homeostasis offer a potential explanation for the conflicting results reported in previous studies.

The study demonstrates that long-term valine supplementation exacerbates high-fat diet-induced metabolic dysfunction, particularly impairing glucose tolerance and insulin sensitivity. This detrimental effect is largely mediated through elevated 3-HIB levels, leading to increased basal glucose uptake, glucotoxicity, and ultimately impaired insulin signaling in skeletal muscle. The findings emphasize the importance of dietary protein quality and highlight the differential effects of individual BCAAs on metabolic health. Further research should focus on the specific mechanisms linking 3-HIB to impaired insulin signaling and explore potential therapeutic strategies targeting this pathway to mitigate the detrimental effects of valine excess.

The study was conducted using a mouse model, and results may not be directly translatable to humans. Although efforts were made to blind investigators for some analyses, potential bias remains a possibility. Future research should expand upon the current model, assessing different dietary contexts and focusing on translational aspects to human populations. Further investigation into human metabolic responses to various valine intake levels and the interplay between valine and other BCAAs is required.