BCAAs acutely drive glucose dysregulation and insulin resistance: role of AgRP neurons

The increasing prevalence of obesity and type 2 diabetes (T2D) has led to increased interest in dietary interventions. High-protein diets, often rich in branched-chain amino acids (BCAAs – leucine, isoleucine, and valine), are popular due to their roles in protein synthesis, hormone regulation, fat metabolism, immune function, and physical fitness. BCAA supplementation is used clinically to prevent muscle wasting. However, paradoxically, elevated circulating BCAAs are consistently observed in obesity, insulin resistance, and T2D, serving as an early predictor of future diabetes risk. BCAA supplementation disrupts glucose metabolism and induces insulin resistance, while BCAA restriction improves these parameters. Existing studies primarily examine long-term effects, making it difficult to determine if these are direct effects of BCAAs or secondary to other physiological changes. This study aimed to determine the acute effects of BCAAs on glucose regulation to establish a direct causal relationship. Previous research has shown that a single gavage of isoleucine improves glucose tolerance, but investigating the effects of all three BCAAs together is more physiologically relevant, as most BCAA-rich sources contain them in a combined ratio. This study examined the acute effects of BCAA exposure and breakdown on glycemic control and insulin sensitivity in male and female mice using in vivo techniques, including catheter-guided frequent sampling, glucose tolerance tests, insulin tolerance tests, and hyperinsulinemic-euglycemic clamps. The role of agouti-related protein (AgRP) neurons, which disrupt glucose homeostasis and elevate plasma BCAAs, was also investigated through chemogenetic manipulation.

A substantial body of literature links elevated circulating BCAAs to obesity, insulin resistance, and type 2 diabetes. Studies have shown that BCAA supplementation leads to glucose dysregulation and insulin resistance, while interventions to reduce plasma BCAA levels, including dietary restriction, pharmacological treatments, and surgical interventions, improve insulin sensitivity and glycemic control. However, the long-term nature of these studies obscures whether the effects are direct or secondary to other physiological changes. Some studies have examined the acute effects of individual BCAAs, but a comprehensive analysis of the combined acute effects of all three BCAAs is lacking. The role of the hypothalamus, particularly AgRP neurons, in glucose homeostasis and BCAA regulation has also been a recent area of investigation.

Three-month-old male and female C57BL/6J mice were used. A cohort of mice were also placed on a high-fat diet for 8 weeks. For chemogenetic studies, AgRP-IRES-Cre and C57BL/6J WT mice were used. Jugular catheters were implanted for frequent blood sampling. BCAAs were administered intravenously or intraperitoneally at a physiological dose equivalent to a single mouse meal. 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (BT2), a BCAA-lowering compound, was used to enhance BCAA catabolism. Hyperinsulinemic-euglycemic clamps were performed to assess whole-body insulin sensitivity. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted. For chemogenetic experiments, AgRP neurons were stimulated using clozapine-N-oxide (CNO) after stereotaxic injection of AAV containing a stimulatory DREADD. Plasma BCAAs, insulin, glucagon, C-peptide, and corticosterone were measured. Western blotting was used to assess protein expression of pAKT and BCKDH in various tissues. Immunohistochemistry confirmed DREADD expression. Statistical analysis included Welch's t-test, ANOVA, and repeated measures ANOVA.

Intravenous BCAA infusion acutely elevated blood glucose and plasma insulin in male mice, but not in females. Pre-treatment with BCAAs did not affect glucose tolerance, but impaired the counterregulatory response to hypoglycemia in males. Hyperinsulinemic-euglycemic clamps showed that BCAAs acutely impaired whole-body insulin sensitivity in males, evidenced by lower glucose infusion rates needed to maintain euglycemia. A single injection of BT2 effectively prevented the fasting-induced rise in plasma BCAAs and significantly improved glucose tolerance in high-fat-fed male mice, but not in females. Chemogenetic overactivation of AgRP neurons impaired glucose tolerance, an effect completely reversed by acute BCAA reduction with BT2. This effect was also only observed in males. Individual BCAA injections showed that valine acutely elevated blood glucose in males.

The study's findings demonstrate that BCAAs can acutely impair glucose homeostasis and insulin sensitivity, providing a mechanistic explanation for their long-term detrimental effects observed in obesity and diabetes. The sex-specific effects suggest hormonal influences on BCAA metabolism. The significant improvement in glucose tolerance in high-fat-fed male mice following BT2 treatment underscores the potential of BCAA-lowering strategies for metabolic disorders. The role of AgRP neurons in mediating BCAA's effects on glucose regulation provides a novel mechanism for brain control of glucose homeostasis. While the study focuses on acute effects, the findings have implications for understanding the chronic metabolic consequences of high BCAA intake. The mechanism of BCAA-induced insulin resistance, whether through impaired hepatic glucose production or peripheral glucose disposal, requires further investigation. The study also suggests a role for BCAAs in disrupting the counter-regulatory response to hypoglycemia.

This study provides strong evidence that BCAAs acutely impair glucose homeostasis and insulin sensitivity, primarily in male mice, through mechanisms potentially involving hepatic insulin resistance and impaired counter-regulatory response to hypoglycemia. The complete normalization of AgRP neuron-induced glucose intolerance by BCAA reduction highlights a novel mechanism linking the brain to peripheral glucose metabolism. Future research should investigate the underlying mechanisms and explore the therapeutic potential of BCAA-lowering interventions for metabolic disorders, considering sex differences.

The study primarily used mouse models, limiting the direct translatability to humans. While the BCAA dose was chosen to mimic a physiological single meal, the continuous infusion during the clamp studies may not entirely reflect natural dietary intake. The investigation of AgRP neurons focused on male mice and further studies incorporating female mice are needed. The mechanisms underlying the sex differences in BCAA metabolism and their impact on metabolic outcomes require further exploration.