Introduction and aims:

The central nervous system contributes to glucose homeostasis by integrating hormonal signals such as insulin to regulate hepatic glucose production (HGP). The nucleus of the solitary tract (NTS) within the dorsal vagal complex is an important site of central insulin action, yet the specific cellular mechanisms mediating this effect remain unclear. Astrocytes are increasingly recognized as active metabolic sensors in the brain. This study investigated whether insulin acts on NTS astrocytes to regulate glucose metabolism and explored the downstream signalling pathways involved.

 

Methods:

Male Sprague–Dawley rats were used to examine insulin signalling in the NTS. Cell-type–specific insulin receptor expression and activation were assessed using RNAscope, FITC-labelled insulin, and c-Fos immunohistochemistry. Astrocytic insulin signalling was selectively disrupted using a GFAP-Cre–dependent insulin receptor knockdown. Hepatic glucose production and glucose uptake were measured using pancreatic euglycemic and hyperinsulinemic clamp techniques. Endozepine release from primary astrocytes was quantified by ELISA, and GABAa receptor signalling was modulated pharmacologically via targeted NTS infusions.

Statistical analysis: Data are presented as mean ± SEM. Sample sizes ranged from n = 3–4 animals for histological and RNAscope analyses to n = 5–12 animals per group for in vivo clamp and pharmacological studies. In vitro experiments using primary astrocyte cultures were performed with n = 3–6 independent preparations. Statistical significance was assessed using unpaired Student’s t-tests or one- and two-way ANOVA with appropriate post hoc comparisons. A p value < 0.05 was considered statistically significant.

Ethical approval: All animal experiments were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986, approved by the University of Leeds Ethical Review Committee, and are reported in compliance with ARRIVE guidelines.

Results:

Insulin receptors were predominantly expressed in NTS astrocytes, with fewer receptors detected in neuronal populations. Insulin administration in the NTS induced widespread neuronal activation, while direct insulin signalling occurred mainly in astrocytes. Astrocyte-specific insulin receptor knockdown abolished insulin-dependent suppression of hepatic glucose production without affecting glucose uptake. Insulin stimulated the release of endozepines from astrocytes, and direct NTS infusion of endozepines mimicked insulin’s metabolic effects. Blockade of the benzodiazepine binding site of GABAa receptors prevented insulin- and endozepine-mediated suppression of HGP, while GABAa receptor antagonists reproduced the effect of insulin. Importantly, endozepine or GABA antagonist administration restored NTS control of glucose production in high-fat-diet–induced insulin-resistant rats.

 

Conclusion:

These findings identify NTS astrocytes as key mediators of central insulin action on glucose metabolism. Insulin signalling in astrocytes promotes endozepine release, which reduces GABAergic inhibition to suppress hepatic glucose production. This astrocyte-dependent pathway provides new insight into brain–liver communication and suggests potential therapeutic targets for insulin resistance and type 2 diabetes.