Abstract Disclosure: T.S. Faniyan: None. J. Robles: None. X. Zhang: None. S. Bathina: None. P.S. Brookes: None. R.J. Perry: None. K.H. Chhabra: None. We recently reported the contribution of renal GLUT2 (glucose transporter 2) to systemic glucose homeostasis using a mouse model in which we can knockout renal Glut2 gene at a desired time. Renal Glut2 knockout mice have improved glucose tolerance and are protected from high-fat diet or streptozocin-induced diabetes because they excrete excess blood glucose in urine. Interestingly, despite the profound loss of glucose in urine, renal Glut2 knockout mice have normal fasting blood glucose levels. These findings indicate that an increase in endogenous glucose production may be compensating for the loss of glucose in urine. To determine the mechanisms responsible for evoking the increase in endogenous glucose production, we focused on studying the hypothalamic-pituitary-adrenal (HPA) axis because of its established role in elevating blood glucose levels. We used ELISA to measure circulating adrenocorticotrophic hormone (ACTH) and corticosterone, which are major hormones of the HPA axis. Moreover, we used fluorescence in situ hybridization to assess the expression of hypothalamic corticotropin releasing hormone gene (Crh). We also performed in vitro metabolomics analysis of the mouse liver using mass spectrometry. Finally, we measured glucose production from pyruvate in vivo in the liver and kidney using mass isotopomer distribution analysis. Renal Glut2 knockout mice had increased (p<0.05) levels of hypothalamic Crh (100 ±13 vs 142 ±9 %, control vs experimental), circulating ACTH (120 ±8 vs 213 ±12 pg/ml), and corticosterone (30.4 ±5.5 vs 115 ±15.2 ng/ml). In addition, we found that metabolites such as hepatic glucose-6-phosphate (19.2 ±1.5 vs 15 ±1 arbitrary unit, control vs experimental), glucose-1-phosphate (20 ±1.5 vs 15.5 ±1), and fructose-6-phosphate (16 ±1.2 vs 12.4 ±0.8) were decreased in renal Glut2 knockout mice. We further observed that glucose production was increased in both the liver (15.1 ±0.4 vs 18.4 ±0.5 mg/kg/min) and kidney (1.7 ±0.2 vs 2.6 ±0.3 mg/kg/min) in renal Glut2 knockout mice. Our results demonstrate that loss of renal Glut2 causes hyperactivation of the HPA axis coupled with decrease in hepatic glucose metabolism and increase in hepatic as well as renal glucose production. These findings may explain how the renal Glut2 knockout mice prevent hypoglycemia despite massive glycosuria. Altogether, our study identifies a novel crosstalk between the kidneys, hypothalamus, and adrenal glands to regulate systemic glucose homeostasis. This information may be useful in either optimizing the efficacy of drugs that enhance glycosuria to treat diabetes or addressing the side effects of such drugs on the HPA axis. Presentation: Thursday, June 15, 2023