9237 Loss of Glucose in Urine Reprograms Mitochondrial Function and Increases Mannose Production in the Kidneys to Maintain Adequate Supply of Energy in Renal GLUT2 Knockout Mice

Abstract Disclosure: N. Rashid: None. K.H. Chhabra: None. Glucose transporters such as GLUT2 and SGLT1/2 are responsible for reabsorption of glucose in kidneys. Inhibition of SGLT2 or lack of renal Glut2 induces massive loss of glucose (energy) in urine in humans and rodents. Despite this massive glucose loss, both the humans and rodents are able to maintain normal supply of energy even when they are fasted. Molecular basis of the compensatory mechanisms responsible for maintaining the normal blood glucose levels despite high glycosuria remains unknown. Therefore, here we aimed to determine how renal Glut2 KO mice maintain normal energy and glucose homeostasis at baseline despite massive loss of glucose in urine. Given the role of mitochondria in energy homeostasis and cellular metabolism, we hypothesized that quantifying mitochondrial proteome and metabolome in the kidneys would identify novel adaptive pathways to explain this phenotype. To test the hypothesis, we used 8-10 weeks old male renal Glut2 KO and their littermate control mice produced on the C57BL/6 genetic background. We isolated mitochondria from kidneys and adopted 2D fluorescence difference gel electrophoresis followed by MALDI TOF/TOF mass spectrometry to quantify mitochondrial proteome. In addition, we used untargeted metabolomics to determine changes in metabolite levels in kidneys. We also performed western blot to validate the results obtained from the proteomic analyses. Mitochondrial proteomics revealed significant alterations (1.5 up/down fold change; p ≤ 0.05) in proteins involved in mitochondrial respiratory chain and energy homeostasis. Using western blot, we validated the downregulation of pyruvate carboxylase involved in gluconeogenesis (CON vs KO: 100 ± 9.9 vs 33.1 ± 5.6; % of control, p<0.05) and glycine amidinotransferase, involved in amine, polyamine and creatine biosynthesis (CON vs KO: 100 ± 30.4 vs 3.9 ± 0.5; p<0.05). These results indicate the adaptive responses of renal Glut2 KO mice to maintain their energy homeostasis. Remarkably, kidney metabolomics demonstrated that mannose levels were 18 fold higher in the KO mice as compared to control group. Mannose is known to enter glycolysis pathway to provide adequate supply of energy when glucose is unavailable. We further confirmed the involvement of mannose metabolism as an alternative source of energy by observing increased levels of phosphomannose isomerase (CON vs KO: 100 ± 9.9 vs 248 ± 39.7; p<0.05) which is an essential enzyme to interconvert fructose-6-phosphate to mannose-6-phosphate and thereby produce mannose. Our data shows that loss of glucose in urine causes reprogramming of mitochondrial function to increase the efficiency of ATP synthesis and enhances mannose production in renal Glut2 KO mice. The findings identify novel compensatory pathways that make up for the glucose loss in urine and can thus be targeted to improve the efficiency of SGLT2 inhibition in treatment of diabetes mellitus. Presentation: 6/2/2024