Renal tubule mTORC2 deletion increases gluconeogenesis and urinary glucose excretion
Authors: Demko John , Saha Bidisha , Takagi Enzo , Manis Anna , Richman Phillip , Pearce David 
Published: May 2023

Methods: Rictor is a critical component of the mTORC2 complex. Tubule-specific Rictor knockout (TRKO) mice were made using doxycycline inducible Pax8-Cre Rictor-flox. Male and female TRKO mice and their control littermates were used for all experiments. Tolerance tests were performed with intraperitoneal glucose (1g/kg), insulin (0.75U/kg), and pyruvate (2g/kg) after overnight fasts. Refeeding at the time of sacrifice to induced insulin signaling; mice were fasted for 18 hours then refed 4 hours. Whole kidney relative mRNA was measured via RT-PCR. Kidney plasma membrane and cytosolic proteins were separated using the BioVision Plasma Membrane Protein Extraction Kit, and protein abundance was measured with western blotting. Results: There were no differences in serum glucose during refeeding experiments, glucose tolerance tests, or insulin tolerance tests between TRKO and control mice at any timepoints (n=9 per group). However, the mean ± standard error of mean (SEM) urine glucose concentration was 472.5±181.2mg/dL in TRKO mice compared to 30.8±5.0mg/dL in control animals during refeeding (n=9 per group; p<0.01). Serum glucose was higher in TRKO mice compared to controls (n=7 per group) after giving the gluconeogenic substrate pyruvate at 60 (234.4±15.0 vs 189.4±9.1mg/dL; p<0.01) and 90 minutes (194.3±7.4 vs 148.9±5.9mg/dL; p<0.01). TRKO mice (n=8) also had elevated hemoglobin A1c (HbA1c) compared to control mice (n=6 per group) after 3 months on a 0.5% K+ diet (6.00±0.21% vs 5.23±0.11%; p<0.01). Refed TRKO mice kidneys compared to controls had significantly higher relative mRNA of PEPCK (3.74±0.76 vs 1.18±0.15AU; p<0.01) and G6Pase (4.24±1.02 vs 1.37±0.29AU; p<0.01) (n=15 per group). Refed TRKO mice kidneys compared to controls also had elevated protein abundance of PEPCK (0.54±0.05 vs 0.28±0.03AU; p<0.001) but no difference in G6Pase (0.62±0.03 vs 0.49±0.05AU; ns). Kidneys from refed TRKO and control mice showed no differences in relative mRNA of SGLT2, SGLT1, or GLUT2 (n=15 per group). Kidneys from TRKO and control mice also showed no difference in plasma membrane protein abundance of SGLT2 and GLUT2 (n=8 per group). Conclusion: This study demonstrates that insulin signaling through mTORC2 is critical for suppression of renal GNG and complete reabsorption of glucose. Increased serum glucose during pyruvate tolerance testing, increased HbA1c, increased gluconeogenic gene transcription (i.e., PEPCK, G6Pase), and increased PEPCK protein abundance all support increased renal GNG in TRKO mice. Glycosuria was present in TRKO mice despite no difference in serum glucose between TRKO and control mice, suggesting that mTORC2 is important for both renal GNG and glucose reabsorption. Future studies will use TRKO mice to further evaluate glucose transporters and elucidate the mechanism of glycosuria. NIDDK Support, T32DK007219; Diacomp, 5U24DK115255-040 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.