A Novel Function of FOXO4 and mTORC2 for Regulation of Kidney Gluconeogenesis

Background: The proximal tubule of the kidney and the liver are the only organs capable of gluconeogenesis (GNG). Insulin signaling through mTORC2 and Akt2 regulates GNG in the liver, and our recent work confirmed that mTORC2 coordinates regulation of GNG and glucose transport in the kidney. FOXO1 is a well-established transcription factor for GNG in the liver. However, the major gluconeogenic transcription factor in the kidney has not been identified. We initially assumed it was FOXO1, but western blot anomalies suggested a FOXO family member with different molecular weight. Methods: Rictor deletion causes selective disruption of the mTORC2 complex. Male and female mice with inducible tubule-specific Rictor knockout (TRKO) were made with the Pax8-rtTA TetOCre Rictorflox/flox system. Mice were fasted then refed to induce insulin signaling prior to sacrifice and tissue collection. Whole kidney phosphoprotein abundance was normalized to respective total protein abundance by western blotting. Results: Our prior work has shown that TRKO mice have situational hyperglycemia due to increased GNG and overexpression of gluconeogenic enzymes. In the current study, we found no difference in phosphorylation of FOXO1 between TRKO and WT mice after refeeding (6.77±0.81 vs 8.25±0.82AU; not significant [ns] by t-test; n=10 per group) or fasting (5.29±1.39 vs 7.19±2.00AU; ns; n=6 per group). However, there was a dramatic reduction in phosphorylation of FOXO4 in TRKO compared to WT mice after both refeeding (0.068± 0.017 vs 12.44±2.35AU; p<0.001) and fasting (0.40± 0.12 vs 15.31±31AU; p<0.01). Phosphorylation of Akt2 at S473, the target of mTORC2, was reduced in TRKO compared to WT mice after refeeding (4.91± 0.25 vs 6.40± 0.53AU; p<0.05) and fasting (2.98±0.28 vs 10.86±1.47AU; p<0.001). Reduced phosphorylation of FOXO4 and Akt2 followed the same pattern. Conclusion: These data suggest that FOXO4, rather than FOXO1, is the mTORC2-dependent transcription factor that regulates GNG in the kidney. Akt2 is activated by mTORC2, and Akt2 is likely to directly phosphorylate FOXO4 and FOXO1 given their sequence homology. We propose that FOXO4 plays plays a unique role in the kidney compared to the liver because GNG and glucose transport occur in the same cells of the proximal tubule. Additional experiments are required to confirm and further explore the role of FOXO4 in renal glucose homeostasis. Funding: NIDDK Support, Private Foundation Support