The Endosomal pH Regulator NHE6 is Required for Insulin-Stimulated Glucose Uptake in Adipocytes

Objective: Trafficking of GLUcose Transporter isoform 4 (GLUT4) from the adipocyte vesicular pool to the cell surface is tightly regulated by insulin to maintain glucose homeostasis in response to changing demands in energy consumption. Previously, endosomal Na+/H+ exchanger 6 (NHE6, gene name SLC9A6) was identified in adipocyte plasma membranes and shown to associate with GLUT4-positive vesicles. However, the functional contribution of NHE6 to GLUT4 trafficking and glucose uptake remained uncharacterized. Methods: NHE6 was knocked down in 3T3L1 cells either before or after differentiation into adipocytes using lentiviral delivery of shRNA. Uptake of [3H] 2-deoxyglucose into adipocytes was quantified in response to insulin. We used epitope tagged constructs to distinguish between localization of vesicular and surface levels of NHE6 and GLUT4 proteins. Protein and transcript levels of components of the glucose signaling pathway were monitored by qPCR and Western analysis, respectively, in response to NHE6 knockdown and/or treatment with chemical modulators of endosomal pH. Results: We show that insulin-stimulated glucose uptake in adipocytes is severely impaired upon NHE6 depletion. Correspondingly, insulin-stimulated surface expression of GLUT4 at the adipocyte plasma membrane was diminished in NHE6 knockdown cells due to a post-transcriptional decrease in basal GLUT4. Metformin response of GLUT4 was also muted in the absence of NHE6. Further, we demonstrate diminished activation of the GLUT4 translocation pathway in the absence of NHE6 via reduced expression of the insulin receptor and reduced phosphorylation of the downstream effector kinase Akt. Components of GLUT4 storage vesicles, including GLUT4, LRP1 and sortilin were downregulated in NHE6-knockdown adipocytes under basal conditions. Proteostatic control of key components of the insulin signaling pathway (insulin receptor, GLUT4) could be restored by chemical bypass of NHE6 using the V-ATPase inhibitor bafilomycin or the Na+/H+ exchanger mimetic monensin. Conclusions: Thus, NHE6 is critical for proper expression and trafficking of GLUT4. Basal expression of both insulin receptor and GLUT4 in NHE6 knockdown cells could be restored by bafilomycin-inhibition of the H+-ATPase or the H+ ionophore monensin pointing to pH dysregulation as the underlying defect. We suggest that NHE6 is a component of GLUT4 storage vesicles where it regulates proteostasis. These findings establish NHE6 as a novel contributor to glucose homeostasis and energy metabolism with implications for Christianson syndrome patients who carry loss of function mutations in the SLC9A6 gene. ### Competing Interest Statement The authors have declared no competing interest.