Effect of Inhibiting Vacuolar Acidification on Insulin Signaling in Hepatocytes

Abstract

Previous studies have shown that the endosomal apparatus plays an important role in insulin signaling. Inhibition of endosomal acidification leads to a decrease in insulin-insulin receptor kinase (IRK) dissociation and insulin degradation. Thus, vacuolar pH could function as a modulator of insulin signaling in endosomes. In the present study we show that in primary hepatocytes pretreated with bafilomycin, there is an inhibition of vacuolar acidification. Incubation of these cells with insulin was followed by an augmentation of IRK activity but an inhibition of phosphatidylinositol 3-kinase/Akt activity and a decrease in insulin-induced DNA and glycogen synthesis. Bafilomycin treatment inhibited IRK recycling to the plasma membrane without affecting IRK internalization. Impaired IRK recycling correlated with a decrease in insulin signaling. We suggest that inhibiting vacuolar acidification sequesters activated IRKs in an intracellular compartment(s) where signaling is inhibited. This implies that endosomal receptor trafficking plays a role in regulating signal transduction.

Epidermal Growth Factor-induced Vacuolar (H+)-ATPase Assembly

“…Bafilomycin was previously observed to inhibit insulin-stimulated mitogenesis in primary hepatocytes (44), Swiss 3T3 cells (45), BNL (murine embryonic liver) cells (46), and eight different human cancer cell lines (47). In this study we showed that EGF-stimulated DNA synthesis in primary rat hepatocytes was also inhibited by bafilomycin using both 10 and 100 nM EGF (Fig.…”

Involvement of Vesicular H+-ATPase in Insulin-Stimulated Glucose Transport in 3T3-F442A Adipocytes

“…Thus, in our experiments, it is very unlikely that bafilmycin A 1 inhibits the insulin-stimulated 2-DG uptake by perturbing the alkalinizing effect of insulin. Another possibility is that bafilomycin A 1 may inhibit insulin signaling cascades as it did in rat hepatocytes [40]. Then again, Malikova et al [41] demonstrated that bafilomycin A 1 and concanamycin A, another specific inhibitor of VATPase, do not affect insulin signal transduction in adipocytes even though they inhibit insulin-stimulated glucose transport.…”

Tetanus Toxin Is Transported in a Novel Neuronal Compartment Characterized by a Specialized pH Regulation

“…These features are particularly important, because acidic pH triggers a conformational change in TeNT and other CNTs, allowing the active subunit to translocate through the endosomal membrane into the cytosol (9,10). Moreover, acidic pH is known to drive the dissociations of ligands, including growth factors, from their receptors and influence their signaling and intracellular targeting (11,12). We sought to address these important questions by preparing chimeras of TeNT H C and ratiometric pHluorin, a pH-sensitive green fluorescent protein mutant (13).…”