THE INSULIN-MIMETIC AGENT VANADATE PROMOTES RECEPTOR ENDOCYTOSIS AND INHIBITS INTRACELLULAR LIGAND-RECEPTOR DEGRADATION BY A MECHANISM DISTINCT FROM THE LYSOSOMOTROPIC AGENTS

Vanadate (sodium orthovanadate) is an insulin-mimetic agent and phosph otyrosine phosphatase inhibitor that has been proposed as a potential therapeutic agent for diabetes, We previously reported that vanadate d ecreased the number of cell-surface insulin receptors but inhibited re ceptor degradation in cultured lymphocytes (IM-9) (1). To determine wh ether vanadate affected receptors without intrinsic tyrosine kinase ac tivity, its effects on LDL and transferrin receptors and their ligands were examined, Vanadate exposure resulted in a dose-and time-dependen t decrease in LDL binding to cultured human fibroblasts associated wit h a decrease in cell surface receptor number while total solubilized c ell LDL receptors increased, Vanadate also inhibited the LDL-mediated downregulation of total cellular LDL receptors in the absence and pres ence of cycloheximide consistent with an inhibition of LDL receptor de gradation, In the case of the ligand, vanadate augmented the accumulat ion of intact I-125-LDL associated with an inhibition of up to 80% of the ability of LDL to decrease cholesterol synthesis, Since these acti ons were similar to the effects of lysosomotropic agents, we examined the effect of vanadate on intraendosomal pH using the fluorescent prob e acridine orange, In contrast with chloroquine and NH4Cl, vanadate di d not neutralize the pH of the acidic intracellular compartment, Furth ermore, after a transient insulin-like effect, chronic exposure to van adate diminished I-125-diferric transferrin binding to rat adipocytes. In contrast with the inhibitory action of NH4Cl, intracellular Fe-59 uptake remained unaffected and was proportional to cell-surface bindin g capacity in the presence of vanadate, These data demonstrate a chron ic effect of vanadate to promote the accumulation of intracellular rec eptors and to inhibit Ligand and receptor degradation, The latter effe ct is not mediated by pH changes, appears to be localized to a late en dosomal/lysosomal compartment, and suggests a possible role for tyrosi ne dephosphorylation in the regulation of receptor-ligand degradation.