ACTION OF INSULIN ON THE SURFACE-MORPHOLOGY OF HEPATOCYTES - ROLE OF PHOSPHATIDYLINOSITOL 3-KINASE IN INSULIN-INDUCED SHAPE CHANGE OF MICROVILLI

In previous studies we have shown that the insulin-responding glucose transporter isoform of 3T3-L1 adipocytes, GluT4, is almost completely located on microvilli. Furthermore, insulin caused the integration of these microvilli into the plasma membrane, suggesting that insulin-ind uced stimulation of glucose uptake may be due to the destruction of th e cytoskeletal diffusion barrier formed by the actin filament bundle o f the microvillar shaft regions [Lange et al. (1990) PEES Lett. 261, 4 59-463; Lange et al. (1990) FEES Lett. 276, 39-41]. Similar shape chan ges in microvilli were observed when the transport rates of adipocytes were modulated by glucose feeding or starvation. Here we demonstrate that the action of insulin on the surface morphology of hepatocytes is identical to that on 3T3-L1 adipocytes; small and narrow microvilli o n the surface of unstimulated hepatocytes were rapidly shortened and d ilated on top of large domed surface areas. The aspect and mechanism o f this effect are closely related to ''membrane ruffling'' induced by insulin and other growth factors. Pretreatment of hepatocytes with the PI S-kinase inhibitor wortmannin (100 nM), which completely prevents transport stimulation by insulin in adipocytes and other cell types, a lso inhibited insulin-induced shape changes in microvilli on the hepat ocyte surface. In contrast, vasopressin-induced microvillar shape chan ges in hepatocytes [Lange et al. (1997) Exp. Cell Res. 234, 486-497] w ere insensitive to wortmannin pretreatment. These findings indicate th at PI 3-kinase products are necessary for stimulation of submembrane m icrofilament dynamics and that cytoskeletal reorganization is critical ly involved in insulin stimulation of transport processes. The mechani sm of the insulin-induced cytoskeletal reorganization can be explained on the basis of the recent finding of Lu et al. [Biochemistry 35(1996 )14027-14034] that PI 3-kinase products exhibit much higher affinity f or the profilin-actin complex than the primary products, PIP and PIP2. Thus, activated PI 3-kinase may direct a flux of profilin-actin compl exes to the membrane locations of activated insulin receptors, where, due to the release of actin monomers after binding of profilactin to P I(3,4)P-2 and PI(3,4,5)P-3, massive actin polymerization is initiated. As a consequence, PI 3-kinase activation initiates a vectorial reorga nization of the cellular actin system to membrane sites neighboring ac tivated insulin receptors, giving rise to local membrane stress as vis ualized by extensive surface deformations and shortening of microvilli . In addition, extensive high-affinity binding of F-actin-barbed endca pping proteins enhances the cytoplasmic concentration of rapidly polym erizing filament ends. Consequently, the actin monomer concentration i s lowered and the (cytoplasmic) pointed ends of the microvillar shaft bundle depolymerize and become shorter. The observations presented str engthen the previously postulated diffusion-barrier concept of glucose -and ion-uptake regulation and provide a mechanistic basis for explain ing the action of insulin and other growth factors on transport proces ses across the plasma membrane. (C) 1998 Academic Press.