An endogenous membrane-bound substrate of the insulin receptor 13-subunit tyrosine kinase in liver, ppl2O, has been identified as HA4, a 110-kDa membrane glycoprotein localized primarily to the bile canalicular domain of the hepatocyte. HA4 has been implicated in bile salt transport and cell adhesion. Monoclonal antibodies to HA4 were used to identify it as a substrate of the insulin receptor kinase. Anti-ppl20 and anti-HA4 were found to cross-react, and phosphopeptide maps for each of the corresponding antigens were identical. The identification of ppl20 as HA4 serves to link insulin action through the receptor tyrosine kinase activity to bile metabolism and raises questions pertaining to the intracellular site(s) of action of the insulin receptor.Several potential mechanisms of action have been proposed to explain the pleiotypic actions of insulin in tissues such as liver. These have included the generation of second messengers (1-3), interaction with guanine nucleotide-binding proteins (4), the activation of phosphoinositol turnover (5), and change in protein phosphorylation (1-3). The last has become an attractive hypothesis following the discovery that the 95-kDa ,-subunit of the insulin receptor possessed tyrosine kinase activity (2) against itself (autophosphorylation) as well as exogenous and endogenous substrates (2, 6-11). Several membrane-bound and cytosolic substrates for insulin receptor P-subunit tyrosine kinase activity have been identified in a number of tissues (1). However, in most cases neither the functions of the endogenous proteins nor the effect of insulin-stimulated phosphorylation on them is known. An exception is ppl5, a soluble substrate in adipocytes that is thought to act as the intermediary in insulin-stimulated glucose transport (11).One of the endogenous substrates present in liver is a protein called ppl20. It is found in a plasma membraneenriched fraction, is detergent-soluble, and binds to wheat germ agglutinin (WGA), indicating that it is a glycoprotein with complex oligosaccharide chains. ppl20 is also phosphorylated on tyrosine residues by physiological levels of insulin in vitro and intact hepatoma cells (6-9). However, the identity and precise cellular location of ppl20 in vivo are not known. In this study, we have identified ppl20 as HA4, an integral plasma membrane glycoprotein that is largely restricted to the bile canalicular membrane domain in vivo.MATERIALS AND METHODS Preparation of Antibodies. Antibodies to ppl20 were raised in New Zealand White rabbits (6), and antiinsulin receptor antiserum B-d was obtained from a human patient with extreme insulin resistance (12). Mouse monoclonal antibodies to rat liver membrane proteins HA301, HA201, and HA4 were obtained as described by Hubbard et al. (13,14) and coupled to Sepharose beads.Membrane Protein Preparations. Plasma membrane sheets were prepared from livers of fasted rats (13) and the integral membrane proteins were extracted (15). ppl20 and the insulin receptor were partially purified from solubilized rat ...

Fetal hyperglycemia and hyperinsulinemia, as induced by administration of streptozotocin to pregnant rats, during late gestation resulted in the onset of the major period of hepatic glycogen synthesis and accumulation at days 19–20 of gestation (22 days = term) rather than at days 20–21 as for normal fetuses. In addition, sustained high levels of liver synthase phosphatase and phosphorylase phosphatase activities prevented the normal term increase in activation of phosphorylase and inactivation of synthase in hyperglycemic/hyperinsulinemic fetuses. The suppression of term fetal changes in phosphorylase activation in particular contributed to the maintenance at term of fetal liver in a condition favoring glycogenesis rather than glycogenolysis.

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The development of insulin receptors and insulin-stimulated receptor autophosphorylation were studied in livers of prenatal and neonatal rats. Insulin receptors were present in mid-gestation, as early as day 14 in fetal development (full term is 22 days in the rat), with ligand-activated receptor kinase present. In contrast, insulin-stimulated phosphorylation of a M_r 120 kd glycoprotein derived from rat liver membranes, known as ppl20/HA4 and more recently identified as ecto-ATPase, was not observed in fetal liver until day 17 of gestation. Thereafter, phosphorylation of pp120/HA4 increased throughout late gestation. The data suggest that maturation of the insulin receptor kinase occurs soon after initial appearance of the receptor in mid-gestation, but insulin-stimulated phosphorylation of endogenous substrate(s) is dependent on the appearance of specific substrates, such as pp120/HA4.

Type II fucosidosis in an autosomal recessive disease. The paper presents a case of a patient with alpha-L-fucosidase of whom a skin specimen was examined under the electron microscope. Storage material was observed mainly in endothelial cells of blood capillaries and Schwann cells surrounding small peripheral nerves of papillary dermis. Within both cells two different kinds of inclusions were revealed: (1) clear vacuoles and (2) dense bodies with an internal structure prevalently lamellar. All these ultrastructural alterations were observed long before the appearance of clinically defined angiokeratoma at cutaneous level. Hence, they present the same alteration found in the absence of angiokeratoma in type I fucosidosis.