Paul F. Pilch

HEXOSE TRANSPORT IN ADIPOCYTES: STIMULATION BY INSULIN IN THE ABSENCE OF INTACT RECEPTOR

Adipocytes treated with trypsin exhibit an elevated rate of hexose transport as measured by conversion of D-[ 1 -14C]glucose to 14C01. This insulin-like effect of trypsin is half maximal at 10 pg/ml protease. Addition of increasing concentrations of insulin to adipocytes treated with 10 pglml trypsin results in a further increase in glucose oxidation until the full stimulation is achieved, and the same dose-response relationship is observed compared to that seen in untreated fat cells (ED,, = 10 pUlml). Insulin binding studies with untreated and trypsin-treated adipocytes reveal that fat cell proteolysis with 10 pglml trypsin results in decreased receptor number but increased receptor affinity for the insulin ligand as compared to the control cells. Decreased receptor number is therefore compensated by increased receptor affinity for insulin such that little change in receptor occupancy is observed after proteolysis. [1251]Insulin can be cross-linked to fat cells and the high-affinity insulin receptor can be identified by autoradiography subsequent to membrane isolation, solubilization, and dodecylsulfate-polyacrylamide gel electrophoresis ( PILCH, P. F. & M. P. CZECH. 1979. 3. Biol. Chem. 254: 3375. 1980. Ibid. 255: 1722). When this procedure is employed after adipocytes have been treated with 10 &mi trypsin, then trypsin inhibitor, the native receptor (M,., 350,000) is virtually absent but receptor fragments (Mr, 120,000-180,000) are seen which retain the ability to specifically bind and cross-link [1251]insulin. The Coomassie blue and Shiff’s Reagent profiles of membrane proteins are unchanged after treatment with 10 pglml trypsin and slight proteolysis of adipocyte membrane proteins is seen only at 2000 pglml of proteolytic enzyme. It is therefore likely that the insulin-like effects of trypsin are due to specific proteolytic perturbation of the insulin receptor such that a signal is generated which results in stimulated glucose transport. Furthermore, complete integrity of the insulin receptor peptide chain is not required for insulin action since proteolytic cleavage, which virtually abolishes the ability of insulin to cross-link to the native receptor (Mr, 350,000) leads to minimal disruption of insulin binding and action. It is likely that either “nicked” receptor is functionally active or that receptor fragments can mediate insulin action.