Abstract
The glucose transport activity associated with the plasma membrane-rich and Golgi-rich fractions of fat cells was determined after they were reconstituted into egg lecithin liposomes. When the two subcellular fractions were isolated under conditions that would minimize their cross-contamination, the transport activity in the plasma membrane-rich fraction was found to be increased 6.3- to 8.6-fold by insulin, which was added to cells before homogenization, and that the activity in the Golgi-rich fraction was reduced approximately to one-half. In this study, the glucose transport activity in the plasma membrane-rich fraction (either in the basal or plus insulin state) was solubilized, reconstituted, and assayed with an overall efficiency of 25-35%. Four agents known to have insulin-like effects on the glucose transport activity in intact fat cells (hydrogen peroxide, sodium vanadate, trypsin, and p-chloromercuriphenyl sulfonate) not only increased the transport activity in the plasma membrane-rich fraction, but also decreased the activity in the Golgi-rich fraction. The effect of hydrogen peroxide, unlike that of insulin, was not abolished when the insulin receptor was modified proteolytically. Upon administration of insulin to fat cells, and subsequent elimination of the hormone, the glucose transport activities associated with the plasma membrane-rich and Golgi-rich fractions were affected almost concomitantly towards opposite directions. It is proposed as a working hypothesis that translocation of the glucose transport system to the plasma membrane from the Golgi-rich fraction is the major, if not the sole, mechanism by which insulin stimulates glucose transport in fat cells.
Highlights
Four agents known to have insulin-like effects on the glucosetransport activity in intact fat cells increased the transport activity in the plasma membrane-rich fraction, and decreased the activity in the Golgi-rich fraction
The effect of insulin observed in both of the above studies were highly significant; the hormone-dependent increase in the transport activity detected in our reconstituted plasma membrane-rich fraction was 2.0- to 2.5-fold (1, 2), and the corresponding value reported by Cushman and Wardzala was 3.1-fold (3)
ApparentInsulin Effects Observed in IsolatedSubcellular Fractions-Figs1. and 2 show typical results of fractionation carried out by Method A (Fig. 1) and Method B (Fig. 2) as described under "Materials and Methods." These figures indicate that the glucose transport activities associated with the plasma membrane-rich fraction (Fractions 3-7) and the Golgirich fraction (Fractions 13-17) were well separated from each other, especially by Method B
Summary
Four agents known to have insulin-like effects on the glucosetransport activity in intact fat cells (hydrogen peroxide, sodium vanadate, trypsin, and p-chloromercuriphenyl sulfonate) increased the transport activity in the plasma membrane-rich fraction, and decreased the activity in the Golgi-rich fraction. Upon administration of insulin to fat cells, and subsequent elimination of the hormone, the glucose transport activities associated with the plasma membranerich and Golgi-rich fractions were affected almost concomitantly towards opposite directions. The effect of insulin observed in both of the above studies were highly significant; the hormone-dependent increase in the transport activity detected in our reconstituted plasma membrane-rich fraction was 2.0- to 2.5-fold (1, 2), and the corresponding value reported by Cushman and Wardzala was 3.1-fold (3). These values are considerably less than that routinely observed in the glucose transport activity in intact fat cells, which is 5- to 10-fold (e.g. see Ref. 5). It was yet to be ascertained whether the proposed translocation is the major mechanism, or merely represents a small fraction of the multiple mechanisms, by which insulin stimulates glucose transport in fat cells
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