The glucose transport activity of fat cells was assayed in a cell-free system. The activity was solubilized and incorporated into egg-lecithin liposomes. The carrier-mediated glucose transport activity was estimated by subtracting the cytochalasin B-insensitive component from the total glucose uptake activity of the modified liposomes. When a crude microsomal preparation from fat cells was fractionated by sucrose density gradient centrifugation, two transport activities (peaks A and B) were separated. Peak A coincided with the peak of 5'-nucleotidase, a marker of the plasma membrane. Peak B appeared to coincide with the peak of UDPGal:N-acetylglucosamine galactosyltransferase, a marker of the Golgi apparatus. Peak A was considerably smaller than peak B under basal conditions. When cells were exposed to 1 nM insulin for 5 min before homogenization, the height of peak A increased whereas that of peak B decreased. Insulin had no significant effect on the galactosyltransferase activity. The Km values of glucose transport facilitated by the activities in peaks A and B were both approximately 10-15 mM. These results imply that insulin facilitates.translocation of the transport activity from an intracellular storage site to the plasma membrane. We previously reported that fat cells rapidly internalize cellbound insulin (1). The internalized hormone is associated with a subcellular structure that is separable from the plasma membrane by sucrose density gradient centrifugation. Because both the insulin receptor and the hormone-sensitive glucose transport mechanism are thought to be localized in the plasma membrane, we felt it of interest to explore the possibility that cells might internalize the transport mechanism along with the insulin-receptor complex. As a result of this study, we obtained preliminary data indicating that the glucose transport activities of fat cells are associated with two different subcellular structures. Our present study was initiated to carry out further investigation on the nature of these two glucose transport activities. MATERIALS AND METHODSCytochalasin B was purchased from Aldrich, blue dextran from Pharmacia, and crude l-a-phosphatidyl choline of egg yolk (type IX-E, approximately 60% pure, lot no. 88C-7070, referred to as egg lecithin in this report) from Sigma. The sources of other materials are described elsewhere (1).Isolated fat cells were prepared by the collagenase method (2) from epididymal and perirenal adipose tissues of Sprague-Dawley rats (180-280 g). Freshly prepared cells were incubated with gentle shaking (30 cycles per min) for 30 min at 37'C in Krebs-Henseleit Hepes buffer (3) containing fraction V bovine serum albumin at 20 mg/ml and 2 mM glucose. The purpose of this incubation was to stabilize the basal transport activity (4). The incubation was continued 5 more min either in the presence or in the absence of 1 nM insulin. The cells were then homogenized and the crude microsomal fraction was separated by differential centrifugation as described (1), except that ...

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To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.

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