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Besides their function of lipid storage, the adipose cells secrete a number of proteins of physiopathological importance. To get further insights into this function, which remains poorly characterized, we sought to compare the mechanisms and regulation of secretion of two individual proteins in the same cells. Leptin and angiotensinogen were chosen and assessed by radioimmunoassay and quantitative immunoblotting, respectively, in primary culture of epididymal adipose cells from young obese Zucker rats. Leptin was secreted at a steady rate of 4 ng/106 cells/h over 2–6 h. Despite secretion, leptin cellular content remained stable at 3 ng/106 cells. In contrast, the rate of angiotensinogen secretion decreased regularly from 45 arbitrary units/106 cells/h at 2 h, to half this value at 6 h, although cell content remained constant at 100 arbitrary units/106 cells. Inhibition of protein synthesis by cycloheximide depleted the cells from leptin, but not from angiotensinogen for up to 6 h. Insulin increased leptin secretion (+75%) and cell content (+70 %), without affecting angiotensinogen. Secretion of both proteins was inhibited by Golgi-disturbing agents, brefeldin A and monensin. The presence of brefeldin A led to a specific rise in leptin cell content, an effect inhibited by cycloheximide and enhanced by insulin (+80%). These data show that leptin and angiotensinogen are both secreted through Golgi-dependent pathways and that their respective intracellular pool exhibit distinct turn-over rate and insulin sensitivity. These characteristics might account for the differential response of these adipose proteins to variations in the systemic environment. J. Cell. Biochem. 82: 666–673, 2001. © 2001 Wiley-Liss, Inc.

We previously reported that in cultured adipose cell lines insulin increased selectively the expression of Glut 1, in contrast to in vivo regulation where variations in insulinemia have been shown to affect only GLUT 4. We have addressed here the question of the long-term regulation of GLUT 1 and GLUT 4 in fat cells by using primary cultures of rat adipocytes. Epididymal fat cells were isolated by collagenase and cultured 4 days in DMEM supplemented with BSA 1%, FCS 1%, and glucose 10 mM. GLUT 1 and GLUT 4 proteins were assessed in total cellular membranes by Western blotting, using specific antibodies against their respective C-terminal peptides. GLUT 1 steadily increased over culture time to reach at day 3, a level 3-fold higher than the initial value. In contrast, GLUT 4 decreased sharply and stabilized at day 3, at 30% of the initial value. The changes in GLUT 1 and GLUT 4 mRNAs with culture time were parallel to changes in the corresponding proteins, suggesting a pre-translational level of regulation. The expression of the lipogenic enzyme, fatty acid synthetase (FAS), highly expressed in fat cell, decreased over time following a pattern closely parallel to that of GLUT 4. Chronic exposure to insulin added at day 2 had no effect on GLUT 4 expression but increased the expression of GLUT 1 and FAS by 70% and 36%, respectively. Glucose consumption was stable over 4 days of culture, while lactate production increased from 24 to 36% of glucose utilization, in agreement with the loss in FAS. Glucose consumption increased only slightly with insulin (+160%), in good keeping with the low levels of expression of both GLUT 4 and FAS in these cultured cells. These data indicate that culture alters oppositely the expression of GLUT 1 and GLUT 4 in rat adipocytes and suggest that factor(s) other than insulin predominate in their regulation in vivo.