Abstract
Adipocytes route internalized insulin through two major pathways, a degradative pathway and a retroendocytotic pathway. To examine whether sorting of incoming insulin-receptor complexes can be altered, we assessed the effect of vanadate on the intracellular processing of both insulin and insulin receptors. After cells were pretreated with vanadate (1 mM for 30 min at 37 degrees C), 125I-insulin was loaded into the cell interior. When the net efflux of insulin from cells into the medium was then monitored, vanadate was found to slow the efflux of insulin from a t1/2 of 6.2 min (controls) to 11 min. Since efflux reflects both the rapid extrusion of intact insulin and the slower release of degradative products, we proposed that vanadate diverts more insulin into the degradative pathway. Further evidence in support of this idea included the following: 1) when intracellular degradation of insulin was impaired by chloroquine, undegraded insulin accumulated faster within vanadate-treated cells, consistent with greater flux through a degradative pathway; 2) vanadate increased the percentage of degraded insulin released from cells from 61 and 72%; and 3) under steady-state binding conditions, more insulin resided in the cell interior of vanadate-treated cells (44.8% versus 34.5%), and the time required for the intracellular pool to reach equilibrium was prolonged (t1/2 of 5.5 min versus 4.0). Neither insulin internalization nor degradation was impaired by vanadate alone. In related studies Tris was found to inhibit insulin-mediated receptor recycling by only 10%, whereas in the presence of vanadate (plus Tris) almost all incoming insulin receptors were prevented from recycling. Vanadate alone had no effect on the ability of insulin receptors to recycle. Based on these results we conclude that: 1) vanadate shunts incoming insulin from a more rapid retroendocytotic pathway to a slower degradative pathway and diverts insulin receptors from a Tris-insensitive recycling pathway to one that can be completely inhibited by Tris; 2) these effects are selective, in that vanadate impairs neither insulin degradation nor receptor uptake and recycling. Considered together, these findings support the idea that a sorting mechanism exists for the intracellular routing of incoming insulin-receptor complexes.
Highlights
These pathways were found to differ in several ways
Based onthese results we conclude that: 1) vanadate shunts incoming insulin from a more rapid retroendocytotic pathway to a slower degradative pathway and diverts insulin receptors from a Tris-insensitiverecyintracellular itinerary of insulin receptors [3, 4], it appears likely that thereceptors themselves traverse two pathways; the first pathway returns both insulin receptors and intact ligand to thecell surface through a chloroquine-insencling pathway to onethat can be completely inhibited sitive recycling pathway, while the second pathway returns by Tris; 2) these effects are selective, in that vanadate unoccupied insulin receptors tothe cell surface after the impairs neither insulin degradation nor receptor up- intracellular dissociation of insulin-receptor complexes
We preloaded '251-insulininto the cell interior of control and vanadate-pretreated cells (1 m ~ / 3 0min/37 "C)andthen monitored both the rate of insulin efflux and the composition of insulin released to the medium
Summary
L The abbreviations used are: zineethanesulfonic acid; MEM, Hepes, 4-(2-hydroxyethyl)-l-piperaDulbecco's modified Eagle's medium. Composition of Internalized Insulin- Fig. 4 can be interpreted to mean that vanadate treatment shumnotrse insulin toa chloroquine-sensitivedegradative pathway, there remained an alternative explanation.Namely, vanadate may tiate that intact insulinis released at the same rate in itself be a lysosomotropic agent thatcould act synergistically control and vanadate-treatedcells and toconfirm that vana- with chloroquineto more effectively block insulin degradation dat,e reduces the amount of insulin processed through the and cause a greater increase in the amountof cell-associated retroendocytoticpathway, we performedtheexperiment '251-insulin.
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