Inhibition of Insulin Receptor Binding by Phorbol Esters

Abstract

Phorbol esters inhibit the binding of insulin to its receptors on U-937 monocyte-like and HL-60 promyelocytic leukemia human cell lines. Within 20-30 min, exposure of these cells to 12-0-tetradecanoylphorbol 13-acetate (TPA) at 37 "C results in a 50 % reduction of the specific binding of '251-insulin. Half-maximal inhibition occurs at 1 nM TPA. Other tumor-promoting phorbol esters also inhibit '251-insulin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA does not alter the degradation of the hormone nor does it induce any shedding of its receptors in the medium. The effect of phorbol esters is dependent on temperature and cell type. It is less prominent at 22 "C than at 37 "C. It is reversible within 2 h at 37 "C. TPA reduces the binding of insulin predominantly by increasing its dissociation rate. This effect results in an accelerated turnover of the hormone on its receptors.Phorbol esters are tetracyclic diterpenes that are potent tumor-promoting agents in some tissues (e.g. mouse skin) [1,2] while they induce differentiation in others [3-51. They bind to specific receptors on the cell surface [6] and evoke multiple biological and biochemical changes when added to cultured cells ([7] for review). The nature of the responses initiated by these compounds in vivo as well as in vitro has many similarities with those of growth-stimulating polypeptide hormones such as epidermal growth factor (EGF). Recently is has been demonstrated that phorbol esters reduce the number [8] or the affinity [9,10] of EGF receptors, while they clearly potentiate the biological effects of EGF [lo-131.In addition, these tumor promoters possess insulin-like activity (stimulation of glucose transport and oxidation) in a number of cell types [I41 and they facilitate the growthpromoting action of insulin on 3T3 fibroblastic cells [ l l -131. The binding of insulin to its receptors has been reported to be unaltered by phorbol esters in 3T3 [9] and Hela cells [15]. Since this has also been the case for the binding of nerve growth factor, concanavalin A, multiplication-stimulating activity and low-density lipoproteins to their receptors [9], it was thought that tumor promoters specifically alter the EGF binding. This observation might be related, however, to the cell types under investigation. Thus, in his study of EGF and phorbol receptors in mouse OTT-6050 embryonal carcinoma cells [16], Salomon incidentally reported that TPA (12-0-tetradecanoylphorbol 13-acetate) inhibits the binding of '" I-insulin.In the present investigation we have examined the effect of phorbol esters on the insulin-receptor interaction in the U-937 monocyte-like [5] and the HL-60 promyelocytic leukemia [3,4] human cell lines. The HL-60 cells possess specific receptors for insulin [17-191 and phorbol esters [20] and they are sensitive to the action of these agents [14,21].Ahhreviutions. Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; TPA, 12-0-tetradecanoylphorbol 13-acetate; EGF, epidermal growth factor.Ou...

Diacylglycerols modulate phosphorylation of the insulin receptor from human mononuclear cells

“…A variety of responses have been found, depending on the source of the receptors and experimental conditions employed. Some workers showed inhibition of specific insulin binding by TPA at 37 )C (to U-937, IM-9, HL-60 cells, rat adipocytes) [14][15][16]331. Insulin binding at lower temperatures have not been generally altered (rat hepatoma Fao cell line [19], unknown receptor source [32]).…”

Tumor-promoting phorbol ester stimulates tyrosine phosphorylation in U-937 monocytes.

“…In intact U-937 cells, PMA markedly and rapidly inhibits binding of insulin to its receptor at 37TC (9)(10)(11). The binding subunit of the insulin receptor (a subunit) has an apparent molecular weight of 125,000-135,000 by several criteria (19).…”

“…Two events are of interest with respect to insulin and phorbol 12-myristate 13-acetate (PMA) action on the U-937 monocyte-like cell: (i) insulin stimulates phosphorylation of the 95,000-dalton subunit of the U-937 insulin receptor, and this same preparation contains a kinase activity that phosphorylates exogenous substrates such as casein and specific tyrosine-containing synthetic copolymers (8); (ii) PMA, the most potent tumor promoter, has a major effect on the affinity of insulin for its receptor in the U-937 cell and the IM-9 lymphocytes as well as of EGF for its receptor on HeLa cells (9)(10)(11)(12).…”

Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation.

“…Biologically active phorbol esters and other tumor prorooters, like mezerein, saccharin, and cyclamate, have previously been shown to diminish the binding of several other growth factors such as epidermal growth factor (4,13,28), insulin (6,31), nerve growth factor (10), and transferrin (17). The decreased binding has been attributed to either reduced receptor affinity (4,6,10,28) or decreased number of receptors (13,17).…”