Development of insulin and epidermal growth factor receptors during the differentiation of rat preadipocytes in primary culture.

An homogeneous cell population isolated from the inguinal tissue of 3-day-old rats is able to proliferate in primary culture. In the presence of a physiological concentration of insulin (1.5 nM) it converts into cells exhibiting the morphology and the biochemical characteristics of adipocytes. Insulin and epidermal growth factor (EGF) receptors were studied during both the exponential growth and the adipose conversion phases of these cells. Binding experiments with 125I-labelled peptides were performed directly in the culture dishes. The number of high affinity insulin binding sites increased, during the entire culture period studied, reaching 18 days after plating the value of 10,600 x 2360. Control cells (cultured in the presence of anti-insulin antibody) exhibited an increase of the concentration of insulin binding sites from no more than 500 sites/cell to 6880 +/- 1710 sites/cell between dat 0 and 9 (corresponding to the exponential growth phase); this increase was followed by a rapid reduction in insulin receptors during the stationary phase. The density of EGF binding sites increased between day 0 and 4 (one cell cycle), whether the cells were maintained or not with insulin, and plateaued thereafter. Mature adipocytes freshly isolated from the inguinal tissue of 3-day-old rats had no detectable EGF binding sites, but their content in high affinity binding sites for insulin was similar to that of cells after complete adipocyte conversion in primary culture.

Characterization and repartition of epidermal growth factor-urogastrone receptors in gastric glands isolated from young and adult guinea pigs.

Physiological studies indicate that epidermal growth factor-urogastrone (EGF) acts on stomach epithelium as mitogen and modulator of acid secretion. Here, we studied the binding of 125I-EGF to gastric glands isolated from the guinea-pig fundus (acid-secreting part) and antrum. At 20 degrees C, the association of 125I-EGF to gastric glands was time-dependent (plateau at 90 min) and reversible (75-85% dissociation in 1 h). No degradation of the peptide was detected, but a time-dependent loss of binding capacity was observed. At apparent equilibrium (90 min, 20 degrees C) unlabelled EGF (80 pM to 80 nM) competed with 125I-EGF-binding in the same manner in antrum and fundus (50% inhibition, with 0.6 nM EGF). Whereas kinetics properties were similar in antrum and fundus, the binding capacity was 40-55% lower in fundus than in antrum in young animals (6-8 weeks). By contrast, in adult animals (20-30 weeks), binding was the same in both parts of stomach. Scatchard analysis showed that two orders of binding sites were present in all cases (Ki 0.34-0.47 nM, Ki 2.2-3.4 nM), and that the differences observed were only accounted for by number of binding sites. These results show that EGF possess high affinity binding sites on gastric epithelium. These sites, dependent upon the age of the animals, may be related to the modulations by EGF of gastric trophism and secretions.

[Effect of experimental diabetes and its treatment with insulin on the translation of RNAs and protein synthesis in rat enterocytes]. / Effet du diabète expérimental et de son traitement à l'insuline sur la traduction des RNAs et la synthèse protéique dans les entérocytes de rat.

We examined the effect of diabetes on protein synthesis in intestinal epithelial cells. Isolated enterocytes of diabetic rats display a marked decrease (50%) in 3H-leucine incorporation into intestinal proteins as compared to normal cells. Treatment of diabetics with insulin restores the level of protein synthesis up to normal values. Similarly, in vitro translation products of isolated RNA are much lower in diabetics than in normal or treated diabetics. It is concluded that in intestinal cells, insulin can play an important role in regulating the biosynthesis of proteins by modulating the translation of specific RNAs.

Growth-promoting effect, biological activity, and binding of insulin in human intestinal cancer cells in culture.

The biological action and binding of insulin were tested in two human intestinal cancer cell lines originating from the duodenum (HUTU 80) and the colon (HT 29). After serum deprivation for 24 hr, insulin stimulated cell division and the incorporation of labeled precursors into RNA, protein, and DNA for both cell lines. The action on the RNA and protein was rapid and significantly different (1.5 to 2 times that of control) 1 hr after adding insulin. These effects were dose dependent, present at physiological concentration in vivo (10(-10) M), and independent of the transport of precursors. For thymidine incorporation, the stimulation was delayed up to 8 hr and culminated with cell division 20 hr later. As previously shown for HT 20, HUTU 80 cells exhibited insulin-specific binding sites. Binding of 125I-insulin was saturable; reversible; and time, temperature, and pH dependent. Scatchard analysis of the binding data of the two cell lines gave curvilinear plots. Assuming the presence of two independent binding sites, the high-affinity constants were 6 to 8 X 10(8) M-1, and the number of high-affinity receptors was similar and accounted for 2000 to 3000 receptors/cell. For both cell lines, the effect of insulin on protein and RNA synthesis was significantly different from control at 1 hr when binding reached a maximum at 37 degrees. The biological action of insulin on growth and macromolecular synthesis was dose dependent and maximum at about 10(-8) M insulin, which corresponds to 70% displacement of 125I-insulin binding. Furthermore, the binding and the biological action of proinsulin were about 2% that of native insulin in the two cell lines studied. These results show that insulin acts as a growth factor for these two cell lines and that these effects are probably mediated by the interaction of insulin with specific receptors.

Evidence for the presence of insulin binding sites in isolated rat intestinal epithelial cells.

Insulin receptors have been demonstrated in isolated rat intestinal epithelial cells. The specific binding of 125I-insulin was time--and temperature--dependent, the optimal temperature of study being 15 degrees. Dissociation of bound 125I-insulin by an excess of unlabelled hormone was rapid and attained 66 +/- 2% in 2 h. When initiated by dilution, the dissociation attained 35 +/- 4% in 2 h, and 72 +/- 1% in 2 h when 10(-7) mol/l unlabelled insulin was added. The pH optimum for the binding process was between 7.5 and 8, and the binding increased proportionally to cell protein concentration up to 1.5 mg/ml. Under standard conditions (2 h at 15 degrees) the degradation of the labelled hormone in the medium accounted for 20--50% of total tracer, depending on the concentration of cells. At apparent equilibrium (2 h at 15 degrees), unlabelled insulin in the range of 10(-10) to 10(-7) mol/l inhibited competitively the binding of 4.3--7 X 10(-11) mol/l 125I-insulin; fifty per cent inhibition was obtained with 3 X 10(-9) mol/l native insulin. Scatchard analysis, after correction for degradation, gave curvilinear plots, that may be explained by two orders of binding sites, with 2,000 +/- 200 sites/cell of high affinity (Ka = 2.2 +/- 0.2 X 10(9) l/mol) and 39,000 +/- 3,000 sites/cell of low affinity (Ka = 5.6 +/- 1.6 X 10(7) l/mol). The potency of proinsulin to compete with 125I-insulin for the binding site was 3% that of insulin, unrelated peptides were inactive. Such results give a molecular basis to different reports suggesting that the intestine could be a target-tissue for insulin.