Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes.

The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated by the use of labelled ovine prolactin) was increased 2-3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.

Down regulation of masked and unmasked insulin receptors in the liver of transgenic mice expressing bovine growth hormone gene.

The interaction of insulin with its receptor was studied in microsomes from livers of transgenic mice expressing the bovine growth hormone gene with mouse metallothionein-1 promoter (MT/bGH) and in their normal (non-transgenic) littermates. Specific binding of 125I-insulin was detected in hepatic microsomes from normal and transgenic mice with an apparent Kd of 8 and 200 nM, for high and low affinity sites, respectively. The transgenic MT/bGH mice had a marked hyperinsulinism without significant elevation of plasma glucose levels. Under identical conditions of preparation and incubation, microsomes from the transgenic male and female mice bound 39% and 34% less insulin than those from their litter mates. Scatchard's analysis indicates that this decrease in binding is due to a decrease in the number of receptor sites. In contrast to the marked decrease in insulin binding to unmasked receptors, the levels of masked (also called cryptic) insulin receptors were similar (or slightly increased) in transgenic mice microsomes as compared to those of their normal litter mates.

Identification of somatogenic binding sites in liver microsomes from normal mice and transgenic mice expressing human growth hormone gene.

Somatogenic binding sites were detected and characterized in microsomal preparations from livers of normal mice and mice expressing metallothionein-I/hGH (mMT/hGH) hybrid gene, using 125I-labelled bovine or human GH, or a photoreactive derivative of hGH (125I-AP-hGH1). Specific binding of 125I-bGH was detected in liver microsomes from both normal and transgenic mice with an apparent Kd of 2 nM. 125I-hGH was partially displaced by bGH. 125I-AP-hGH1 was covalently bound to the microsomal preparations, and bGH prevented the formation of the 130 kDa species with no appreciable effect on 63 kDa and 70 kDa lactogenic complexes.

Isolation, purification and primary structure of insulin from the turtle Chrysemys dorbigni.

Insulin A and B chains from pancreas of the turtle Chrysemys dorbigni have been purified to homogeneity, and their primary structures have been determined. The sequence of the A chain is G-I-V-E-Q-C-C-H-N-T-C-S-L-Y-Q-L-E-N-Y-C-N, and that of the B chain is A-A-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-S-P-K-A. The amino acid sequence of Chrysemys insulin is identical to that of another turtle (Pseudemys scripta), the chicken, and turkey. When compared with alligator insulin, it has three conservative substitutions in the B chain. However, there are seven substitutions when compared with the insulin of the rattlesnake.

Modulation of insulin induced ornithine decarboxylase by putrescine and methylputrescines in H-35 hepatoma cells.

The effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min. 1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with Ki values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a Ki = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 microM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 microM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.

Proteins associated with somatogenic and lactogenic receptors in microsomal membranes and intact rat hepatocytes.

Lactogenic and somatogenic receptors present in rat liver have been examined by cross-linking with a derivative of human somatotropin (AP-hGH1) followed by SDS-polyacrylamide gel electrophoresis. AP-hGH1, which has a content of 2.2 azidophenacyl groups per molecule, mainly linked to half Cys-182 and half Cys-189, exerted a specificity similar to that of the native hormone (hGH), with an ability of 46% with respect to hGH to compete with the radiolabelled hormone for the binding sites of microsomal preparations. Photolysis of the 125I-labelled derivative bound to the lactogenic receptors present in either microsomal membranes or Triton X-100 solubilized preparations gave rise to a 63 kDa species. In addition, 30% of the covalent complexes formed in microsomal membranes belonged to a species with a molecular mass of 70 kDa. Incubation of viable rat hepatocytes with the radiolabelled derivative at either 0 degrees C for 3 h or 15 degrees C for 1.5 h and subjection to irradiation, yielded covalent complexes of molecular masses estimated at 130, 73, 63, 45 and 35 kDa. Experiments performed in the presence of 1 mM NaCN, gave rise to the previous species in a similar yield as that obtained in the absence of cyanide. The 130 kDa complex is related to the somatogenic binding sites, since it was not visualized in the presence of unlabelled bovine somatotropin, while the 70-73, 63, 45 and 35 kDa bands disappeared when the incubations were performed in the presence of unlabelled ovine prolactin.

Identification of insulin binding sites in isolated cells from rat submaxillary gland.

Isolated cells from rat submaxillary gland bound 125I-labelled insulin in a time-dependent process that reached a maximum at 30-40 min at 25 degrees C. The radioactivity bound to cells could be dissociated by dilution of the binding site-hormone complex with the incubation buffer. The presence of unlabelled insulin in the incubation buffer inhibited 125I-labelled insulin degradation according to the amount of hormone added. After 10 min of incubation at at 25 degrees C, radioactivity associated to cells was almost exclusively identified as intact 125I-labelled insulin. With increasing times, a greater contribution of final products of degradation in total radioactivity bound to cells was observed; nevertheless, in the presence of unlabelled insulin the radioactivity associated to low molecular weight products markedly decreased. Equilibrium binding data analysis gave rise to a non-linear Scatchard plot, whose high affinity component showed a dissociation constant of 6.6 +/- 0.4 nM. These observations are consistent with the presence of insulin binding sites in rat submaxillary gland cells which are similar in their characteristics to those identified in other tissues.

Effect of proteolytic enzymes on masked insulin receptors in rat submaxillary gland microsomes.

Trypsin and alpha-chymotrypsin effects on masked insulin receptors were studied. Phospholipase C treatment, incubation in a high ionic strength buffer or solubilization were used as alternative procedures for the unmasking of insulin receptors. These three methods expose receptor structures which are inaccessible to insulin in the current experimental conditions of binding assays without any significant change in binding affinity. Both exposed and masked receptors proved to be equally sensitive to trypsin and alpha-chymotrypsin degradation. At 25 degrees C, about 5 micrograms trypsin/ml for 50 min or 80 micrograms alpha-chymotrypsin/ml for 200 min were necessary in each case to cause a 50% inhibition of the binding of 125I-iodo insulin to microsomes. The results suggest that masked receptors are only nonfunctional to bind insulin but they are not located in compartments inaccessible to molecules present in the medium.

Membrane methylation in isolated rat testis interstitial cells unmasks functional luteinizing hormone receptors.

Treatment of intact isolated rat testis interstitial cells with S-adenosylmethionine as methyl donor, increases substantially the number of LH human CG receptors (100-200%) without modifying the equilibrium dissociation constant. The increase in binding capacity was associated with an augmentation in the sensitivity of the rat testis interstitial cells to produce testosterone in response to LH, suggesting a functional role of the unmasked receptors. The amount of S-adenosylmethionine necessary to obtain an increase in LH binding capacity and preserve cell viability was 25-50 micrograms/ml per 1.6 X 10(7) cells. 10 mM MgCl2 in addition to the Mg2+ present in the medium was necessary to maintain cell viability. 3H-labelled methyl groups were incorporated mainly into the lipid fraction (208 fmol/10(6) cells) when 3H-S-adenosylmethionine was incubated with the cells for 2 h at 30 degrees C. Our results are consistent with the conclusion that early action of LH may involve an activation of methyltransferase activity, phospholipid methylation, an increase in LH binding capacity and an increase in receptor function.

Characterization of the insulin-binding sites in turtle thyroid microsomes.

The characteristics of the specific binding of labelled insulin to turtle thyroid microsomes were investigated. Binding experiments were performed in Krebs-Ringer bicarbonate buffer (pH 7.4) at 25 or 4 degrees C for different periods of time. Dissociation of the labelled insulin from the binding sites was also evaluated. It was found that the binding is dependent on time, temperature and microsomal protein concentration, with an optimum pH of 8.0. Unlabelled insulin and pro-insulin competed with the labelled insulin, binding in direct proportion to their biological activities, while glucagon and growth hormones did not compete for the binding sites. Scatchard plot analysis established the presence of binding sites of high and low affinities, and the rate of dissociation of bound insulin was considerably increased by the addition of unlabelled insulin. Both results are compatible with a negative co-operativity site-site interaction model. Trypsin abolished the insulin binding. These findings indicate that the microsomes from the turtle thyroid gland contain specific binding sites for insulin. However, pre-incubation of microsomes with phospholipase C or S-adenosyl-L-methionine (SAM), or incubation in the presence of 2 mol NaCl/l did not increase the specific insulin binding. Therefore, the binding properties are similar to those observed in mammalian insulin-responsive tissues except for the absence of the effects of 2 mol NaCl/l, phospholipase C or SAM, which suggests the absence of masked insulin-binding sites.

Specific binding sites for insulin in the rat pituitary gland.

Studies were carried out in order to characterize specific insulin binding sites in the rat pituitary gland. Binding of labeled insulin by pituitary microsomes reached equilibrium after 4 h at 4 degrees C and remained stable over 16 h; at 25 degrees C the plateau was reached in 20 min. Equilibrium binding data analysis of competitive displacement of bound 125I-iodo insulin by unlabeled insulin yielded a non-linear Scatchard plot. At 25 degrees C the Kd for the high affinity component was 2.8 +/- 0.1 X 10(-9) M and the receptor concentration was 260 +/- 80 fmol/mg of microsomal protein. A Kd value of 4.6 +/- 0.4 X 10(-8) M and a binding capacity of 800 +/- 200 fmol/mg microsomal protein were obtained for the low affinity sites. Insulin binding to microsomes was enhanced 2.7 times by increasing the ionic strength of the incubation medium with 2 M NaCl, and was abolished when the microsome preparation was preincubated with trypsin prior to binding measurements. Other hormones, such as bovine thyrotropin, ovine follitropin, human somatotropin and ovine prolactin did not interact with the insulin receptor. Proinsulin displaced the labeled hormone in direct proportion to its insulin-like biological activity.

Specific uptake, dissociation, and degradation of 125I-labeled insulin in isolated turtle (Chrysemys dorbigni) thyroid glands.

Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with 125I-insulin in the presence or absence of unlabeled insulin, in order to study its specific uptake. At 24 degrees, the specific uptake reached a plateau at 180 min of incubation. The dose of bovine insulin that inhibited 50% of the 125I-insulin uptake was 2 micrograms/ml of incubation medium. Most of the radioactive material (71%) extracted from the gland, after 30 min incubation with 125I-insulin, eluted in the same position as labeled insulin on Sephadex G-50. Only 24% eluted in the salt position. After 240 min incubation, increased amount of radioactivity appeared in the Na125I position. When bovine insulin was added together with the labeled hormone, a substantial reduction of radioactivity was observed in the insulin and Na125I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with 125I-insulin either at 24 or 6 degrees. The percentage of trichloroacetic acid (TCA)-soluble radioactive material in the dissociation medium increased with incubation time at both temperatures. However, the degradation activity was lower at 6 than at 24 degrees. The addition of bovine insulin to the incubation buffer containing 125I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with 125I-hGH or 125I-BSA showed values of uptake, dissociation, and degradation similar to those experiments in which an excess of bovine insulin was added together with the labeled hormone. Thus, by multiple criteria, such as specific uptake, dissociation, and degradation, the presence of insulin-binding sites in the turtle thyroid gland may be suggested.

Unmasking of insulin receptors in rat submaxillary gland microsomes: effect of high ionic strength, phospholipase C and S-adenosyl-L-methionine.

The specific binding of [125I]insulin to submaxillary gland microsomes was significantly enhanced by increasing the ionic strength of the incubation medium. This effect was neither related to changes in receptor or hormone degradation nor in the polymerization of the tracer. When equilibrium binding data from competition curves of unlabelled insulin versus [125I]insulin were analyzed, a marked increase in total binding capacity in high ionic strength was observed (from 890 to 2440 fmol/mg protein), with no change in binding affinity. Phospholipase C digestion was also able to increase specific [125I]insulin binding to microsomes. These results suggest the presence of masked receptors in submaxillary gland microsomes. Methylation of rat submaxillary gland microsomes by using S-adenosyl-L-methionine as the methyl donor significantly increased [125I]insulin binding. Scatchard analysis of the equilibrium binding data showed that addition of S-adenosyl-L-methionine (0.46 mM) to microsomes resulted in an enhancement of the total binding capacity (from 990 to 1520 fmol/mg protein) with no change in the affinity constants, which suggests the exposure of masked insulin receptors under such conditions. Both the methyl group incorporation into membrane phospholipids and the effect on insulin binding were dependent on the S-adenosyl-L-methionine concentration used and were partially suppressed in the presence of S-adenosyl-L-homocysteine, a specific competitive inhibitor of the methyltransferases activity. When microsomes were treated with S-adenosyl-L-[methyl-3H]methionine, the 3H-labelled methyl groups incorporated were found mainly in the lipid fraction associated to phosphatidylcholine, suggesting in this case that the unmasking of insulin receptors could be a consequence of alterations produced in membrane composition. The effects of phospholipase C, S-adenosyl-L-methionine and high ionic strength on insulin binding were not additive, suggesting that these procedures unmask receptors from the same pool.

Influence of divalent cations on the detection of somatogenic and lactogenic binding sites in mouse liver cells.

Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM.

In vivo specific uptake of labeled insulin by turtle (Chrysemys dorbigni) thyroid gland.

Insulin labeled with 125I was injected into turtles to study its specific uptake by the thyroid gland. The gel filtration behavior of labeled material in blood and thyroid gland was determined in order to ascertain if the uptake is specific. Most animals were pretreated with KI to saturate the gland with iodide. Maximum specific uptake of radioactivity by the thyroid gland was only detected in animals pretreated with KI. A significant dose-related reduction (ED50 = 0.5 micrograms/kg) was observed when unlabeled insulin was administered simultaneously with 125I-insulin. Prolactin, glucagon and growth hormone (2 mg/kg) did not affect 125I-insulin uptake. Most of the radioactive material extracted from the turtle thyroid 15 min after 125I-insulin injection coeluted with 125I-insulin on Sephadex G-50. This peak decreased as a function of time after 125I-insulin administration. Similar elution patterns were found for thyroid extracts from turtles previously treated with KI. The labeled hormone in the gland was rapidly degraded or processed to both higher and lower molecular weight compounds. Prior administration of KI suppressed the former, whereas when unlabeled insulin was injected simultaneously with 125I-insulin the amount of degradation products was reduced. The demonstration of radioactive degradation products is consistent with the intracellular receptor-mediated degradation hypothesis. These findings indicate the presence of specific insulin-binding sites in the thyroid gland.

Characteristics of insulin receptors in a clonal line of rat pituitary tumor cells in culture.

The presence of insulin binding sites in a clonal line of cells from rat pituitary tumor (GH1 cells) is described. The binding of insulin was a reversible and specific process. Binding of 125I-insulin to GH1 cells was inhibited by unlabeled insulin and to a minor extent, by proinsulin and desalanine insulin in direct proportion to their biological activities. Trypsin abolished the insulin binding to the cells. Twelve hours incubation of cells with insulin (5 microM) reduced in a 45% of the number of binding sites for the hormone. 125I-insulin bound to GH1 cells dissociated with a t 1/2 of 8 min. At 24 C labeled insulin was degraded by GH1 cells. No degradation was detected at 6 C. Unlabeled insulin inhibited most of the degradation of 125I-insulin, suggesting that degradation resulted from a saturable process. At steady-state, radioactive degradation products as well as 125I-insulin were recovered from GH1 cells.

Characteristics of insulin receptors in a clonal line of rat pituitary tumor cells in culture.

The presence of insulin binding sites in a clonal line of cells from rat pituitary tumor (GH1 cells) is described. The binding of insulin was a reversible and specific process. Binding of 125I-insulin to GH1 cells was inhibited by unlabeled insulin and to a minor extent, by proinsulin and desalanine insulin in direct proportion to their biological activities. Trypsin abolished the insulin binding to the cells. Twelve hours incubation of cells with insulin (5 microM) reduced in a 45

of the number of binding sites for the hormone. 125I-insulin bound to GH1 cells dissociated with a t 1/2 of 8 min. At 24 C labeled insulin was degraded by GH1 cells. No degradation was detected at 6 C. Unlabeled insulin inhibited most of the degradation of 125I-insulin, suggesting that degradation resulted from a saturable process. At steady-state, radioactive degradation products as well as 125I-insulin were recovered from GH1 cells.