Biomedical subjects
Publications and source records attributed to B I Posner.
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Insulin binding to Golgi and plasmalemma fractions from the liver of obese (ob/bo and db/db) and lean control mice was studied. The specficiity of binding in Golgi fractions (Golgi vesicles and Golgi cisternae) was identical to that observed previously in rat liver Golgi fractions and plasmalemma. Binding to plasmalemma was much lower in obese compared to lean mice, but binding to Golgi cisternae was either not changed (ob/ob) or slightly increased (db/db) in obese compared to lean littermates. These observations suggest that intracellular (Golgi cisternae) insulin receptors are regulated differently from those of the plasmalemma. We propose that insulin affects hepatic receptor content in two different ways. On the one hand plasmalemma receptor loss is accelerated, and on the other hand intracellular receptor production is increased. This model can explain in one framework the effect on insulin in reducing and prolactin in augmenting the level of their respective hepatic receptors.
An insulin radioreceptor assay (RRA) using human placental microsomal membranes was used to measure insulin-like activity (ILA) extracted from human plasma concentrates (Cohn fraction IV-4) by acid ethanol. The soluble activity (ILAs), chromatographed on Sephadex G-75 in 1 M acetic acid, migrated as a small molecule (fractional elution volume, 0.56) ahead of insulin (fractional elution volume, 0.70), whereas at neutral pH, ILAs migrated as a large molecular weight species. The ILAs peak from acid gel filtration on Sephadex was further purified by chromatography on carboxymethyl cellulose (CMC). The ILAs peak from both Sephadex and CMC diluted parallel to the porcine insulin standard in the insulin RRA and was totally unreactive in an insulin RIA. The CMC-purified material was iodinated and purified by binding to and elution from human placental membranes. The binding of [125I]ILAs to human placental membranes was inhibited only minimally by insulin and proinsulin and not at all by epidermal growth factor, nerve growth factor, glucagon, or lactogenic hormones, including human growth hormone. Multiplication-stimulating activity (MSA) inhibited in a manner parallel to ILAs. A Scatchard plot of the binding data was nonlinear. Sephadex ILAs was subjected to isoelectric focusing. The fractions assayed in both insulin and ILAs RRAs yielded comparable results. Peaks of ILA were observed at pHs 5.3, 6.6, and 8.4. When CMC-ILA was subjected to isoelectric focusing in polyacrylamide, a single peak of activity migrating between pH 6.2-6.8 was seen. [125I]ILAs focused at exactly the same pH. Electrophoresis of CMC-ILAs in acid-urea revealed a sharp peak of activity migrating with one of the five protein bands seen after staining. Again, [125I]ILAs comigrated with unlabeled ILAs. The molecular weight of ILAs, as determined on a calibrated Sephadex G-150 column at neutral pH, was 9,000-10,000 daltons. CMC-ILAs stimulated [14C]glucose incorporation into triglycerides of rat adipose tissue and augmented [3H]thymidine incorporation into human fibroblasts, chicken embryo fibroblasts, and BALB 3T3 cells as well as [35S]sulfate incorporation into macromolecules of rabbit chondrocyte culture medium. In summary, ILAs isolated on the basis of a RRA for insulin is a slightly acidic peptide with some of the biological activities expected of a somatomedin.
Chondrocyte cultures were prepared from rabbit growth plate (GPC) and articular (ARC) chondrocytes. These two cell types have distinct morphological characteristics. The cells reached maximum numbers by days 10 and 21 for ARC and GPC, respectively. The proteoglycans (PG) contained in the cellular pool were extracted and purified by DEAE cellulose chromatography. The effect of a partially purified somatomedin peptide with insulin-like activity on [35S]sulphate incorporation into PG was evaluated. In both ARC and GPC a significant stimulation of [35S]sulphate uptake into PG subunits was obtained with 1 ng Eq./ml of somatomedin peptide. In order to obtain the same stimulatory effect with porcine insulin, a 1000-fold greater concentration was required. The electrophoretic patterns of the PG subunits on acrylamide-agarose electrophoresis were identical on control incubations and after stimulation with the somatomedin peptide. These data demonstrate in vitro biological activity of this peptide on well differentiated articular and epiphyseal growth plate chondrocytes in culture. These cultures appear to provide a sensitive biological assay for somatomedin peptides.
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The rationale of the specific-binding assay was applied to the detection of the liver insulin receptor in vivo. Quantitative electron microscope radioautography indicated that, 3 min after an intraportal injection, 125I-insulin was exclusively located to the hepatocyte plasmalemma.
The specific binding of 125I-labeled insulin, human hormone ([125I]hGH), bovine growth hormone ([125I]bGH), and ovine prolactin ([125I]oPRL) was studied in mouse liver membranes. [125I]hGH and [125I]oPRL bound to adult liver membranes. Pregnancy increased the specific binding of [125I]hGH but not that of [125I]oPRL. [125I]hGH was displaced from membranes of pregnant mice by hGH, oPRL, and bGH, but only by hGH and oPRL from liver membranes of nonpregnant mice. Significant specific binding of [125I]bGH was seen only in pregnancy. The binding of [125I]bGH to pregnant mouse liver membranes increased with increasing concentration of either membrane protein or [125I]bGH. Both the specific binding and dissociation of [125I]bGH were greatly influenced by the time and temperature of incubation. Binding of [125I]bGH was inhibited by growth hormones, including hGH and rat GH, and not by lactogenic hormones (various prolactins and human placental lactogen), ACTH, glucagon, or insulin. The inhibition of [125I]hGH binding by hGH and bGH, in the presence of excess (2 mug/ml) of PRL, was very similar to that seen with [125I]bGH. Scatchard plots of displacement dose-response curves obtained under steady state conditions of 4C were nonlinear and very similar with either [125I]bGH or [125I]hGH. This contrasted with the linear Scatchard plots obtained from displacement dose-response curves of either [125I]oPRL or [125I]hGH in the presence of excess (2 mug/ml) bGH. Termination of pregnancy, either naturally or by hysterectomy, reduced [125I]bGH specific binding to nonpregnant levels by 24 to 36 h. Estrogen administration did not increase [125I]bGH binding in hepatic membranes. Nonpregnant mice possess hepatic lactogen binding sites which are uninfluenced by pregnancy. GH specific binding sites are markedly augmented during pregnancy. The close correlation between the level of these sites and pregnancy suggests that they are regulated by a product of the fetoplacental unit.
Regulation of the lactogen specific binding sites of rat liver was studied in several different high lactogen states. The specific binding of [125I]iodo-hGH was determined as a measure of these sites. Hypophysectomy of pregnant rats produced a much smaller decrease in hepatic binding of [125I]iodo-hGH than was seen in nonpregnant females. Three different prolactin secreting tumors (MtT/F4, MtT/F45, MtT/W5) produced greatly elevated levels of both rPRL and hepatic binding of [125I]iodo-hGH in both male and female rats. The increased binding reflected an increase in the concentration of lactogen specific membrane binding sites. There was no change in the apparent affinity of binding. Hypophysectomy of tumor-bearing rats was not followed by a decrease in the concentration of hepatic sites. Pituitary transplants beneath the kidney capsules of hypophysectomized (hypox) rats produced elevated rPRL levels and increased [125I]iod-GH specific binding to hepatic membranes. The elevation of serum rPRL preceded the increase in hepatic binding. There was a direct correlation between rPRL levels attained by 8 days after implantation and the level of hepatic binding. Estrogen treatment did not alter this correlation. Hormonal replacement with prolactin in combination with various hormones failed to induce the lactogen specific hepatic sites in estrogen treated hypox males, and did not prevent the marked decrease in hepatic binding observed in females following hypophysectomy. It is suggested that chronic elevation of prolactin plays a role in inducing lactogen specific binding sites in rat liver. Estrogen's inductive action seems to be largely effected at the pituitary level.
A prolactin receptor, present in adult female rat liver, can be induced in males by estrogen. Hypophysectomy diminished receptor levels in the female and rendered males unresponsive to estrogen. A renal pituitary implant blunted the decrease in hypophysectomized females and induced the receptor in hyophysectomized males. The increased receptor level in hypophysectomized males with a renal pituitary implant was preceded by a sustained elevation of circulating prolactin. Our observations suggest that prolactin induces its own receptor.
Major developments in the area of polypeptide hormone receptors have been reviewed. Receptors are high affinity, high specificity binding sites which appear to be located largely, if not entirely, on the plasma membrane of cells. Receptors are proteins intimately associated with and influenced by lipids. Receptor sites and degrading sites appear to be readily distinguishable entities. The binding of hormone to receptor is distinct and has been dissociated from subsequent steps leading to hormonal response. There is no direct relationship between receptor occupancy and the magnitude of target response to hormone. So called 'spare' receptors can be viewed thermodynamically as enhancing target tissue sensitivity to hormone. The binding of hormone to receptor appears to be a point at which regulation of tissue sensitivity can be influenced either through altering the affinity for hormone or the number of receptors. One factor apparently involved in the regulation of receptor levels is the hormone itself. Receptors have been used to develop assay procedures which have significantly complemented the bioassay and radioimmunoassay. Finally, the measurement of receptor levels in disease has provided new insights into pathophysiology.
We have previously shown that specific binding sites for lactogenic hormones are present at much greater levels in the liver membranes of female than of male rats. In the present studies [125I]iodo-hGH was used to study binding sites specific for lactogenic hormones in liver membranes. In male rats, a single injection of 2 mg estradiol valerate induced these binding sites. The induction was maximal by 9-12 days and was dose-dependent. Ovariectomy significantly reduced the specific binding of [125I]iodo-hGH from 9.7 +/- 0.7% in shamoperated to 6.9 +/- 0.3% in experimental rats (P less than 0.01) without a change in affinity. Fluctuations in specific binding of [125I]iodo-hGH were observed at different stages of the estrous cycle. Binding at estrus and diestrus I was significantly greater than at diestrus II and proestrus (P less than 0.05). The disappearance of binding sites following hypophysectomy was rapid, declining from 13.2 +/- 1.2% in intact rats to 6.0 +/- 0.8% and 2.2 +/- 0.4% 14 and 48 h, respectively, after surgery. In contrast, binding of insulin was slightly increased after hypophysectomy. Anti-estrogens (clomiphene, ICI 46,474, and nafoxidine) prevented the induction of binding sites in male rats given estradiol (E2). A single injection of 200 mug cycloheximide 11 days after an injection of 2 mg E2-valerate reduced binding by more than 90% in 3 h with a return to control levels by 48 h. The maximal decline in insulin binding was 54% during this entire period. These studies suggest that endogenous estrogen plays a role in regulating hepatic binding sites for lactogenic hormones. The level of these binding sites is critically dependent on the presence of an intact pituitary. The possible rapid turnover of these sites suggests that regulatory influences at the tissue level may have an important role in modulating hormone action.
A receptor exists in female rat liver with high specificity for lactogenic hormones. Previous work showed the receptor level increased at the time of puberty in female but not male animals. Pregnancy caused a further substantial increase. Here we show that estrone (50 mug/day) administration to male rats induced a 10- to 30-fold increase in specific binding of ovine prolactin and human growth hormone after 8-12 days with a significant increase first seen after 4 but not 2 days of injection. In females, this regimen increased binding to pregnancy levels. In prepuberal (20-days-old) male and female rats, estrone was also markedly stimulatory. The binding sites for ovine prolactin and human growth hormone were of high affinity in liver membranes from both female and estrone-treated male rats (K(a) = 0.6 to 1.4 x 10(9) M(-1)). Estrone and estradiol were equally effective in inducing the lactogenic receptor. Estriol (50 mug/day), progesterone (500 mug/day), human placental lactogen (1 mg/day), and testosterone (100 mug/day) were without influence. Hypophysectomy drastically decreased the levels of lactogenic receptor in mature female rats, and estrogen treatment failed to restore receptor levels to normal. Hypophysectomized male rats were also unresponsive to estrogen. Throughout these studies the specific binding of (125)I-labeled insulin remained relatively constant. This work demonstrates estrogen induction of a lactogenic receptor. The pituitary gland appears to have a critical, though presently undefined, role in the induction process.
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