somatomedin-C Receptors Research Articles

Association of Acromegaly and Multiple Myeloma: A Case Report

Abs tract Malignancy is an important cause of mortality in acromegaly. Hematological malignancies are very rare in acromegaly. Here, we report an 80-yearold patient with acromegaly and multiple myeloma. Patient died within a month of diagnosis. Previous studies have shown that growth hormone and somatomedin-C activate B lymphocyte and somatomedin-C receptors are found in multiple myeloma cells. Possible effects of growth hormone and somatomedin-C on multiple myeloma progression are discussed in the light of the relevant literature. Turk Jem 2013; 17: 75-8

Insulin, somatomedin-C, human chorionic gonadotropin, and forskolin enhance glucose oxidation by granulosa cells.

The long exposure times required to observe stimulatory effects of insulin on steroidogenesis and protein synthesis in granulosa cells suggested that these effects might be secondary to stimulation of another metabolic process. The present studies examined the effects of insulin, the insulin-like growth factor somatomedin-C (Sm-C), human chorionic gonadotropin (hCG), and forskolin, a compound that activates adenylyl cyclase independently of a receptor, on glucose metabolism. Granulosa cells from preovulatory porcine ovarian follicles were incubated at 37 degrees C in Dulbecco's phosphate-buffered saline supplemented with bovine serum albumin, vitamins, amino acids, and glucose (0.01-20 mM). Cells were incubated with [14C]glucose for up to 23 h with or without a prior 20-h preincubation. Oxidation of glucose, assessed by quantitation of 14CO2 produced, was dependent on time and concentration of glucose. Optimal glucose concentrations for glucose oxidation were 3 mM in the absence or presence of insulin and correlated well with the measured glucose concentrations in follicular fluid (3 mM). After a 20-h preincubation in the absence or presence of insulin (1 microM), the rates of CO2 production were 10.6 and 21.6 pmol/micrograms DNA/h for control and insulin-treated cells, respectively. Insulin had an EC50 of 164 nM. Sm-C and hCG were more potent stimulators than insulin with EC50s of 768 pM and 161 pM, respectively. The greater sensitivity of granulosa cells to Sm-C than to insulin supports the concept that insulin exerts its effect via reactivity with the Sm-C receptor. The effect of hCG may have been mediated by cyclic adenosine 3',5'-monophosphate (cAMP), since forskolin also enhanced 14CO2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of Leydig cell functions by culture with Sertoli cells or with Sertoli cell-conditioned medium: effect of insulin, somatomedin-C and FSH

The potential regulatory action of Sertoli cells on Leydig cell functions has been investigated by using a coculture system of Leydig and Sertoli cells obtained from immature pig or by culturing Leydig cells with Sertoli cell-conditioned medium (SCCM). Coculture of Leydig and Sertoli cells for 48 h in the absence of insulin or somatomedin-C (Sm-C), produced a small but significant increase in both hCG receptors and hCG-induced testosterone production. Addition to the medium of pFSH (100 ng/ml), insulin (5 μg/ml) or somatomedin-C (50 ng/ml) produced a marked increase in these two parameters of Leydig cell function. A further significant increase was observed when pFSH was associated with insulin or Sm-C. In contrast, coculture of Leydig cells with aortic endothelial cells decreased both the hCG receptor number and the hCG responsiveness. SCCM stimulated Leydig cell testosterone production following a 4 h incubation. The stimulation depended upon the amount of SCCM used and the conditions in which Sertoli cells were cultured. The most active was the medium from cells cultured in the presence of pFSH and insulin at high concentrations. Since pig Sertoli cells have specific somatomedin-C receptors, but not insulin receptors, it is likely that the effect of micromolar concentrations of insulin are exerted through Sm-C receptors. In addition, SCCM produced a long-term effect after 48 h incubation. SCCM from cells cultured in the absence of insulin and pFSH inhibited both the hCG receptor number and hCG responsiveness. A similar inhibition was observed with SCCM medium from cells cultured without insulin but with pFSH. However, in this case, the inhibition was overcome by Sm-C. SCCM from cells treated with insulin and pFSH had a slight chronic effect but much less so than in the coculture system. Conditioned medium from aortic endothelial cells was always inhibitory. These results suggest that Sertoli cell secretory products have two effects on Leydig cells: acute stimulation of steroidogenesis and long-term trophic effects the nature of which depends upon the in vitro model used and the hormonal supplementation of the culture medium. Whether the factors responsible for the acute and the chronic effect are identical remains to be investigated.

Glucocorticoids enhance somatomedin-C binding and stimulation of amino acid uptake in human fibroblasts.

The regulation of growth by the GH-dependent mitogen somatomedin-C (Sm-C) may involve not only changes in circulating levels of Sm-C, but also alterations in cellular sensitivity to Sm-C induced by humoral factors. Glucocorticoids have been reported to enhance the stimulatory effect of Sm-C on DNA synthesis and cell replication in cultured human fibroblasts, but the cellular alteration responsible for this effect was not fully defined. Using an assay for cellular sensitivity to Sm-C based on stimulation of uptake of the amino acid analog aminoisobutyric acid, a 20-h preincubation with 100 nM dexamethasone was found to enhance both the sensitivity and the maximal responsiveness of human fibroblasts to a 3-h incubation with Sm-C. This effect was found using fibroblasts from multiple normal donors of different ages, and dexamethasone was approximately 10-fold more potent than hydrocortisone. The glucocorticoid antagonist RU486 (100 nM) largely reversed the enhancing effect of 100 nM dexamethasone on Sm-C action. Binding of [125I]Sm-C to intact fibroblast monolayers or trypsin-dispersed cells could be increased by 60-80% by glucocorticoid preincubation, and this increase correlated well with enhanced stimulation of [3H]aminoisobutyric acid uptake, suggesting that the enhancement of Sm-C action in glucocorticoid-treated cells may be mediated at the level of the Sm-C receptor.

Somatomedin-C substitutes for insulin for the growth of mammary epithelial cells from normal virgin mice in serum-free collagen gel cell culture.

We investigated the effect of somatomedin C (SM-C) on the growth of mouse mammary ductal epithelial cells in collagen gel culture. Epithelial cells, isolated by collagenase digestion of whole glands, were placed into primary serum-free collagen gel cell culture for 10-12 days, during which SM-C was added alone or in combination with other growth-promoting factors. Previous work has shown that these cells require a superphysiological concentration of insulin (10 micrograms/ml) for optimum growth in serum-free medium (a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's medium) containing epidermal growth factor (EGF). When SM-C (1-250 ng/ml) alone was added to serum-free basal medium containing EGF, it stimulated growth (at concentrations greater than 25 ng/ml) to at least the same extent as insulin at 10 micrograms/ml. There was no additive stimulation of growth when optimal concentrations of insulin and SM-C were added together. The nonadditive stimulation at optimal concentrations of these hormones may indicate that the previous requirement for a superphysiological concentration of insulin for maximum growth was due to low affinity binding of insulin to the SM-C receptor. Rat insulin-like growth factor II (Collaborative Research) at 50-200 ng/ml did not stimulate growth in the presence or absence of insulin. SM-C could not stimulate growth alone. The presence of EGF or mammogenic hormones (progesterone and PRL) was required.

SOMATOMEDIN-C (Sm-C) RECEPTORS IN CULTURED PIG LEYDIG CELLS (LC): CHARACTERIZATION AND REGULATION. ROLE OF Sm-C ON LC FUNCTIONS

Using affinity cross-linking we have shown the presence of Sm-C receptors (Cm-C-R) in cultured pig LC. The Sm-C-R were further characterized by competition binding (KD 1.10−9 M). When LC were cultured in a chemically defined medium without hormones, the number of Sm-C-R varied from 10000 to 15000 sites/cell. Treatment of LC with hCG induced a 2 to 3 fold increase in Sm-C-R. This effect was time-(maximum effect after 48 h) and dose-dependent (ED50 = 10−10 M). Moreover LC treatment with Sm-C for 3 days induced an increase of hCG receptors (4-6 fold) and of hCG responsiveness (cAMP and testosterone production) (8-10 fold) with ED50 of about 2 ng/dl. In addition, Sm-C stimulated LC DNA synthesis (ED50 ≃ 10 ng/ml). These results show : 1) LC contain specific Sm-C-R which are positively regulated by hCG; 2) Sm-C increased both hCG receptors number and hCG steroidogenic responsiveness of LC. Since Sertoli cells secrete Sm-C (see Chatelain et al) our data strongly suggest that Sm-C might play a role in the paracrine regulation of LC. Therefore Sm-C could play a key role on the delayed puberty observed in children with isolated growth hormone deficiency.

Interaction of the Monoclonal AntibodiesαlR-1 andαIR-3 with Insulin and Somatomedin-C Receptors

alpha IR-3, a monoclonal antibody that interacts with the somatomedin-C receptor, inhibited the binding of somatomedin-C, but not of insulin, to human placental membranes and intact IM-9 cells. alpha IR-1, a monoclonal antibody that interacts with the insulin receptor, did not inhibit the binding of either hormone. Inhibition of somatomedin-C binding by alpha IR-3 was mainly due to a decrease in its affinity. 125I-Labeled alpha IR-3 bound specifically to placental membranes and intact IM-9 cells and was inhibited by concentrations of unlabeled alpha IR-3 that were lower than those required to inhibit somatomedin-C binding. [125I]alpha IR-3 binding was also inhibited by somatomedin-C and insulin, but only at very high concentrations. A410, a rabbit antiserum that reacts with both receptors for insulin and somatomedin-C, also inhibited labeled alpha IR-3 binding. alpha IR-I did not. These results help to define the epitopes with which these antibodies interact.

Somatomedin C-binding and action in fibroblasts from aged and progeric subjects.

Aging is associated with diminished cell growth, which has been ascribed in part to decreased cellular responsiveness to serum mitogens. To investigate whether there is an age-related loss of responsiveness to somatomedin-C (SM-C), we studied SM-C binding and action in early passage fibroblasts from normal donors, aged 7-96 yr, and one progeric subject. SM-C stimulated [3H]thymidine incorporation 4- to 16-fold in young cells, 4- to 17-fold in aged cells, and 4- to 11-fold in progeric cells. SM-C was synergistic with 0.25% human hypopituitary serum in stimulating [3H]thymidine incorporation in all cell lines. Dose-response curves for SM-C stimulation of thymidine incorporation were not significantly altered in aged or progeric cells. Half-maximal responses occurred at 5-15 ng/ml SM-C for all cell lines. [3H]Thymidine incorporation results were supported by cell replication studies. In addition, binding of [125I] SM-C was virtually identical in all cell lines, with 50% displacement at 2-5 ng/ml SM-C. Thus, in vivo aging does not appear to be associated with either an alteration in SM-C receptors or a diminished cellular responsiveness to SM-C's mitogenic effects.

Interaction of circulating cell-derived and plasma growth factors in stimulating cultured smooth muscle cell replication.

Multiple growth factors that circulate in plasma have been shown to stimulate cellular growth in vitro. The plasma growth factors appear to stimulate DNA synthesis in cultured fibroblasts only after prior exposure of cell growth factors derived from circulating cell types, such as platelets and macrophages. The purpose of these studies was to investigate the role of the plasma growth factors in stimulating smooth muscle cell replication following exposure to platelet-derived growth factor (PDGF). Following transient exposure to PDGF, insulin stimulated smooth muscle cell replication but only when supraphysiologic concentrations were used (i.e., greater than 1.0 micrograms/ml). Somatomedin-C (Sm-C), in contrast, was found to stimulate a 320% increase in [3H]thymidine incorporation when concentrations that are present in extracellular fluids were used (i.e., 0.5-10 ng/ml). Epidermal growth factor (EGF), an important mitogen for multiple cell types, caused a 70% increase in [3H]thymidine incorporation when added to quiescent cells following PDGF exposure, and EGF caused a substantial increase in the absolute level of [3H]thymidine incorporation when coincubated with Sm-C. When EGF (1 ng/ml) was incubated simultaneously with concentrations of Sm-C between 1 and 10 ng/ml plus Sm-C-deficient plasma, maximal [3H]thymidine incorporation was 2.1-fold greater in the presence of EGF. In contrast, insulin (20 ng/ml), when coincubated with Sm-C under similar conditions, had no enhancing effect on the cellular response to Sm-C. None of the plasma factors tested was an effective stimultant of replication when incubated either in serum-free medium or in the presence of Sm-C-deficient plasma without prior PDGF exposure. Hydrocortisone was shown to inhibit smooth muscle cell replication in concentrations between 10(-7) and 10(-5) M. In summary, multiple plasma growth factors can stimulate the smooth muscle cell replication, and Sm-C appears to be most effective of those tested. Insulin and EGF appear to work by different mechanisms; that is, EGF can facilitate the cellular response to Sm-C, whereas insulin is effective only at supraphysiologic concentrations at which it will directly bind to Sm-C receptors.

Inhibition of the mitogenic effects of plasma by a monoclonal antibody to somatomedin C.

Immunoneutralization studies with a monoclonal antibody to somatomedin C (Sm-C) were undertaken to further determine the role of this peptide in cellular proliferation. For our model we used density-arrested cultures of BALB/c 3T3 cells. Transient exposure of these cells to platelet-derived growth factor enables them to respond to platelet-poor plasma by progressing through the G1 stage and undergoing renewed DNA synthesis. In this system, the combination of nanogram concentrations of Sm-C and epidermal growth factor can fully substitute for plasma, and microgram concentrations of insulin can substitute for Sm-C by crossreacting with the Sm-C receptor. We now show that a monoclonal antibody to Sm-C, which in defined medium blocks the mitogenic effect of Sm-C but not insulin, also blocks the stimulation of DNA synthesis by human plasma or calf serum. Furthermore, by adding the antibody at progressively later times during G1, we show that these cells escape from their dependence on Sm-C for DNA synthesis after traversing G1 to a point at or near the G1/S boundary.

Affinity-labeled somatomedin-C receptors and binding proteins from the human fetus.

We investigated the ontogeny of the human placental membrane somatomedin-C (Sm-C)/insulin-like growth factor I (IGF-I) receptor by affinity labeling with the cross-linking agent disuccinimidyl suberate (DSS). Specific Sm-C receptors, identified from as early as 6 weeks gestation, demonstrated no apparent structural changes through the course of gestation. Second trimester human fetal brain membranes cross-linked to [125I]Sm-C exhibited an identical pattern of receptor binding. These findings in both placenta and fetal brain membrane are consistent with the proposed heterotetrameric structure of the Sm-C/IGF-I receptor. Using a similar DSS cross-linking technique, we identified Sm-C-binding proteins in second trimester amniotic fluid [apparent molecular mass (Mr), 38,000 and 35,000] and term cord plasma (Mr, 41,000, 38,000, and 35,000). Identically sized binding components (Mr, 35,000-45,000) were also found in membrane preparations of preterm placenta and brain after cross-linking. Evidence that these binding species represent contamination of preterm membrane preparations with soluble amniotic fluid and/or fetal plasma Sm-C-binding proteins and that they are not derived from membrane receptors is as follows: (1) these binding species, like amniotic fluid and cord plasma binding proteins, were insensitive to competition with insulin (in concentrations as high as 0.6 mg/ml), a characteristic not shared with membrane receptor binding components; (2) these binding species were largely removed from placental membranes by extensive washing and appeared in the supernate when membrane preparations were incubated at 4 C for 18 h, indicating that they were soluble and not an integral part of the particulate membrane; (3) limited proteolysis of placental membrane preparations did not result in the appearance of similar binding species; and (4) preparation of preterm placental membranes in the presence of protease inhibitors did not eliminate these binding species. Use of traditional methodology to study binding of somatomedin to receptors in membranes prepared from preterm human tissues may be misleading because of contamination by amniotic fluid and/or plasma-derived binding proteins.

The somatomedin-C receptor of human placenta

The binding and physical characteristics of the human placenta somatomedin-C (Sm-C) receptor were investigated and compared with those of the placental insulin receptor. These receptors were found to have many similar properties, but several distinctions exist between them. The two receptors have similar binding properties. Although distinct receptors, as evidenced by specificity studies, the Sm-C and insulin receptors possess similar affinities (10 × 10 9 L/mol and 3 × 10 9 L/mol, respectively) and receptor concentrations (0.4 pm/mg and 0.2 pm/mg) in placental membranes. There is also a low-affinity, high-capacity receptor for each hormone. The intact receptors are indistinguishable in size by three methods. The Stokes radius is 7.2 nm by gel filtration, the sedimentation coefficient is 11S, and the SDS polyacrylamide gel apparent molecular weight is approximately 350,000 daltons. The two receptors differ in their pH optimum for ligand binding, their isoelectric points, and their subunit size. The pH optimum for Sm-C binding is 8.2, and for insulin binding the optimum is 7.9. The isoelectric point for the Sm-C receptor is 4.6, whereas the isoelectric point for the insulin receptor is 4.2. Although the whole receptor and the half receptors for Sm-C and insulin cannot be distinguished by size on SDS gels, the binding subunit for the Sm-C receptor appears to be 8000 daltons smaller than that of the insulin receptor (134,000 v 142,000 daltons). We conclude from these studies that the insulin and Sm-C receptors of human placenta are similar in structure but are distinct entities.

Size discrepancy between somatomedin-C and insulin receptors.

Somatomedin-C (Sm-C) and insulin receptors have similar size and structure. Each receptor is a heterotetramer composed two alpha- and two beta-subunits. Sodium dodecyl sulfate (SDS)-polyacrylamide gel autoradiographic studies of affinity labeled receptor preparations demonstrated that each has a whole receptor of greater than 350,000 daltons, a half-receptor of 220,000 daltons, and binding subunit (alpha-subunit) of about 140,000 daltons. Using SDS-polyacrylamide disc gels and double labeling techniques, we demonstrated that the 125I affinity labeled alpha-subunit of the Sm-C receptor from human placenta was 8,000 daltons smaller than the 131I affinity labeled insulin receptor alpha-subunit. Further double label studies demonstrated that [125I]insulin and [131I]insulin cross-linked to placental insulin receptors precisely comigrated on SDS-disc gels, indicating that the difference between insulin and Sm-C alpha-subunits is not an artifact of the system. The difference in ligand size (Sm-C, 7,500 daltons; insulin, 5,700 daltons) would minimize the observed difference and is clearly not the cause of the difference in size observed. These studies provide further evidence in support of two functionally and physically distinct receptor molecules for insulin and Sm-C in human placenta.

Somatomedin-C stimulates the phosphorylation of the beta-subunit of its own receptor.

Phosphorylation of the somatomedin-C receptor was investigated both in intact IM-9 cells and in IM-9 cells that had been solubilized with Triton X-100. Intact IM-9 cells were incubated with [32P]H3PO4 for 1 h and for an additional 5 min in the absence or presence of insulin or somatomedin-C. The cells were then solubilized and subjected to wheat germ agglutinin Sepharose chromatography. The extent of phosphorylation of insulin and somatomedin-C receptors was assessed by immunoprecipitating the wheat germ agglutinin Sepharose eluates with monoclonal antibodies specific for each receptor and analyzing the immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The beta-subunits of both receptors were phosphorylated in the absence of hormone, and the extent of phosphorylation of each receptor was enhanced by both hormones. However, each hormone was more potent than the other in enhancing phosphorylation of its own receptor. The beta-subunit of the somatomedin-C receptor was also phosphorylated when solubilized IM-9 cells that had been purified on wheat germ agglutinin Sepharose were incubated with [gamma-32P]ATP. In this soluble preparation, phosphorylation occurred on tyrosyl residues and was enhanced by concentrations of somatomedin-C in the range of 2.5 to 250 ng/ml, which is consistent with its receptor affinity. Tyrosyl phosphorylation of the somatomedin-C receptor also occurred when highly purified receptor, prepared by wheat germ agglutinin Sepharose affinity chromatography followed by immunoprecipitation, was incubated with [gamma-32P]ATP. This indicates that the responsible tyrosyl kinase activity is intrinsic to the receptor or tightly associated with it.

Characterization of a specific somatomedin-c receptor on isolated bovine growth plate chondrocytes.

The interaction of somatomedin (Sm) with growth plate chondrocytes (GPCs) is believed to be the primary stimulus of skeletal growth. Using techniques designed to disrupt as little as possible the phenotypic characteristics of GPCs, we have been able to obtain 3-4 x 10(8) viable cells from the major physes of one newborn calf. The availability of these cells plus essentially pure Sm-C/insulin-like growth factor I, the most GH-dependent Sm, has now made possible detailed studies of the interaction of this radiolabeled peptide with the GPC receptor and of the subsequent processing of this hormone by these cells. The enzymatic methods required to free GPCs from their matrix led to loss of receptors, followed by rapid receptor regeneration by de novo synthesis in suspension cultures. Binding of [125I]iodo-Sm-C to GPCs was time dependent and saturable, with optima at 15 C and pH 7.8. At 37 C, binding peaked at 90 min and declined thereafter. Multiplication-stimulating activity, insulin, and nerve growth factor were less potent than unlabeled Sm-C in competition with [125I]iodo-Sm-C for its receptor. Human GH, epidermal growth factor, and fibroblast growth factor failed to show competition even at 10(-6) M. Analysis of the fate of [125I]iodo-Sm-C bound to GPCs at 37 C provided evidence that this hormone is internalized and extruded from the cell in a partially degraded form. Scatchard analysis of [125I]iodo-Sm-C binding to GPCs and to chondrocytes isolated from articular cartilage revealed similar Ka values, but reproducibly 2-6 times more receptors on growth plate than on articular chondrocytes.

Monoclonal antibodies to receptors for insulin and somatomedin-C.

Three monoclonal antibodies, designated alpha IR-1, alpha IR-2, and alpha IR-3, were prepared by fusing FO myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of insulin receptors from human placenta. These antibodies were characterized by their ability to immunoprecipitate solubilized receptors labeled with 125I-insulin or 125I-somatomedin-C in the presence or absence of various concentrations of unlabeled insulin or somatomedin-C. alpha IR-1 preferentially immunoprecipitates insulin receptors and also less effectively immunoprecipitates somatomedin-C receptors, while alpha IR-2 and alph IR-3 preferentially immunoprecipitate somatomedin-C receptors, but may also weakly immunoprecipitate insulin receptors. These three monoclonal antibodies, as well as A410, a rabbit polyclonal antibody, were used to immunoprecipitate insulin and somatomedin-C receptors from solubilized human lymphoid (IM-9) cells and human placenta membranes that had been 125I-labeled with lactoperoxidase. Analysis of the immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both receptors are composed of alpha and beta subunits. The beta subunit of the insulin receptor (immunoprecipitated by alpha IR-1 and A410) has a slightly more rapid mobility than the corresponding subunit of the somatomedin-C receptor (immunoprecipitated by alpha IR-2 and alpha IR-3). Interestingly, the alpha subunit of the placenta somatomedin-C receptor has a slightly faster mobility than its counterpart from IM-9 cells. Immunoprecipitation of receptor that had been reduced and denatured to generate isolated subunits indicates that alpha IR-2 and alpha IR-3 interact with the alpha subunit of the somatomedin-C receptor while A410 interacts with both subunits of the insulin receptor. alpha IR-1 failed to react with reduced and denatured receptors.

Plasma somatomedin-C in fasted and refed rats: close relationship with changes in liver somatogenic but not lactogenic binding sites.

Circulating levels of somatomedins in man and rats are reduced by fasting and restored by refeeding. To determine the mechanisms for these alterations in somatomedin levels, the affinities and binding capacities of 125I-labelled bovine GH (somatogenic binding sites) and of 125I-labelled ovine prolactin (lactogenic binding sites) were assessed in liver homogenates from fasted and refed rats. Correlations were made with plasma immunoreactive somatomedin-C (Sm-C) and plasma insulin. During fasting and refeeding there was a close temporal relationship between the fall and the rise of plasma levels of Sm-C and insulin, and the number of hepatic GH binding sites. After fasting for 1 day, plasma Sm-C dropped by 68% and plasma insulin by 76% when compared with values before fasting. At the same time, GH binding capacity was significantly reduced (7·3 ± 2·8 (s.e.m.) pmol/liver v. controls, 20·3 ± 2·1 pmol/liver; P < 0·01). Refeeding for 24 h normalized plasma insulin levels and restored GH binding capacity to values before fasting (13·2 ± 2·4 v. 20·3 ± 2·1 pmol/liver; P > 0·05). Plasma Sm-C rose significantly with refeeding and returned to initial values at day 4 of refeeding (0·82 ± 0·10 v. 0·77 ± 0·07 units/ml; P > 0·05). In contrast, changes in prolactin binding sites correlated poorly with changes in plasma Sm-C. There was a modest decline with fasting significant only after 72 h (6·8 ± 0·6 v. 13·9 ± 3·2 pmol/liver; P < 0·05), and refeeding for 24 h did not restore prolactin binding (5·7 ± 1·2 pmol/liver). Since a number of reports suggest that fasting induces a state of tissue insensitivity to GH, our findings suggest that the reduction in hepatic GH binding capacity might be a mechanism for the fasting-induced reduction in Sm-C. The reduction in plasma insulin, which accompanies fasting, might play a permissive role in the intracellular metabolic events involved in Sm-C and GH receptor regulation.

Monensin blocks the maturation of receptors for insulin and somatomedin C: identification of receptor precursors.

Cultured human lymphoid (IM-9) cells were labeled with [(35)S]methionine in the presence and absence of monensin, a carboxylic ionophore that inhibits post-translational protein maturation. Labeled receptors for insulin and somatomedin C were immunoprecipitated with antibodies specific for each receptor. Monensin inhibits the biosynthesis of mature alpha and beta subunits of both receptors and leads to the accumulation of immunoreactive polypeptides with molecular weights of 180,000. These 180,000 molecular weight polypeptides exist as disulfide-linked dimers and may be biosynthetic precursors of both alpha and beta subunits. In the presence of monensin, small amounts of immunoreactive polypeptides with molecular weights 115,000 and 89,000 also are produced. These may be abnormally processed forms of the alpha and beta subunits lacking residues normally added during terminal glycosylation. In cells treated with monensin, the polypeptides of molecular weights 180,000 and 115,000 can be affinity-labeled with (125)I-labeled insulin. These labeled polypeptides are immunoprecipitated by antibodies specific for insulin receptors but not by antibodies specific for somatomedin-C receptors. This indicates that the putative precursors for insulin and somatomedin-C receptors are distinct polypeptides, although they have similar molecular weights and similar modes of processing. A possible structural relationship between the precursors for these receptors and the type II insulin-like growth factor receptor is discussed.

Release of somatostatin-like immunoreactivity from incubated rat hypothalamus and cerebral cortex. Effects of glucose and glucoregulatory hormones.

Somatostatin (SRIF) is localized in the hypothalamus, extrahypothalamic brain, and throughout the gastrointestinal tract. Release of gastrointestinal SRIF-like immunoreactivity (SRIF-LI) is under nutrient regulation but the effect of nutrients on neural SRIF-LI is unknown. The present studies examined the effects of glucose uptake and metabolism and hormones influencing glucose disposition on SRIF-LI release from medial basal hypothalamus (MBH) and cerebral cortex (Cx) incubated in Krebs-Ringer bicarbonate containing bacitracin. After a preincubation to achieve stable secretion, tissues were incubated for 20 min in 14 mM glucose (basal) and then, for 20 min in fresh medium with test materials. MBH SRIF-LI release was inversely related to medium glucose concentration with release in the absence of glucose (235+/-42 pg/MBH per 20 min) more than five times that in the presence of 25 mM glucose (46+/-4 pg/20 min). In the presence of 14 mM glucose MBH SRIF-LI release was stimulated above basal by agents interfering with glucose uptake including 3-O-methyl-d-glucose (42 mM; 70+/-5 vs. 42+/-3 pg/20 min, P < 0.05), phlorizin (50 mM; 351+/-63 vs. 29+/-2 pg/20 min, P < 0.001) or cytochalasin B (20 muM; 110+/-7 vs. 22+/-2 pg/20 min, P < 0.001). Inhibition of glucose metabolism by 2-deoxy-d-glucose resulted in dose-related stimulation of MBH SRIF-LI release (maximal at 28 mM; 201+/-28 pg/20 min vs. 32+/-4 pg/20 min, P < 0.001). Viability of MBH was unimpaired by incubation in the absence of glucose or following exposure to 2-deoxy-d-glucose as determined by retention of SRIF-LI responsiveness to stimulation by potassium (60 mM) or neurotensin (5 muM). In contrast, Cx SRIF-LI release was slightly inhibited by decreases in medium glucose and unaffected by inhibition of glucose uptake or metabolism. These results provide evidence for nutrient regulation of MBH but not Cx SRIF-LI release and may explain inhibition of growth hormone seen in the rat in response to hypoglycemia. Insulin (10 nM-1 muM) stimulated MBH but not Cx SRIF-LI release while glucagon was without effect. Our previous demonstration that MBH SRIF-LI release was stimulated by somatomedin-C, but not insulin at physiologic concentrations, is consistent with an action of insulin through the somatomedin-C receptor at the doses studied. Our studies indicate a regional specificity for the control of SRIF secretion within the brain and suggests the possibility of a role for hypothalamic SRIF in metabolic regulation.

Structural Similarity of Human and Bovine Somatomedin Receptors and Human Insulin Receptor: Analysis by Affinity Labeling

The growth promoting and insulin-like actions of somatomedin-C (Sm-C) are thought to be mediated respectively by interactions with the Sm-C and insulin (In) receptors. Since Sm-C and In are structurally homologous, we compared the size and subunit structure of their respective radiolabeled receptors in human placental membranes (hPM) and bovine chondrocytes (bC) by crosslinking with disuccinimidyl suberate (DSS). The labeled hormone-receptor complexes (RCs) were analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and autoradiography. Specificity of the respective RCs was demonstrated by competitive inhibition studies. Both hPM RCs had apparent molecular weights of > 250,000 daltons. Reduction with 2-mercaptoethanol yielded subunits of ~ 140,000 daltons. Limited proteolysis with trypsin, chymotrypsin and staphylococcal V-8 protease of the labeled RCs gave similar products. The Sm-C RC In bC isolated from calf epiphyseal plates also contained a 140,000 dalton binding subunit indistinguishable from the HPM receptor. Conclusions: 1) the hPM receptor for Sm-C has a 140,000 MW binding subunit linked by disulfide bonds. 2) By this technique, the hPM Sm-C receptor is structurally indistinguishable from either the Sm-C receptor in bC or the In receptor in hPMs, 3) The sizes of In and Sm-C receptors and their subunits differ from that reported for MSA and certain other hormones.