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.     

Regulation of hepatic growth hormone receptors by insulin.

Induction of diabetes in the rat with streptozotocin caused a decrease in the specific binding of human growth hormone to liver receptors. The decrease was due to a loss of binding sites, with no change in the affinity constant for growth hormone (5.6 × 10⁹M^?1). A highly significant correlation was seen between serum insulin levels and hepatic growth hormone binding. Specific insulin binding to hepatic receptors was increased in diabetes, with a highly significant negative correlation between serum insulin levels and insulin binding. The loss of growth hormone receptors was reversed by treating diabetic rats with insulin. Since hormones which bind to “lactogenic” binding sites in the liver are reported to regulate somatomedin levels, the insulin dependence of human growth hormone receptors might account for the decrease in serum somatomedin in diabetes.     

Differences in hepatic growth hormone receptor binding during development of normal and dwarf chickens

The aim of this study was to compare the growth hormone (GH) receptor in liver microsomal fractions of normal chickens (Dw) and chickens carrying the dwarf gene (dw). Specific binding of GH to its hepatic receptor was significantly higher for Dw embryos from d 14 till d 20 of incubation than for dw embryos. The difference in binding was due to a decreased binding capacity but not affinity in the livers of the dwarf embryos. The same binding pattern was found in livers of adult chickens: lower binding was again caused by a lower number of GH receptors and at this stage the difference was even clearer than during embryonic development. Binding studies on livers of growing chicks demonstrated that binding was low for both genotypes, but a small though significant difference between them remained. The cause of this decrease in number of GH receptors in dwarf birds has yet to be determined but may be due to the primary action of the dwarf gene.     

The defect in insulin receptors in obese-hyperglycemic mice: a probable accompaniment of more generalized alterations in membrane glycoproteins.

Although the liver plasma membranes of obese-hyperglycemic (ob/ob and db/db) mice bind less insulin than the membranes from thin litter mates, nearly equally depressed binding is observed for the general plasma membrane markers, concanavalin A and wheat germ agglutinin. Glucagon binding is virtually unchanged, and adenylate cyclase activity from obese mice is more sensitive to glucagon. Isolated kidney cells from obese mice have reduced insulin binding, but the decrease in WGA and con A binding is even more profound. Cultured (48 to 72 hours) spleen lymphocytes also have grossly reduced WGA and con A binding. The apparent change in insulin binding in obese animals may be but a minor part of generalized alterations in membrane glycoproteins. Factors complicating comparisons of hormone binding in different metabolic states are considered.     

Decreased binding of insulin to liver plasma membrane receptors in hereditary diabetic mice.

The interaction of insulin with its receptors was studied in liver plasma membranes of the young non-obese hereditary diabetic mouse (KK strain). Under identical conditions of preparation and incubation, the membranes of the KK mouse bind only 55-70% as much insulin per mg of protein as those of the control mouse (Swiss albino). Scatchard analysis suggests that this decrease in binding is due to a decrease in the number of receptor sites in the membrane of the diabetic mouse. However, the membranes of diabetic and control mice do not exhibit significant differences in hexosamine and sialic acid contents, enzyme activities, and protein and glycoprotein analysis. The decrease in insulin receptors in the KK mouse seems to correlate with the insulin resistance which they exhibit.     

Ontogeny of ovine fetal liver and kidney plasma membrane insulin receptors and fetal growth

This investigation was performed to define certain characteristics of insulin-receptor interaction during the last 2 months of gestation in fetal sheep liver and kidney. Twenty-one sheep carrying a total of 46 fetuses were sacrificed at various gestational ages from 94 days to term; fetal and maternal livers and kidneys were analyzed by a radioreceptor assay for insulin binding characteristics. Specific binding of insulin to partially purified ovine fetal liver and kidney plasma membranes increased as gestation approached term, at which time specific binding was two- to fourfold greater to fetal than to maternal tissues. Associated with increased specific binding were late gestational increases in affinity of insulin for receptors in both fetal liver and kidney and an earlier increase in insulin receptor concentration in fetal kidney. These observations in fetal sheep liver and kidney are similar to reported observations in other species. However, the increase in specific binding of insulin to male fetal liver membranes was exponential; in contrast, there was no apparent increase in specific binding to female fetal liver membranes during the gestational interval surveyed. Both the weights and the vertebral column lengths of these fetuses were shown by multivariate analysis to be significantly affected by the interaction between specific binding of insulin and fetal sex. However, in 30 additional sheep fetuses we observed no difference between male and female fetuses in the increase with time in liver glycogen content. The lack of sex difference in this postreceptor event is consonant with the demonstrated dissociation between liver insulin receptors and glycogen synthesis in the late fetal rat. Our observations suggest that late gestational differences between male and female sheep fetuses in insulin specific binding to liver and, possibly, to other tissues such as cartilage, muscle, and/or fat, that are coupled to postreceptor events may account for differences in fetal growth between the sexes.     

Tissue-specific expression and dietary regulation of a chimeric phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice

A series of transgenic mice was produced by microinjection of a segment of DNA, containing 460 base pairs of the phosphoenolpyruvate (P-enolpyruvate) carboxykinase promoter-regulatory region ligated to the bovine growth hormone structural gene, into the male pronucleus of fertilized mouse eggs. Founder animals which contained the gene were selected for further analysis and for breeding. The concentration of bovine growth hormone in the serum of animals which were shown to contain the gene ranged from a low of 5 ng/ml serum to approximately 2300 ng/ml serum. Mice with high levels of bovine growth hormone had growth rates double that of their litter mates which did not contain the transgene. The transgene was expressed only in the liver and kidney of the animals studied, and the level of specific mRNA for bovine growth hormone in these tissues could be regulated by diet in a manner similar to the endogenous P-enolpyruvate carboxykinase gene. Feeding the animals a diet high in carbohydrate for 1 week caused a 90% decrease in the concentration of bovine growth hormone in the blood, suggesting that the expression of the chimeric P-enolpyruvate carboxykinase/bovine growth hormone gene is sensitive to insulin. When the same animals were then refed a diet high in protein, but devoid of carbohydrate, the concentration of bovine growth hormone in their blood was induced 30-fold within a week. The administration of dibutyryl cyclic AMP to the transgenic mice caused a 2-fold induction in the level of bovine growth hormone in the serum within 90 min. Thus the region between -460/+73 in the P-enolpyruvate carboxykinase promoter-regulatory region contains sequences which can direct the tissue-specific expression, as well as hormonal and dietary responsiveness, of a linked structural gene.     

Involvement of glycoconjugates in insulin-receptor interactions Studies in liver plasma membranes of control and diabetic mice

The involvement of glycoconjugates in the insulin-receptor interactions in mouse liver is tested by digestions of membranes with various enzymes. Trypsin decreased the binding of [¹²⁵I]insulin to liver membranes. After digestion with β-galactosidase no “high affinity” receptor sites could be detected. The effects observed with plant lectins confirm the involvement of galactoconjugates in the insulin binding process. Sophora japonica and Ricinus communis lectins (with galactose specificity) and concanavalin A largely inhibit the binding process of insulin and those effects concern the “high affinity” receptor sites. Other lectins (wheat germ agglutinin, Dolichos) and enzymes (α-l-fucosidase, $\text{β-N-}\text{acetyl-hexosaminidase and neuraminidase}$) are without effect on insulin binding.Comparative studies performed on diabetic mouse liver membrane (KK mice), previously characterized by decreased number of insulin receptors, are in good agreement with qualitatively similar receptor sites in both non-diabetic (control) and diabetic mice. Effects of enzymes and lectins yielded same results as compared to control membranes. Plasma membrane proteins and glycoproteins in both types of mouse are indistinguishable with respect to enzymic and chemical analysis. Sodium dodecyl sulphate acrylamide gel electrophoresis shows identical patterns. Moreover, the decrease in the number of insulin receptors is easily reversed with diet restriction. These data are consistent with the similarity of receptor sites in control and diabetic liver membrane.     

The relationship between age and genotype and circulating concentrations of triiodothyronine (T3), thyroxine (T4), and growth hormone in commercial meat strain chickens

The presence of the sex-linked dwarf gene (dw) in homozygous male (dw/dw) and female (dw/-) meat strain chickens is associated with a significant reduction in circulating levels of triiodothyronine (T3). Heterozygous (Dw/dw) male broiler strain chickens have T3 concentrations similar to those in homozygous (Dw/Dw) male broilers. Genetically normal (Dw/Dw) but significantly slower growing roaster strain male meat chickens had consistently higher T3 than the faster growing broilers at all ages in one experiment but only at 8 weeks in a second experiment. Age and not growth rate appears to have a greater influence on serum T3 concentrations in the slow- and fast-growing normal strains. Growth hormone levels were significantly higher in the dwarf chickens at all ages and in all three experiments. The heterozygous and homozygous broilers had similar GH levels and the slow-growing, genetically normal roasters had intermediate concentrations between the broiler and dwarf lines. GH was influenced to a greater extent by the rate of body weight gain than by increasing age in the genetically normal fast and slow growing strains.     

Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T₄) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T₄ was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T₄ also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T₄ and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development.     

Down-regulation of prolactin receptors in the liver, mammary gland and kidney of female virgin rat, infused with ovine prolactin or human growth hormone

Down regulation of prolactin (PRL) receptors resulting from i.v. infusion of oPRL or human growth hormone (hGH) into female virgin rats was demonstrated. A decrease of over 85% in the number of free receptors was achieved within 15 - 30 min using infusion of oPRL or hGH at 25 micrograms/h and remained at this level until the end of infusion. Ovine growth hormone or recombinant bovine growth hormone at ten-fold higher concentration had no effect at all. The decrease in the specific binding resulted from a lower number of binding sites and not from change in the dissociation constants. The decrease in the total receptors in the liver was more gradual and leveled off at 40 - 50% of the initial value. Our results suggest that a change in blood PRL or hGH level may lead to a new steady state in the number, occupancy and distribution of prolactin receptors.     

Alterations in GHRH binding and GHRH receptor mRNA in the pituitary of adult dw/dw rats

Lewis dwarf (dw/dw) rats exhibit growth hormone (GH) deficiency and growth retardation linked to a malfunction of GHRH signaling. In this study, GHRH-receptor (GHRH-R) binding and mRNA in the pituitary of adult male dw/dw and age-matched normal Lewis rats was measured by radioligand binding assay and real-time PCR. Only one of nine pools of dw/dw pituitary membranes revealed detectable binding of [His(1), 125I-Tyr(10), Nle(27)]hGHRH(1-32) amide (B(max); 4.3 fmol/mg protein). In contrast, GHRH-R binding was 22.4 +/- 2.60 fmol/mg protein in normal Lewis rats. mRNA for GHRH-R was detectable in all dw/dw rat pituitaries examined, averaging 21% that of Lewis rats. Low expression of GHRH-R reflects reduced GHRH-R mRNA as well as a possible reduction in translation of the receptor protein.     

Reduced levels of thyroid hormones, insulin, and glucose, and lower body core temperature in the growth hormone receptor/binding protein knockout mouse

The mechanisms that are responsible for the extension of lifespan in the mouse with targeted disruption (knockout [KO]) of the growth hormone (GH) receptor/binding protein (GHR-KO) are unknown. However, in the long-living Ames dwarf mouse, blood glucose and body core temperature (Tco) are consistently lower than in normal mice. In addition, insulin levels are reduced and corticosterone levels are elevated in male dwarfs. These functional alterations, similar to those seen in animals under caloric restriction, have not been proven to be causally related to the extension of lifespan, but they do provide some insight into what traits may be necessary for long life. Therefore, to investigate which of these parameters are similarly affected in two genetically unrelated, yet similarly long-living mouse models, we measured Tco, thyroid hormones (triiodothyronine [T3] and thyroxine [T4]), and insulin, in addition to morning and afternoon levels of glucose and corticosterone, in young adult male and/or female GHR-KO mice and their normal siblings. Tco in GHR-KO mice was numerically reduced throughout the 24-hr period; however, these differences were only significant 4 hr prior to lights-off (14:00 hr), immediately after lights-off (18:00 hr), and during the 3 hr preceding lights on (03:00 to 06:00 hr). GHR-KO mice had significantly reduced levels of T3 and T4, while the ratio of these hormones was similar to that in normal mice. Insulin levels in GHR-KO mice were lower than in normal mice; levels in male GHR-KO mice were below the detectable limits of the assay used. Glucose levels in GHR-KO mice (male and females) were lower than in normal mice in measurements taken in both morning and afternoon; however, these differences arose from consistent reductions in males, as morning glucose levels in GHR-KO females were similar to those of normal mice. Corticosterone levels measured in blood plasma collected under basal (nonstressed) conditions showed sex-related alterations. Basal corticosterone levels in female GHR-KO mice were similar to normal females, while those in male GHR-KO mice were higher than in normal males in the afternoon. Corticosterone levels in stressed GHR-KO females were similar to those measured in stressed normal females. These data show that the long-living GHR-KO mouse shares a reduction in glucose, insulin, thyroid hormones, and Tco with the Ames dwarf mouse. Reductions in these parameters may be important to the underlying mechanisms of delayed aging in these animals.     

Effects of estrogen and growth hormone on steroid sulfatase activity and estrogen binding in rat liver

It is now well established that the activity of certain liver enzymes displays sex differences and that administration of human growth hormone to male rats alters the liver metabolism in a "female" direction. In this work we studied steroid sulfatase activity and binding of estradiol-17 beta in livers from intact rats and found a sex difference, with considerably higher enzyme activity in male as compared to female liver tissue. Continuous infusion of native and recombinant human growth hormone and estradiol-17 beta to male rats reduced sulfatase activity to "female" levels. A specific binding of estradiol-17 beta with receptor properties was found in the rat livers, but the concentration of binding sites did not change after administration of growth hormone or estradiol in this group of intact animals. Our data confirm previous reports that continuous administration of human growth hormone "feminize" liver metabolism, and since estradiol was found to have an identical effect on sulfatase activity it is suggested that the effect of estradiol-17 beta in this respect may be indirect, mediated via an altered secretory pattern of rat growth hormone.     

Factors Contributing to the Poor Myelination in the Brain of the Snell Dwarf Mouse

Abstract: Conventional histological examination of the pituitary does not distinguish Snell dwarf mutants (dw/dw) from their normal littermates (+/?) in the neonatal stage. However, immunohistochemical examination of pituitaries of litters born to heterozygous Snell parents revealed that in approximately 25% of the glands examined, the number of positive cells was very low in the neonatal stage. We attempted to delineate the events resulting in the poor myelination in the brain of the Snell dwarf mouse, and to devise an immunohistochemical method for identifying the mutant neonate. Differences in the brain weights of the dw/dw and +/? mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in CNPase activity was found to be suppressed in the cerebrum and brain stem throughout the developmental stage, but not in the other parts of the brain. The yield of isolated myelin decreased by 58% in the mutant mouse, but CNPase activity was equivalent to that of control myelin. Differences in DNA content per cerebrum from the dw/dw and +/? mice first became apparent on the 10th day of age. Henceforth, the dw/dw mice showed no further increase, although the +/? mice continued to increase. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about 50% of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the mutant cerebrum is a hypomyelination due to reduced oligodendroglial proliferation caused by lack of circulating growth hormone.     

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.     

Association between serum insulin, serum somatomedin and liver receptors for human growth hormone in streptozotocin diabetes

Streptozotocin-induced diabetes in the female rat caused a decrease in the serum level of somatomedin (Sm), measured by radioreceptor assay. The decrease was reversed by insulin therapy. In diabetes of varying severity, serum insulin and Sm levels showed highly significant association up to the insulin concentration (18 microU/ml) corresponding to normal serum Sm (1 U/ml). Similarly, the hepatic binding of human growth hormone (hGH) showed highly significant association with serum Sm levels up to the degree of binding (7% of tracer) corresponding to normal serum Sm. Binding of hGH to normal liver was about 12% of tracer. These results suggest that insulin might regulate serum Sm via its effect on liver lactogenic receptors, and that about half of these receptors are "spare", or in excess of those required to maintain normal serum Sm levels.     

Insulin receptors in virus-induced diabetes mellitus in mice

Quantitative estimations were made of insulin receptors on liver cell membrane of DBA/2 mice infected with M variant of encephalomyocarditis virus. The virus produced an impairment of glucose metabolism on day 3 of infection, which lasted for 5 months. The fasting plasma insulin concentration was markedly decreased on day 14. The specific binding of 125-I insulin to the membrane receptor was significantly decreased on day 3 of infection. The binding inhibitions were stronger in male mice than in females. The number of insulin receptors began to decrease on day 1, was decreased remarkably by day 3, and returned on day 7 to the level before infection. A decrease of receptor affinity was also observed in infected animals. These results seem to show that changes in insulin receptors are one cause of the impairment of glucose metabolism in the initial phase of virus-induced diabetes.     

Ovarian follicle apoptosis in bovine growth hormone transgenic mice

Growth hormone directly or via insulin like-growth factor-I has been shown to inhibit preovulatory follicle apoptosis, which is the underlying mechanism of follicular atresia. We studied the levels of apoptosis in the ovaries of transgenic mice expressing bovine growth hormone. Female bovine growth hormone transgenic mice (n = 10) and nontransgenic litter mates (n = 8) were killed at early proestrus. Ovaries were collected, sectioned, and processed using a nonradioactive in situ method for apoptosis detection. Follicles were classified and counted on the basis of size and level of apoptosis. Our results demonstrate that the percentage of ovarian follicles containing apoptotic cells was lower in transgenic versus normal mice (30% vs. 46%; P < 0.05). The percentage of follicles undergoing heavy apoptosis was lower (P < 0.05) in transgenic versus control animals in preovulatory and early antral follicles, but it was not different in preantral follicles. The percentage of healthy preovulatory follicles was also higher in transgenic versus normal mice (7.4% vs. 4.3%; P < 0.05). These results indicate that growth hormone overexpression in transgenic mice significantly decreases follicle apoptosis, and thus atresia in the mouse ovary, therefore leading to increased propensity for ovulation in these animals.