Mouse Growth Hormone Research Articles

A novel missense mutation in the mouse growth hormone gene causes semidominant dwarfism, hyperghrelinemia, and obesity.

The SMA1-mouse is a novel ethyl-nitroso-urea (ENU)-induced mouse mutant that carries an a-->g missense mutation in exon 5 of the GH gene, which translates to a D167G amino acid exchange in the mature protein. Mice carrying the mutation are characterized by dwarfism, predominantly due to the reduction (sma1/+) or absence (sma1/sma1) of the GH-mediated peripubertal growth spurt, with sma1/+ mice displaying a less pronounced phenotype. All genotypes are viable and fertile, and the mode of inheritance is in accordance with a semidominant Mendelian trait. Adult SMA1 mice accumulate excessive amounts of sc and visceral fat in the presence of elevated plasma ghrelin levels, possibly reflecting altered energy partitioning. Our results suggest impaired storage and/or secretion of pituitary GH in mutants, resulting in reduced pituitary GH and reduced GH-stimulated IGF-1 expression. Generation and identification of the SMA1 mouse exemplifies the power of the combination of random mouse mutagenesis with a highly detailed phenotype-analysis as a successful strategy for the detection and analysis of novel gene-function relationships.

Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin, and insulin-like growth factor I levels and increased life span.

GH participates in growth, metabolism, and cellular differentiation. To study these roles, we previously generated two different dwarf mouse lines, one expressing a GH antagonist (GHA) and the other having a disrupted GH receptor and binding protein gene (GHR -/-). In this study we compared the two dwarf lines in the same genetic background (C57BL/6J). One of the most striking differences between the mouse lines was their weight gain profile after weaning. The weights of the GHA dwarfs gradually approached controls over time, but the weights of the GHR -/- dwarfs remained low throughout the analysis period. Additionally, fasting insulin and glucose levels were reduced in the GHR -/- mice but normal in the GHA mice. IGF-I and IGF binding protein 3 (IGFBP-3) levels were significantly reduced, but by different degrees, in both mouse lines, but IGFBP-1 and -4 levels were reduced and IGFBP-2 levels increased in GHR -/- mice but unaltered in GHA mice. Finally, life span was significantly extended for the GHR -/- mice but remained unchanged for GHA dwarfs. These results suggest that the degree of blockade of GH signaling can lead to dramatically different phenotypes.

Reduced proteolysis of rabbit growth hormone (GH) receptor substituted with mouse GH receptor cleavage site.

GH binding protein (GHBP) is a circulating form of the GH receptor (GHR) extracellular domain, which derives by alternative splicing of the GHR gene (in mice and rats) and by metalloprotease-mediated GHR proteolysis with shedding of the extracellular domain as GHBP (in rabbits, humans, and other species). Inducible proteolysis of either mouse (m) or rabbit (rb) GHR is detected in cell culture in response to phorbol ester and other stimuli, yielding a cell-associated GHR remnant (comprised of the cytoplasmic and transmembrane domains and a small portion of the proximal extracellular domain) and down-regulating GH signaling. In this report, we map the mGHR cleavage site by adenoviral overexpression of a membrane-anchored mGHR mutant lacking its cytoplasmic domain and purification and N-terminal sequencing of the phorbol 12-myristate 13-acetate-induced remnant protein. The sequence obtained was LEACEEDI, which matches the mGHR extracellular domain stem region sequence L265EACEEDI272, indicating that mGHR cleavage occurs in the extracellular domain nine residues outside of the transmembrane domain, in the same region (but at different residues) as the rbGHR cleavage site we recently mapped. We studied the effects on receptor proteolysis and GHBP shedding of replacing rbGHR cleavage site residues with those corresponding to the mGHR cleavage site. We analyzed five separate rodentized rbGHR mutants incorporating mGHR amino acids either at or surrounding the cleavage site. Each mutant was normally processed, displayed at the cell surface, and responded to GH stimulation by undergoing tyrosine phosphorylation. Only the mutants replaced with mGHR cleavage site residues, rather than surrounding residues, exhibited deficient inducible proteolysis and GHBP shedding. These findings suggested that the GHR cleavage sites in the two species differ in their susceptibility to cleavage. This difference may underlie interspecies variation in utilization of proteolysis to generate GHBP.

Alcohol Suppresses Insulin‐Like Growth Factor‐1 Gene Expression in Prepubertal Transgenic Female Mice Overexpressing the Bovine Growth Hormone Gene

Background Alcohol (ALC) delays puberty in female rats and alters the development of a normal menstrual pattern in rhesus monkeys. These actions are associated with depressed serum levels of growth hormone (GH), luteinizing hormone and insulin‐like growth factor‐1 (IGF‐1). The mechanism of this ALC‐induced depression in IGF‐1 is not known, however, could be due to depressed GH and, possibly, to an alteration in the hepatic GH receptor. To assess whether ALC has a direct action at the liver, we used a transgenic mouse model that overexpresses GH, allowing assessment of potential direct actions of ALC on the level of either the GH receptor or the IGF‐1–synthesizing machinery within the hepatocyte.Methods One group of transgenic mice was fed a liquid diet containing ALC. The second group was pair‐fed the companion isocaloric control liquid diet. The third group of transgenic mice was fed Lab Chow and water. The fourth group consisted of normal (nontransgenic) littermates fed Lab Chow and water. Animals received their respective diets for 5 days. Mice were killed during their late juvenile stage of development, and tissues and blood collected and frozen.Results The ALC‐fed transgenic mice showed a decrease (p < 0.01) in hepatic IGF‐1a and IGF‐1b messenger RNA levels compared with transgenic controls, and this paralleled a decrease (p < 0.01) in serum IGF‐1. ALC did not alter the circulating levels of bovine GH held constant by the promotor and did not alter mouse GH receptor protein levels as analyzed by Western blotting.Conclusions Using this transgenic animal model that maintains circulating GH in the presence of ALC, we found that the ability of ALC to suppress prepubertal Igf1 gene expression can also occur independently of any alterations in the level of circulating GH. This direct effect on the hepatocyte is a postreceptor event because the GH receptor protein levels were not altered by ALC exposure.

Regulation of leptin gene expression by insulin and growth hormone in mouse adipocytes.

The role of leptin in the control of obesity, insulin resistance and type II diabetes has been reported, however, the regulatory mechanism of leptin in animals affected by hormones is not clearly understood. In this study, the effects of insulin, epinephrine, growth hormone or dexamethasone on the expression of leptin was examined in mouse primary adipocytes. The leptin expression was also studied in the adipose tissue of the mouse treated with insulin or growth hormone (0.3 or 0.6 units/animal). Insulin (100 nM) or dexamethasone (100 nM) stimulated leptin mRNA transcription while epinephrine (100 nM) alleviated its transcription in mouse primary adipocytes. The level of leptin protein in cultured media of adipocytes treated with insulin or dexamethasone was higher than that of the control group but growth hormone or epinephrine treatment had no effect on them. Insulin administration (0.6 units/mouse) enhanced leptin mRNA as well as leptin protein in mouse adipose tissue but growth hormone administration (0.3 or 0.6 units/mouse) had no effect on them. Leptin protein level in sera of mice injected with insulin or growth hormone was not significantly different from that of control group. These results indicate that both insulin and dexamethasone stimulate leptin gene expression and secretion of its product, whereas, growth hormone has no effect on the expression of leptin gene in mouse adipocytes.

Mouse growth hormone transcription factor Zn-16: unique bipartite structure containing tandemly repeated zinc finger domains not reported in rat Zn-15

Rat Zn-15 is a transcription factor activating GH gene expression by synergistic interactions with Pit-1, named for 15 DNA-binding zinc fingers, including fingers IX, X, and XI that are responsible for GH promoter binding. In this study, a mouse cDNA for Zn-15 was characterized. The predicted 2192-amino acid mouse protein is 89% identical to rat (r) Zn-15 overall, and is 97% similar in the C-terminal domain necessary for binding the GH promoter. However, the mouse cDNA encodes 16 zinc fingers, and sequences of rZn-15 pituitary cDNAs were the same as the mouse (m) Zn-16; the rat sequence in GenBank has a one nucleotide offset of a 17-bp segment in the finger V region. The mouse and corrected rat sequences contain four tandemly repeated fingers in the N-terminus, each separated by seven amino acids, typical of zinc finger proteins of the transcription factor IIIA-type. Analysis of mZn-16 expression by RT-PCR showed that the mRNA is produced at similar levels in normal and GH-deficient Ames dwarf (Prop-1<df–/–>) mouse pituitaries at postnatal day 1. Mouse Zn-16 mRNA also was detected by ribonuclease protection assay in the pre-somatotrophic mouse cell line GHFT1-5. The Zn-16 protein is bipartite in that the N-terminal half displays tandem spacing typical of most zinc finger proteins, while the C-terminal portion contains long linkers between fingers that cooperatively bind to a DNA response element. Expression in early postnatal pituitary and in pre-somatotrophic cells suggests that Zn-16 could play a role in pituitary development prior to somatotroph differentiation.

Growth hormone (GH) and 17beta-estradiol regulation of the expression of mouse GH receptor and GH-binding protein in cultured mouse hepatocytes.

In the present study, primary mouse hepatocytes from 8- to 10-week-old virgin female Swiss-Webster mice were perfused with collagenase (100 U/ml) using the two-step method. Isolated hepatocytes were plated in a rat tail type I collagen sandwich configuration to examine the regulation of GH receptor (GHR) and GH-binding protein (GHBP) expression by GH and 17beta-estradiol (E2). After 48 h of initial plating, hepatocytes were divided into groups of five replicates and treated for 24 h with medium containing no hormones (controls), GH (100 ng/ml), E2 (10(-9) M), E2 (10(-9) M) plus GH (100 ng/ml), or E2 plus GH and ICI 182-780 at different concentrations. Treatment of hepatocytes with GH or E2 alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR messenger RNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2 and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. In the present study we have shown that in cultured hepatocytes from virgin female mice, GH or E2 alone did not affect the concentrations of GHBP and GHR. However, E2 and GH together significantly up-regulated GHR and GHBP expression.

Growth Hormone (GH) and 17 -Estradiol Regulation of the Expression of Mouse GH Receptor and GH-Binding Protein in Cultured Mouse Hepatocytes

In the present study, primary mouse hepatocytes from 8- to 10-week-old virgin female Swiss-Webster mice were perfused with collagenase (100 U/ml) using the two-step method. Isolated hepatocytes were plated in a rat tail type I collagen sandwich configuration to examine the regulation of GH receptor (GHR) and GH-binding protein (GHBP) expression by GH and 17β-estradiol (E2). After 48 h of initial plating, hepatocytes were divided into groups of five replicates and treated for 24 h with medium containing no hormones (controls), GH (100 ng/ml), E2 (10−9m), E2 (10−9m) plus GH (100 ng/ml), or E2 plus GH and ICI 182–780 at different concentrations. Treatment of hepatocytes with GH or E2 alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR messenger RNA concentrations were also up-regulated 2- to 3-fold. ICI 182–780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2 and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. In the present study we have shown that in cultured hepatocytes from virgin female mice, GH or E2 alone did not affect the concentrations of GHBP and GHR. However, E2 and GH together significantly up-regulated GHR and GHBP expression.

Structure and expression of the mouse growth hormone receptor/growth hormone binding protein gene.

The mouse growth hormone receptor/growth hormone-binding protein (GHR/BP) gene produces several distinct mRNA forms through alternative splicing, including mRNAs encoding the membrane-bound growth hormone receptor (GHR) and the soluble growth hormone-binding protein (GHBP). Transcripts are also heterogeneous in their 5' regions due to alternative selection of two major 5' untranslated region (5'UTR) sequences, designated L1 and L2. Here we report the cloning of all mouse GHR/BP coding exons as well as the exon encoding 5'UTR L2, the most widely expressed 5'UTR. The mouse GHR/GHBP gene contains 11 coding exons, 9 of which are homologous in size and sequence to human GHR exons 2-10. The two mouse exons that do not have homologs in the human gene are designated exons 4B and 8A. Exon 4B, located between exons 4 and 5, encodes an 8-amino acid segment of the ligand binding domain that is unique to mouse GHR and GHBP. Analysis by reverse transcriptase-polymerase chain reaction indicated that exon 4B is constitutively present in mouse GHR and GHBP mRNA. Exon 8A encodes the GHBP hydrophilic tail and 3'UTR sequence. 5'UTR L2 is encoded by a single exon located at least 27 kb upstream of exon 2 and at least 12 kb upstream of the exon encoding 5'UTR L1. The transcription start sites of UTR L2 were mapped and the 5' flanking region sequenced. The exon and proximal promoter region are GC rich, and share a high level of conservation with the equivalent exons in the sheep, bovine and human GHR genes. A CCAAT motif and several putative Sp1 motifs are present, and there is no TATA box. Homology between the mouse sequence and other species is limited to a region of 450 bp upstream of the exon due to the insertion of a fragment of a LINE-1 element upstream of the mouse L2 exon. Ribonuclease protection assays were used to confirm that 5'UTR L2 is widely expressed in multiple tissues and is the predominant form of transcript except in the liver during pregnancy, in which 5'UTR L1 is the major form.

Immune function in transgenic mice overexpressing growth hormone (GH) releasing hormone, GH or GH antagonist.

Effects of life-long exposure to high levels of homologous or heterologous growth hormone (GH) and effects of GH resistance on selected parameters of immune function were studied in adult male transgenic mice overexpressing GH releasing hormone (GHRH), bovine (b) GH or an antagonistic bGH analog. In metallothionein I (MT)-bGH transgenic mice with high peripheral levels of bovine GH, there were significant increases in the absolute weight of the thymus and the spleen and in the mitogenic responses of splenocytes to concanavalin A (ConA), lipopolysaccharide (LPS) and phytohemagglutinin (PHA), as compared to age-matched normal animals. There were no significant differences between MT-bGH transgenic and normal mice in splenocyte viability or in delayed-type hypersensitivity measured by the allergic contact dermatitis response to oxazolone. Similar results, including significant stimulation of splenocyte responses to ConA, LPS, and PHA, were obtained in MT-hGHRH transgenic mice in which overexpression of GHRH leads to striking pituitary enlargement and massive elevation of peripheral levels of homologous (mouse) GH. In MT-bGH-antagonist transgenic mice in which overexpression of an antagonistic bGH analog interferes with the actions of endogenous GH, spleen weight was reduced but proliferative responses of splenocytes to ConA, LPS, and PHA were not affected. It is concluded that overexpression of heterologous or homologous GH in transgenic mice can lead to significant stimulation of some parameters of immune function, whereas antagonism of GH action by expression of an antagonistic GH analog does not affect splenocyte responses to mitogens.

RGD sequences in several receptor proteins: novel cell adhesion function of receptors?

In the process of homology modelling of the 3-dimensional structure of alleles of the human histocompatibility protein HLA-DQ, we discovered that its RGD tripeptide ( β167–169) forms part of a loop. A search through protein sequence data bases, revealed this cell adhesion motif in 67 integral plasma membrane proteins (in 48 extracellularly, and in the remaining 19 intracellularly), which are bona fide receptors, and none of them has thus far been considered as a cell adhesion protein. The 3-dimensional structure of one of these, the rat neonatal F c receptor, is known and its extracellular RGD sequence is in an adhesion-like loop, a fact that went unnoticed in the original papers. In a few other cases, e.g. rat and mouse growth hormone receptor, and mouse CD40 ligand, homology modelling by ourselves and others reveals that the said sequences are part of a loop, in similarity to all RGD sequences found in proteins with established adhesion function and known 3-dimensional structure. Likewise, inspection of all known protein 3-dimensional structures containing an RGD sequence, and not having a documented cell adhesion function (total of 65 separate entries) shows that such sequence is mostly (52/65 or 80% of cases) part of a loop. We therefore call attention to these surprising findings, discuss the possible cell adhesion role of these receptor proteins, and draw an analogy from the two well characterised examples, that of soluble IGF binding protein 1 and the transcriptional activator protein Tat of HIV, where their RGD sequences have been shown by site-directed mutagenesis to participate in cell-adhesion interactions, without prior knowledge of the location of the tripeptide, or the 3-dimensional structure of the respective protein.

111 Binding and signalling properties of a GH-enhancing monoclonal antibody

We have recently described in detail the structure of an epitope defined by a monoclonal antibody which enhances in vivo the biological activity of bovine GH @GH) [ 11. Although several theories have been advanced in relation to the mechanism associated with this enhancement phenomenon, there is little conclusive data supporting any hypothesis [2]. To examine the molecular and cellular aspects of this phenomenon more closely, we have undertaken studies using the well characterised GH-responsive 3T3-F442A preadipocyte cell line and the FDCP-I cell line transfected with full length mouse growth hormone receptor (GHR). As a prelude to this, we examined the binding properties of OA15 in relation to the subsequent interaction between bGH and its receptor. Initial examination of the epitope defined on bGH by OA15 suggested that it was removed from binding sites 1 and 2 on GH required for interaction with the GH receptor [3]. Using the recombinant bGH binding protein (bGHBP), which represents the extracellular domain of the receptor, we have demonstrated, using optical evanescent wave biosensor technology, that engagement of bGH by OA15 still allows for the interaction between bGH and bGHBP. Although further experiments are underway to investigate the effects on the affinity of bGH-bGHBP interaction following bGH binding to OA15, at this stage we can tentatively conclude that these real time binding studies agree with our initial epitope mapping work ie that the epitope defined by OA15 is removed from receptor interaction sites on the hormone. The results of these experiments suggested that in cell culture studies tnpartite complexes of antibody-hormone and receptor would exist. In the context of mechanistic studies, this in turn would suggest that OA15 would not inhibit GH signalling events through disruption of GH-GHR interactions. To examine this hypothesis in more detail we used two GH-responsive cell culture systems the 3T3-F442A preadipocyte cell line and IL-3 dependant FDCP-I cell line which has been transfected with the full length mouse GHR [4]. In F442A cells, treatment with GH leads to the rapid tyrosine phosphorylation of proteins of the JAK-STAT-MAP pathway(s). Figure 1 is an antiphosphotyrosine (py) blot of lysates from F442A cells treated with bGH alone or hormone pre-complexed with OA15. As can be clearly seen, GH treatment stimulates the level of a 9lkDa tyrosine phosphorylated protein. This is most likely to be a member of the Stat (signal transducers and activators of transcription) family of transcription factors. The level of tyrosine phoshporylated 9 lkDa

Microcapsules as bio-organs for somatic gene therapy.

Current human gene therapy relies on genetic modification of the patient's own cells. An alternate non-autologous approach is to use universal cell lines engineered to secrete therapeutic products. Protection with immuno-isolation devices would allow the same recombinant cell line to be used for different patients, thus potentially lowering the cost of treatment. The feasibility of this idea has now been demonstrated in vitro and in vivo. Recombinant gene products with potential therapeutic applications (human growth hormone, factor IX, lysosomal enzymes, adenosine deaminase) have been expressed from genetically modified cells after encapsulation with alginate-poly-L-lysine-alginate or hydroxyethyl methacrylate-methyl methacrylate. We have also demonstrated the feasibility of this idea in vivo. After intraperitoneal implantation, genetically modified mouse Ltk- fibroblasts or C2C12 myoblasts encapsulated in alginate-poly-L-lysine-alginate could deliver recombinant gene products (human growth hormone, human factor IX) to the systemic circulation of mice. The clinical efficacy of this novel approach to gene therapy has now been shown in murine models of human diseases. In the Snell dwarf mice deficient in growth hormone production, implantation of encapsulated mouse myoblasts engineered to secrete mouse growth hormone resulted in increases in body weight, length and organ sizes, some to > 25% above those of the controls. In the Gus/Gus mice suffering from the lysosomal storage disease mucopolysaccharidosis type VII due to deficient beta-glucuronidase, implantation of encapsulated mouse fibroblasts engineered to secrete mouse beta-glucuronidase resulted in delivery of normal levels of the enzyme in the plasma and significant correction of the organ histopathology. Hence, delivery of recombinant gene products through bioartificial devices appears to be a promising strategy for the treatment of genetic diseases.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse).

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Purification and Characterization of Recombinant Mouse Growth Hormone Binding Protein Produced in the Baculovirus Expression System

Sf21 insect cells were infected with recombinant baculovirus containing cDNA for the entire coding region of the mouse growth hormone binding protein (mGHBP). Recombinant (r) mGHBP was expressed at a yield of 17.3 mg/liter/3 days. The molecular size (Mr) of the rmGHBP was approximately 33,000 as estimated by SDS–PAGE. Amino-terminal sequence analysis of the recombinant protein yielded two sequences: one identical to amino acids 1–15 and another corresponding to amino acids 14–21 of the GHR/GHBP. Western blot analysis revealed that this is the sameMras that of one of the two majorMrforms of serum mGHBP. Deglycosylation of serum mGHBP and recombinant mGHBP caused a shift in the molecular size of both proteins to that expected after removal of all N-linked carbohydrates. Binding characteristics of the recombinant mGHBP to mouse growth hormone were similar to those for serum GHBP. Scatchard analysis showed an equilibrium association constant (Ka) for rmGHBP of 3.8 × 108± 0.6 × 108M−1(mean ± SEM,n= 3) andKaof 9.2 × 108± 2.0 × 108M−1(mean ± SEM,n= 3) for the serum mGHBP. In conclusion, this expression system should allow a production of relatively large quantities of mGHBP suitable for physiological studies on the role of this protein.

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10(9) cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.

Structural comparison of a portion of the rat and mouse growth hormone receptor/binding protein genes

A portion of the rat growth hormone receptor (GHR) gene was cloned by PCR. Restriction enzyme digestion and DNA sequence analyses revealed that an exon, exon 8A, encoding the carboxy terminus of the rat serum growth hormone binding protein (GHBP) was located between exons 7 and 8 of the rat GHR gene. Two tandem poly-adenylation (poly A) signals for the GHBP mRNA were found in the 3′-untranslated region of this exon. In addition, a GT repeat tract was found adjacent to the 5′-splice donor site of intron 7/8A. The similarity of the nucleotide sequences of the rat and mouse GHR/GP gene suggests that the poly A signals and GT repeat may be involved in the regulation of GHR and GHBP expression.

Structure of the growth hormone-encoding gene and its promoter in mice

The isolation and nucleotide sequence determination of the 5′ flanking region of the mouse growth hormone (mGH)-encoding gene (mGH) is described. The mGH gene consists of five exons and four introns, as is observed in other mammalian species. The second intron in mGH is much smaller than its rat counterpart, thus being similar in size to human, bovine and porcine GH. The transcription start point was determined to be a C residue 62 bp upstream from the start codon, ATG. Analysis of 1767 bp of the 5' flanking region, with respect to putative regulatory elements, revealed a TATA box, two binding sites for growth hormone factor (GHF1), a GC box (SP1), a thyroid-response element ( TRE) and a silencer (SiL) sequence motif. As expected, the mGH promoter shows a higher degree of homology with rat, as compared to the other mammalian species like pig, cattle and human, where an overall homology exists only at the proximal promoter region

Growth retardation--an unexpected outcome from growth hormone gene therapy in normal mice with microencapsulated myoblasts.

Recently, we have succeeded in using nonautologous myoblasts engineered to secrete mouse growth hormone (GH) to correct partially the growth retardation of the Snell dwarf mice, which suffer from pituitary GH deficiency. The allogeneic myoblasts were protected from immune rejection by enclosure in permselective microcapsules fabricated from alginate, thus validating the clinical efficacy of using universal nonautologous cells for somatic gene therapy. Because GH therapy is considered also for treating patients with normal pituitary function, we now apply this protocol to treat normal mice to evaluate the potential consequences of using GH gene therapy in subjects with no demonstrated GH deficiency. When microencapsulated allogeneic myoblasts engineered to secrete mouse GH were implanted into normal male and female mice, contrary to expectation, the treated animals became significantly shorter and lost weight; their internal organs became smaller and their tibial growth plates were less differentiated, indicating reduced skeletal growth. Females were more severely affected than males and 2 animals died by day 13 of unknown cause. By day 70, most of the abnormalities were restored to normal except for body weights, which remained below normal. In conclusion, although somatic gene therapy for GH delivery is beneficial for pituitary dwarfism, it may have adverse metabolic consequences in those with normal hypothalamic-pituitary functions, and the female mice were more severely affected than males.

Resistance of growth hormone secretion to hypoglycemia in the mouse.

Although previous studies have demonstrated that acute hypoglycemia inhibits growth hormone (GH) secretion due to stimulation of hypothalamic somatostatin (SS) neurones in the rat, the effect of hypoglycemia on GH secretion has not yet been elucidated in the mouse. In this study, the effects of insulin-induced hypoglycemia on mouse GH secretion, hypothalamic c-fos expression, GH-releasing hormone (GRH) and SS mRNA levels were investigated in conscious male mice. Seven days after implantation of chronic atrial catheters, blood samples were taken every 20 min from 1200-1600 h under unrestrained conditions. Insulin was administered iv every 20 min from 1200-1240 h to induce moderate hypoglycemia (MH) and severe hypoglycemia (SH), respectively. Expression of hypothalamic c-fos protein was examined 30 min and 60 min after induction of hypoglycemia by immunohistochemistry. Hypothalamic GRH and SS mRNA levels were examined 1 h and 3 h after induction of hypoglycemia by Northern blot analysis. The lowest mean plasma glucose levels after insulin injections were 49.1 +/- 4.1 mg/dl and 34.2 +/- 5.6 mg/dl in conscious mice, respectively. However, pulsatile GH secretion was not significantly altered in either group. Although both MH and SH markedly stimulated c-fos expression in specific hypothalamic nuclei including the paraventricular nucleus, they did not induce c-fos protein in the periventricular nucleus. Neither MH nor SH altered hypothalamic GRH or SS mRNA levels. These results suggest that hypoglycemia does not activate SS neurons which inhibit GH secretion in the mouse.