Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)/bovine growth hormone gene in transgenic mice.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.     

Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.     

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.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the hα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

A protein factor required for activation of phosphoenolpyruvate carboxykinase by ferrous ions.

When rat liver cytosolic P-enolpyruvate carboxykinase is purified, its activity is no longer enhanced by incubation with 30 muM Fe2+. Ferrous ion stimulation of the purified enzyme is restored by the addition of rat liver cytosol. The agent responsible is a cytosolic protein, named P-enolpyruvate carboxykinase ferroactivator, that was readily separated from the enzyme during purification of the latter. A quantitative assay for P-enolpyruvate carboxykinase ferroactivator is described. Subcellular fractionation of livers from fasted rats shows that 98% of the combined mitochondrial and cytosolic P-enolpyruvate carboxykinase ferroactivator activity resides in the cytosol. Fasting does not produce significant change in this cytosolic activity when compared to that of fed animals. Examination of various tissue homogenates shows that the ferroactivator is found in liver, kidney, erythrocytes, adipose tissue, and brain. No activity was detected in blood serum or skeletal muscle. The ability to enhance the activity of purified rat liver cytosolic P-enolpyruvate carboxykinase in the presence of Fe2+ is not species specific. P-enolpyruvate carboxykinase ferroactivator may have an important function in regulating enzyme activity in vivo.     

Presence of cytosolic phosphoenolpyruvate carboxykinase activity in rat mammary gland

The cytosolic P-enolpyruvate carboxykinase activity was measured in mammary gland and liver of animals at all stages of the reproductive cycle. The specific activity of this enzyme was almost absent in the mammary gland of virgin rats. Different pattern of P-enolpyruvate carboxykinase activity was obtained in liver and mammary gland during lactation cycle. The specific activity of the enzyme increased more than 40-fold in mammary gland and 2-fold in liver during the transition from mid-pregnancy to mid-lactation. Weaning of mid-lactating rats for 48 h resulted in an abrupt change in the enzyme activity in mammary gland while there was a small change in liver. In all the experiments performed, the activity of P-enolpyruvate correlates inversely with the plasma insulin levels described for the lactogenic process.     

Transgene expression in mammary glands of newborn rats

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals. © 1996 Wiley-Liss, Inc.     

Dietary and hormonal regulation of some enzyme activities associated with gluconeogenesis in rabbit liver.

1. Starvation increases the activity of cytosolic P-enolpyruvate carboxkinase in rabbit liver some 4-5 fold but does not alter the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase or glucose-6-phosphatase.2. Alloxan-induced diabetes increases the activities of cytosolic P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase approx. 6-, 2- and 2-fold, respectively. Again the activity of mitochondrial P-enolpyruvate carboxykinase is not altered. 3. Administration of mannoheptulose rapidly increases blood glucose levels and also causes a significant increase in cytosolic P-enolpyruvate carboyxkinase activity within 4 h. The activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase are not affected. 4. Administration of hydrocortisone also increases blood glucose levels and the activities of cytosolic P-enolpyruvate carboxykinase and glucose-6-phosphatase are significantly increased within 12h. Again, mitochondrial P-enolpyruvate carboxykinase and fructose-1,6-diphosphatase activities remain unaffected. 5. The observations that (A) the activity of cytosolic P-enolpyruvate carboxykinase responds to more situations conducive to gluconeogenesis than do the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase, and (B) cytosolic P-enolpyruvate carboxykinase activity is rapidly adaptive under appropriate circumstances, suggests that this particular enzyme's activity plays an important role in the regulation of gluconeogenesis in rabbits.     

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of thehα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output.     

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T, injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about ?35–40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly ?25–32 and ?29–41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by ?72–81 and ?51–70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels. © 1995 Wiley-Liss, Inc.     

Is the p29 protein involved in the rapid regulation of phosphoenolpyruvate carboxykinase (GTP)?

It has been postulated that a protein with a molecular mass of 29,000 daltons (p29), which copurifies with hepatic phosphoenolpyruvate (P-enolpyruvate) carboxykinase, forms a complex with the enzyme and stabilizes its sensitivity to Mn2+ activation by protecting critical sulfhydryl groups from oxidation (Brinkworth, R. I., Hanson, R. W., Fullin, F. A., and Schramm, V. L. (1981) J. Biol. Chem. 256, 10795-10802). In this paper we demonstrate that p29 is not only expressed in tissues which contain high amounts of P-enolpyruvate carboxykinase, such as liver and kidney, but also in brain and muscle, which have no gluconeogenic function. Furthermore, p29 is expressed in rat liver prenatally, whereas P-enolpyruvate carboxykinase is induced only after birth. The effect of p29 to protect P-enopyruvate carboxykinase against aerobic oxidation during in vitro incubation was also observed for ovalbumin and bovine albumin. Peptide sequencing of the p29 and search in a protein data bank revealed a high homology to the muscle-specific subunit of human phosphoglycerate mutase (EC 2.7.5.3). Determination of the enzyme activity confirms the identification of the p29 as the rat liver isoform of phosphoglycerate mutase. Taking all these findings together, it is concluded that this protein has no specific effect on P-enolpyruvate carboxykinase.     

Production of transgenic rabbits and mice carrying the gene for bovine growth hormone

The results of experiments on the transfer of bovine gene for growth hormone into mice and rabbits are presented. The gene was transferred by the technique of microinjection into the zygote. In all cases transgene in rabbits occurred to be changed. In two transgenic mice the bovine growth hormone gene represented some tandem arranged copies. One of the mice had accelerated growth. This phenotypic changes is found to be inheritable.     

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10???g/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.     

The human growth hormone transgene: expression in hemizygous and homozygous mice

Female transgenic mice carrying the mouse metallothionein-I/human growth hormone (hGH) fusion gene are sterile. Transmission of the transgene has been limited to the male germ line, resulting in the production of hemizygous (He) progeny containing only a single (paternal) copy of the gene. Using ovary transfer, we have developed procedures for producing homozygous (Ho) TG mice, viz., male TG mice were mated with control (non-TG) females carrying ovaries donated by female TG mice. In both He and Ho TG animals, serum levels of hGH were higher (1.5-fold) in males than in females, tended to decrease with age of the animal, and were increased (about 5-fold) by zinc induction. However, in comparison to He animals of the same sex, the Ho TG mice attained a greater body weight and had more than 2-fold higher levels of liver hGH-mRNA and serum hGH, both under basal conditions and in response to zinc induction. That is, the expression of the transgene was qualitatively similar in He and Ho TG mice, but the level of transgene activity was greater in the Ho animals. We interpret this to indicate that both copies (maternal and paternal) of the transgene were active and expressed additively (or cooperatively) in the Ho TG animal.     

Effect of prolactin and glucocorticoids on P-enolpyruvate carboxykinase activity in liver and mammary gland from diabetic and lactating rats

Summary The administration of 2 bromo--ergocryptine, to reduce serum prolactin decreased the activity of cytosolic P-enolpyruvatc carboxykinase (GTP) (EC4.1.1.32) about 50% in both liver and mammary gland of lactating animals. Adrenalectomy had similar effects to those of bromo-a-ergocryptine. In contrast, there was a 50% increase in enzyme activity in the mammary gland of diabetic, lactating rats and a 10-fold increase in liver as compared with normal rats. P-enolpyruvate carboxykinase activity in mammary gland as liver is coordinately regulated by prolactin, glucocorticoids and insulin.     

The regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat kidney cortex. The role of acid-base balance and glucocorticoids.

The effects of metabolic acidosis and of hormones on the activity, synthesis, and degradation of renal cytosolic P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) were studied in the rat using isotopic -immunochemical procedures. At normal acid-base balance, the synthesis of the enzyme accounted for between 2 and 3.5% of the synthesis of all soluble protein in the kidney cortex. P-enolpyruvate carboxykinase synthesis was selectively stimulated in acute metabolic acidosis, so that the relative rate of synthesis of the enzyme was increased to 7% 13 hours after oral administration of ammonium chloride. The stimulation of P-enolpyruvate carboxykinase synthesis preceded any increase in the assayable activity of the enzyme. The administration of sodium bicarbonate to acutely acidotic rats returned the rate of enzyme synthesis to normal in 8 hours. The effect of acidosis on both the synthesis and the activity of P-enolpyruvate carboxykinase was prevented by actinomycin D, cordycepin, and cycloheximide. The degradation in vivo of pulse-labeled P-enolpyruvate carboxykinase was not affected by acidosis. Thus, the stimulation of P-enolpyruvate carboxykinase synthesis is the major mechanism for the increase in the level of the enzyme observed in metabolic acidosis. The administration of glucocorticoid triamcinolone resulted in an increase in the relative rate of P-enolpyruvate carboxykinase synthesis and a commensurate increase in the activity of the enzyme in the renal cortex. Both changes were abolished by actinomycin D. Fasting was characterized by a high enzyme activity and a rapid rate of enzyme synthesis in the kidney cortex. This high rate of synthesis was reduced after the administration of sodium bicarbonate, but not after glucose feeding. Moreover, the injection of insulin to diabetic rats did not repress P-enolpyruvate carboxykinase synthesis in the renal cortex. Theophylline plus N-6, 0-2'-dibutyryl adenosine 3':5'-monophosphate stimulated P-enolpyruvate carboxykinase synthesis in the kidney of intact rats. However, the latter effect was probably due to glucocorticoid secretion, since it did not occur in adrenalectomized animals. The administration of parathyroid extracts did not result in the induction of the enzyme. Thus, the hormonal regulation of cytosolic P-enolpyruvate carboxykinase synthesis in the kidney differs markedly from that in the liver.     

Rabbit whey acidic protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice

Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.