Human apolipoprotein B (apoB) mRNA: identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine.

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides (nucleotides 6523-6552 of apoB mRNA), designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. Analysis of intestinal apoB mRNA by hybridization with apoB-Stop and apoB-Gln probes and sequence analysis of apoB clones in two independent human small intestinal cDNA libraries established that intestinal apoB mRNA contained both the apoB mRNA that codes for apoB-100 and the apoB mRNA containing the premature in-frame stop codon, which codes for apoB-48. Investigation of hepatic apoB mRNA and two hepatic cDNA libraries by hybridization with the apoB-Stop and apoB-Gln synthetic probes as well as by cDNA sequencing revealed that liver apoB mRNA also contains both the apoB-100 mRNA and the apoB-48 mRNA containing the stop codon. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine.     

Induction of hepatic receptors for growth hormone (GH) and prolactin by GH infusion is sex independent

To determine whether induction of rat liver GH and PRL receptors by GH infusion is dependent upon the sex of the animal or whether or not the pituitary is intact, rat GH (rGH) or rat PRL (rPRL) was infused at approximately 200 micrograms/day for 7 days into male and female, intact and hypophysectomized rats, and the binding of radioiodinated bovine GH (bGH) and ovine PRL (oPRL) to liver microsomal membranes was measured. In females, bGH binding was reduced by hypophysectomy whether or not membranes were MgCl2 treated to remove endogenous ligand. However, in males, hypophysectomy caused an apparent 3-fold induction of bGH binding sites, which was absent in MgCl2-treated membranes, suggesting that the effect was due to receptor occupancy by endogenous rGH in the intact males. Hypophysectomy also lowered oPRL binding in females but had no effect in males. Infusion of rGH significantly induced binding sites for bGH and oPRL in all treatment groups, independently of sex or the presence of the pituitary, whereas rPRL infusion had no effect on either receptor type except for mild induction of bGH binding in hypophysectomized females. Serum somatomedin-C (SM-C), reduced 95% by hypophysectomy, was restored by rGH, but not rPRL, infusion. However, in intact animals of both sexes, rGH infusion significantly lowered SM-C levels by 30-40%; thus both bGH and oPRL binding in individual pituitary-intact rats were negatively correlated with serum SM-C. In contrast, in rGH-treated hypophysectomized rats, induced GH (but not PRL) binding sites showed significant positive correlation with SM-C levels. These results indicate that the induction of GH and PRL receptors by rGH occurs independently of SM-C generation, but suggest that newly induced GH receptors in GH-treated hypophysectomized rats may be involved in SM-C generation.     

Hypophysectomy and growth hormone receptors in liver membranes of male rats

The effects of hypophysectomy on GH binding were studied in liver membranes of male rats. Ten days after surgery, the specific binding of [125I]iodobovine GH and of [125I] iodohuman GH was 2- to 3-fold higher in microsomal membranes of hypophysectomized rats than in membranes of control male animals. The number of receptors rather than the affinity of the binding was affected. A nonspecific membrane effect due to hypophysectomy is unlikely since membrane markers such as 5' nucleotidase, galactosyl transferase, and insulin binding were not different in liver membranes of hypophysectomized and control rats. The somatogenic specificity and the subcellular distribution of the binding sites were not altered by hypophysectomy; the number of the GH binding sites were increased in plasma membranes as well as in Golgi fractions. Hypophysectomy in male rats creates a situation where growth failure, absence of circulating GH, and lack of plasma somatomedin activity are associated with increased concentration of liver somatogenic receptors. The latter finding could explain why livers of hypophysectomized rats are more sensitive to GH than those of normal rats.     

Rat hepatocyte insulin-like growth factor I and binding protein: effect of growth hormone in vitro and in vivo

This study examines the regulation of insulin-like growth factor I (IGF I) and binding protein (BP) release by adult rat hepatocytes in primary culture. Increasing the density of plating of cells had a marked positive effect on the IGF I production rate per mg cell protein, with the highest rate, approximately 4 pmol/mg cell protein X 24 h, seen at densities of 1 X 10(5) cells/cm2 or higher. Cycloheximide (15 micrograms/ml) inhibited both IGF I and BP production by more than 90%, while actinomycin D (0.1 microgram/ml) caused less marked, but still significant, inhibition. Over the insulin range 30 pM-300 nM there was a 45% increase in IGF I production, but no effect on BP production. Bovine GH stimulated production of both peptides, significant effects (up to 50% stimulation) being seen at concentrations from 20-500 ng/ml. Cells from hypophysectomized rats, with serum IGF I and GH levels reduced more than 90% from normal, had IGF I and BP production rates only 7% of normal, measured after 48 h in culture. In vivo replacement with rat GH, 150 micrograms/day for 5 days by osmotic minipump, significantly restored the production of both peptides by hepatocytes. Cells isolated from rats bearing the GH-secreting tumor, MtT/W15, had IGF I and BP production rates approximately twice as high as normal. A significant stimulatory effect of bovine GH (200 ng/ml) in vitro was seen on cells from normal and hypophysectomized, GH-replaced rats, but not from unreplaced hypophysectomized, or tumor-bearing rats. The results in hypophysectomized, animals are consistent with known changes in hepatic GH receptors, while the lack of GH responsiveness in tumor-bearing rats indicates a persistence of maximal stimulation in culture. The reflection of in vivo GH status by hepatocytes cultured for 48 h suggests that messenger RNA turnover for IGF I and BP must be slow. The close parallel in the regulation of the two peptides under most conditions might be indicative of coordinated synthesis.     

Effect of hypophysectomy on hypothalamic growth hormone-releasing factor content and release in the rat

The possible regulation of hypothalamic GH-releasing factor (GRF) by GH was studied in untreated and GH-treated hypophysectomized rats by measurement of rat hypothalamic GRF content and release in vitro with a specific GRF RIA. Two weeks after hypophysectomy, animals not receiving hormone replacement showed a marked reduction in hypothalamic GRF content (to 28% of sham-operated control values; P less than 0.001). Replacement therapy with T4, cortisone, and testosterone for 9 days did not correct the decrease in hypothalamic GRF content in hypophysectomized rats, though the addition of GH therapy partially restored GRF levels (to 55% of control values; P less than 0.001). GRF release from incubated mediobasal hypothalamic fragments of hypophysectomized rats was decreased both basally and in response to 30 mM K+. This defect was completely corrected by prior GH treatment. The results suggest an impairment of GRF synthesis and release in the presence of GH deficiency.     

Growth hormone maintains its own receptors in rat adipocytes

Hypophysectomy decreased the capacity of adipocytes isolated from epididymal fat to bind [125I]human GH [( 125I]hGH) specifically without changing the apparent affinity for hGH. Specific binding of hGH by adipocytes of both normal and hypophysectomized rats appeared saturated when incubated with 75-80 ng/ml or higher concentrations of GH regardless of whether binding was studied for 2 h at 37 C or for 16 h at 0 C. Maximum binding of hGH by normal adipocytes was approximately 0.45 ng/10(6) cells, and that by adipocytes of hypophysectomized rats ranged from 0.15-0.25 ng/10(6) cells. In cells of both normal and hypophysectomized rats, only 25-30% of the hormone specifically bound at 37 was removed by digestion with trypsin, and about 75% was displaced by incubation with 5 M magnesium chloride, suggesting that these adipocytes internalized a significant fraction of bound hormone and that hypophysectomy did not alter the extent of internalization. Previously bound hormone was lost from normal adipocytes with a half-time of about 32 min and from adipocytes of hypophysectomized rats with a half-time of about 45 min, suggesting that hypophysectomy slowed the rate of processing bound hormone. To determine which pituitary hormone(s) might be required to maintain GH binding, we measured the binding of [125I]hGH at 3 or 30 ng/ml by fat cells prepared from hypophysectomized rats after various treatment regimens. Administration of bovine GH ip at a dose of 10 micrograms/rat every 4 h for 24 h doubled the binding of [125I]hGH by adipocytes prepared 4 h after the last injection. Similar results were obtained in fat cells examined 4 h after only one injection of 60 micrograms bovine GH to rats hypophysectomized 2-4 weeks previously. When binding was measured 16-24 h after GH administration, there was no apparent effect on restoration of binding even after treatment with 100 micrograms GH/day for up to 6 days, suggesting that the effects of GH in maintaining receptor number are transient. In accord with the apparently short-lived ability of GH to maintain its receptors on fat cells, GH binding was significantly reduced in adipocytes obtained form both hypophysectomized and sham-operated rats as early as 4 h after surgery, and by 8 h after surgery, declined to a level as low as that in adipocytes of chronically hypophysectomized rats. Twenty-four hours after surgery, GH binding by cells of sham-operated animals returned to normal. Fasting for 24 h also reduced GH binding by adipocytes of normal rats to a level comparable to that in adipocytes of fed hypophysectomized animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth hormone acutely increases glucose output by hepatocytes isolated from hypophysectomized rats

A series of experiments using isolated rat hepatocytes was carried out to establish rat liver cells in suspension as a physiological model for examining GH responses, and to determine whether acute recombinant bovine GH (rbGH) treatment of rat liver cells increased glucose output and/or suppressed fatty acid synthesis from lactate. Rat liver cells were isolated by collagenase perfusion and incubated in short-term (less than 60 min) suspension. The amount of insulin, glucagon or vasopressin required to elicit a half-maximal response was within the physiological range of the circulating hormone. When hepatocytes from normal rats were acutely (less than 60 min) treated with 0, 0.1, 10, 100 or 1000 nmol rbGH/l, rates of hepatocyte glucose output and fatty acid synthesis were unaltered. In addition, acute rbGH treatment (1000 nmol/l) did not alter hepatocyte responsiveness to insulin or vasopressin. However, acute rbGH treatment of hepatocytes isolated from hypophysectomized rats significantly (P less than 0.05) increased the rate of glucose output twofold and moderately (P less than 0.10) enhanced fatty acid synthesis. The accelerated rate of glucose production was not accompanied by an increase in the amount of glycogen phosphorylase-a. The observations with liver cells from hypophysectomized rats are not consistent with a GH receptor-transducing mechanism which is like that for glucagon (adenylate cyclase-linked) or insulin (tyrosine kinase-linked).     

In vitro apolipoprotein B mRNA editing: identification of a 27S editing complex.

Specific apolipoprotein B (apoB) mRNA editing can be performed in vitro on apoB RNA substrates. Native gels and glycerol gradient sedimentation have been used to determine the physical properties of the in vitro editing activity in rat liver cytosolic S100 extracts. ApoB RNA substrates were progressively assembled as 27S complexes for 3 hr with similar kinetics as seen for the accumulation of edited RNA. Assembly was not observed on RNAs from apoB deletion constructs that did not support editing. The 27S complex contained both edited and unedited RNA sequences. Inhibition of 27S complex assembly by vanadyl-ribonucleoside complexes was accompanied by inhibition of editing. Based on these data, we propose that the 27S complex is the in vitro "editosome," A "mooring sequence" model for RNA recognition and editosome assembly has been proposed involving RNA sequences flanking the edited nucleotide.     

The effects of growth hormone on blood pressure and renin secretion in hypophysectomized rats

The effects of hypophysectomy and GH on blood pressure and renin secretion were studied. Within 2 weeks after removal of the pituitary gland of rats, mean arterial blood pressure declined 30%, and heart rate fell 50%. No significant effect of hypophysectomy on blood volume or hematocrit was noted. Within 2 h after the iv administration of 10 micrograms ovine GH to hypophysectomized rats, blood pressure, but not heart rate, was restored to normal. Despite the hypotension, the PRA of hypophysectomized rats was not significantly greater than that of intact animals. This relatively low PRA could not be accounted for by a lack of renin per se, since kidneys of hypophysectomized rats contained at least as much renin as kidneys from intact rats. Neither the PRA of hypophysectomized rats nor the kidney renin content was significantly altered 24 h after a single injection of GH (100 micrograms, ip). When perfusions were performed at 100 mm Hg, the rate of renin secretion by isolated kidneys obtained from hypophysectomized rats was markedly lower than that of kidneys from intact rats. Reduction of mean perfusion pressure to 50 mm Hg or adding isoproterenol to the perfusate produced much smaller increases in renin secretion by kidneys of hypophysectomized rats than that observed in kidneys from intact rats. Kidneys obtained from hypophysectomized rats treated with GH 24 h earlier had a renin content similar to that of kidneys from untreated animals, but secreted renin at a much higher rate. Kidneys from hormone-treated hypophysectomized rats also exhibited a greater renin secretory response to low perfusion pressure or isoproterenol. These data indicate that hypophysectomy severely impairs the renin secretory response of the isolated kidney and that a single injection of GH can reverse this impairment. These observations suggest the possibility that the hypotension characteristic of hypophysectomized rats may, in part, reflect the lack of GH and alterations in the renin-angiotensin system.     

Growth hormone promotes early initiation of hepatocyte growth factor gene expression in the liver of hypophysectomized rats after partial hepatectomy.

GH accelerates hepatic regeneration in the rat. Hepatocyte growth factor (HGF), a potent hepatocyte mitogen in vitro, is considered to be a major regulator of hepatic regeneration. In the present study, the effects of GH and insulin-like growth factor-I (IGF-I) on HGF gene expression in regenerating rat liver was investigated. In hypophysectomized rats treated with GH, hepatic HGF mRNA levels were increased 3 h after partial hepatectomy and reached peak levels after 5 h. In rats with intact pituitaries and in hypophysectomized rats not given GH treatment, HGF mRNA levels in liver were unchanged during the first 5 h following hepatectomy and reached peak levels after 10-18 h. DNA synthesis in the liver of GH-treated rats increased from low levels 10 h after hepatectomy to peak levels after 18 h. In rats without GH treatment the synthesis of DNA was still low 18 h after hepatectomy and was increased after 26 h. Treatment of hypophysectomized rats with IGF-I promoted increases in hepatic HGF mRNA levels and DNA synthesis 3.5 h and 15 h after hepatectomy respectively. HGF mRNA levels were constantly lower after sham-hepatectomy than after partial hepatectomy. In summary, in hypophysectomized rats the responses of hepatic HGF gene expression and DNA synthesis to partial hepatectomy were both accelerated by treatment with GH or IGF-I.     

The influence of growth hormone and thyroxine on iodothyronine deiodinase activity in the liver, kidney and brown adipose tissue in hypophysectomized rats

The effects of GH and T4 substitution on peripheral iodothyronine deiodinase activity in the liver, kidney and brown adipose tissue of hypophysectomized rats were investigated. Animals were treated with GH (140 micrograms hGH/day), T4 (3 micrograms/day), GH plus T4 (same doses), or saline. Rats were killed 0, 4, 7 or 11 days after treatment was started. Non-hypophysectomized, age-matched rats were killed after 0 and 11 days and served as controls. GH plus T4 restored body weight gain to normal, whereas GH alone and T4 alone did not. Tissue deiodinase activity and T3 concentrations were severely depressed in the hypophysectomized rats compared with non-hypophysectomized controls (to less than 10%). GH substitution in hypophysectomized rats led to a slight but significant elevation in tissue iodothyronine deiodinase activity in the liver and kidney, without concomitant increases in T3. Deiodinase activity in brown adipose tissue did not differ from that in saline-treated controls. T4 administration normalized deiodinase activity and tissue T3 content in all the evaluated tissues. GH plus T4 resulted in a lesser increase in deiodinase activity than T4 alone in the liver and kidney (p less than 0.01 at day 11), whereas no significant difference was observed in brown adipose tissue. In conclusion, GH stimulates iodothyronine deiodinase activity of the liver and kidney in hypophysectomized rats. Moreover, when GH is administered together with T4, the T4-stimulated enzyme activity in the liver and kidney is downregulated, suggesting that GH attenuates (or modulates) the T4 effect on this specific enzyme activity.     

Pituitary regulation of postnatal small intestinal ontogeny in the rat: differential regulation of digestive hydrolase maturation by thyroxine and growth hormone

During the third week of postnatal life, dramatic ontogenic changes occur in the morphology and enzymology of the small intestine of the infant rat, enabling the animal to make the transition from milk to solid food. To investigate the roles of T4 and GH in regulation of these changes, infant rats were hypophysectomized on day 6 of life by the transauricular technique. Hypophysectomy resulted in diminution of somatic and intestinal growth as well as abnormal maturation of the disaccharidases lactase, sucrase, and maltase when measured on day 25. Administration of either T4 or GH to hypophysectomized animals resulted in moderately increased intestinal growth, while complete restoration of small intestinal growth resulted from administration of the combination of both hormones. Although T4, GH, or the combination of hormones reduced lactase activities, T4 alone produced normal maturation of sucrase and maltase. Neither hypophysectomy nor hormone replacement affected aminooligopeptidase. The molecular structure of lactase, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was not altered to a major degree in hypophysectomized animals or animals that received hormone replacement, but minor alterations were evident in sucrase structure in hypophysectomy. These studies indicate that 1) T4 and GH actively participate in postnatal regulation of small intestinal ontogeny; 2) thyroid hormones act directly on developing intestinal tissues to independently produce the normal maturation of the disaccharidases by mechanisms that are not likely to involve alterations in processing of the enzyme-protein; and 3) maturation of aminooligopeptidase is not regulated by pituitary hormones, in distinct contrast to the disaccharidases.     

Depletion of serum growth hormone in adult female rats by neonatal monosodium glutamate treatment without loss of female-specific hepatic enzymes P450 2d (IIC12) and steroid 5 alpha-reductase

The sexually dimorphic profiles of pituitary GH secretion play a key role in regulating the expression of several sex-dependent and developmentally controlled P-450 enzymes in rat liver. Current models for P-450 regulation by GH, however, are primarily based on hypophysectomy and GH replacement experiments. The present study examines the effects on hepatic P-450 expression of neonatal injections of monosodium glutamate (MSG), which allows for the nonsurgical suppression of adult GH levels. Furthermore, the levels of other pituitary-dependent hormones, such as testosterone and estradiol, are largely unchanged in the MSG-treated rats. Although hypophysectomy and GH replacement experiments have previously demonstrated that expression of the female-specific hepatic enzymes P-450 2d (gene product IIC12) and steroid 5 alpha-reductase is strikingly dependent on continuous GH exposure, neither enzyme was decreased in adult female rat liver after the elimination of plasma GH (less than 2 ng/ml) by neonatal MSG treatment. Moreover, although the loss of circulating GH appears to be largely responsible for the more than 10- to 20-fold elevation of the male-specific hepatic P-450 forms 2a (gene product IIIA2) and RLM2 (gene product IIA2) in hypophysectomized female rats, no such elevation of the male-specific P-450s was observed in the GH-deficient MSG-treated female rats. In contrast, the female-predominant forms P-450j (gene product IIE1) and 3 (gene product IIA1) were both elevated in adult rat liver after neonatal MSG treatment, in agreement with earlier hypophysectomy studies and demonstrating the suppressive effects that GH can exert on expression of these P-450 forms. Thus, although MSG and hypophysectomy both produce GH depletion, the responsiveness of the hepatic P-450s to these endocrine manipulations differs, allowing for an expanded understanding of the role of GH in P-450 expression.     

Growth hormone modulates serum cholinesterase

Adult male rats have lower serum cholinesterase activity levels than adult female rats and hypophysectomized male rats have higher activity levels than sham-operated males (similar to control females). GH administered to hypophysectomized male rats abolishes the effect of hypophysectomy on serum cholinesterase. Adult male rats treated neonatally with monosodium L-glutamate to induce arcuate nucleus lesions of the hypothalamus have higher serum cholinesterase activities and decreased serum GH concentrations. GH administered to these male rats results in decreased serum cholinesterase activities. These experiments demonstrate that GH is a negative modulator of serum cholinesterase in the male rat.     

Effect of hypophysectomy on growth hormone receptor gene expression in rat tissues

Two cDNA probes derived from the nucleic acid sequence for the rabbit GH receptor were used to study RNA samples from normal and hypophysectomized (hypox) rat tissues by Northern analysis. Results obtained with a probe that contained a nucleotide sequence corresponding to part of the extracellular domain of the GH receptor indicated that rat liver, gastrocnemius muscle, and epididymal fat each contain a 4.4-kilobase (kb) message and one or more shorter messages that appear to be homologous to the rabbit GH receptor message. The other probe, which contained a nucleotide sequence that corresponds to the intracellular domain of the GH receptor, detected only one 4.4-kb message in these rat tissues. These results suggest that rat tissues may synthesize several forms of the GH receptor, but only one form that contains a region homologous to the intracellular domain of the rabbit liver GH receptor. Hypophysectomy increased the abundance of the 4.4-kb message 5-fold in muscle and reduced it by a factor of 2 in adipose tissue. No significant difference was seen between GH receptor message levels of normal and hypox rat liver when the results were expressed as a fraction of the total RNA. The level of the beta-actin message was also measured in liver, muscle, and fat from normal and hypox rats. No significant differences were found when the message levels in normal rats were compared to those for the corresponding tissue in hypox rats. When normalized to the beta-actin message levels, a significant increase was seen in the relative amount of the GH receptor mRNA in muscle and liver of hypox rats. The increased levels of the GH receptor message in muscle and liver and the simultaneous decreased level in fat suggest that GH receptor synthesis may be regulated selectively in these tissues by hormonal factors that are altered by hypophysectomy.