Biosynthesis of the low molecular weight carrier protein for insulin-like growth factors in rat liver and fibroblasts

Biosynthesis of the low mol wt (Mr) carrier protein for insulin-like growth factors (IGFs) was studied in the BRL-3A rat liver cell line, rat embryo fibroblasts (REFs), and fetal rat liver by biosynthetic labeling of intact cells and cell-free translation of extracted RNA. [35S]Cysteine-labeled carrier protein precursors were immunoprecipitated using antibodies raised to the approximately 33,000 Mr carrier protein from BRL-3A cells that recognize the IGF carrier protein present in fetal and neonatal rat serum, but not in adult rat serum. The IGF carrier protein is synthesized as a 35,000 Mr precursor in a reticulocyte lysate translation system directed by RNA from BRL-3A cells or REFs. Supplementation of the translation incubation with microsomal membranes decreases the size of the precursor to 33,000 Mr, presumably by removal of a signal peptide. In continuous labeling or pulse-chase experiments of intact BRL-3A cells or REFs, the 33,000 Mr protein is labeled within 10 min intracellularly, appears in the medium after 40 min, and persists in the medium for 24 h without a change in size. The intracellular carrier protein was biosynthetically labeled in BRL-3A cells with [3H]leucine, [3H]phenylalanine, [3H]arginine, or [35S]cysteine and purified, and its NH2-terminal amino acid sequence was determined. Eleven of 34 residues were identified and correspond to those of mature unlabeled carrier protein purified from conditioned medium, indicating that after removal of the signal peptide, the carrier protein undergoes no detectable further processing at its NH2-terminus. These results establish that although they are regulated coordinately, IGF-II and the fetal IGF carrier protein are synthesized as separate proteins. Finally, RNA extracted from fetal, but not adult, rat liver directs the synthesis of the 35,000 Mr carrier protein precursor, suggesting that the developmental regulation of the carrier protein may occur at the level of RNA abundance.     

Tissue, developmental, and metabolic regulation of messenger ribonucleic acid encoding a rat insulin-like growth factor-binding protein

Insulin-like growth factor-II (IGF-II) is the predominant insulin-like growth factor in fetal and neonatal rat serum and tissues. In serum, it occurs complexed to a 30-kDa nonglycosylated IGF-binding protein (IGFBP) that is immunologically related to the IGFBP in BRL-3A rat liver cells (rIGFBP-2). Levels of rIGFBP-2 and IGF-II decrease in rat serum after birth. Using a recently isolated cDNA clone for rIGFBP-2 as hybridization probe, we now compare the expression of rIGFBP-2 and IGF-II in fetal tissues and the effects of hypophysectomy and fasting on the abundance of these mRNAs in adult rat liver. rIGFBP-2 mRNA is expressed at high levels in term gestation liver and at lower levels in other tissues. The ratio of rIGFBP-2 to IGF-II mRNAs in stomach, kidney, and lung is similar to that seen in liver, whereas IGF-II mRNA is more abundant than rIGFBP-2 mRNA in muscle, intestine, heart, and skin. Both mRNAs are more abundant in fetal tissues than in the corresponding tissues from adult rats. Dexamethasone treatment of 4-day-old rats for 4 days caused a greater (90%) decrease in hepatic IGF-II mRNA than in rIGFBP-2 mRNA (50%), suggesting subtle differences in the developmental regulation of the two mRNAs. Even more striking differences were observed in the regulation of the two mRNAs in adult rats after hypophysectomy or fasting. Hepatic rIGFBP-2 mRNA was increased 10- to 20-fold compared to age-matched control rats, whereas IGF-II mRNA was not increased. A parallel increase in serum rIGFBP-2 was observed, suggesting that this regulation may result at least in part from the increased abundance of rIGFBP-2 mRNA. Thus, in addition to modulating the stimulation of growth and differentiation by IGF-II in fetal tissues, rIGFBP-2 may play a homeostatic role during catabolic states in the adult rat.     

Tissue-specific expression and developmental regulation of the rat apolipoprotein B gene.

Expression of the apolipoprotein B (apoB) gene was examined in a variety of fetal, neonatal, and adult rat tissues by probing RNA blots with a cloned rat apoB cDNA. Among 10 adult male tissues surveyed, small intestine had the highest concentration of apoB mRNA. Its abundance in liver and adrenal gland was 40% and 0.5%, respectively, of that in small bowel, while none was detected in colon, kidney, testes, spleen, lung, heart, or brain. ApoB mRNA is as abundant in 18-day fetal liver as at any subsequent period of hepatic development. In contrast, the concentration of apoB mRNA remains low in fetal intestine until the last (21st) day of gestation, when it increases sharply to levels that are several-fold higher than in the liver. ApoB mRNA levels in fetal membranes harvested during this late gestational period were 10 times greater than in fetal liver. Since the major lipoprotein species in 19-day fetal plasma is low density lipoprotein, these observations suggest that fetal liver, and particularly its functional homologue, the yolk sac, are the principal sites of fetal lipoprotein synthesis at this stage of development. A 20-fold increase in placental apoB mRNA concentrations during the last 48 hr of pregnancy (to a level that is 50% of that encountered in fetal membrane RNA) suggests a specific role for this organ in maternal-fetal lipid transport immediately prior to parturition. Pulse-labeling experiments using 21-day fetal tissue slices showed that the liver synthesizes both apoB-100 (B-PI) and apoB-48 (B-PIII) albeit in somewhat different ratios than the adult organ. Fetal intestine produces almost exclusively the smaller apoB species, while fetal membranes and placenta synthesize only the larger peptide. The postnatal pattern of apoB mRNA accumulation is similar in liver and intestine. Profound decreases were observed during the late suckling and weaning periods, followed by an increase to adult levels. These final concentrations were similar to those encountered at birth. Analysis of these developmental changes offers an opportunity to generate testable hypotheses about the factors that modulate apoB synthesis.     

Expression of the insulin-like growth factor-II/mannose-6-phosphate receptor in multiple human tissues during fetal life and early infancy.

The insulin like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor has been detected in many cells and tissues. In the rat, there is a dramatic developmental regulation of IGF-II/M6P receptor expression, the receptor being high in fetal and neonatal tissues and declining thereafter. We have systematically studied the expression of the human IGF-II/M6P receptor protein in tissues from 10 human fetuses and infants (age 23 weeks gestation to 24 months postnatal). We have asked 1) whether there is differential expression among different organs, and 2) whether or not the human IGF-II/M6P receptor is developmentally regulated from 23 weeks gestation to 24 months postnatal. Protein was extracted from human tissues using a buffer containing 2% sodium dodecyl sulfate and 2% Triton X-100. Aliquots of the protein extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an anti-IGF-II/M6P receptor antiserum (no. 66416) and 125I-protein A or an immunoperoxidase stain. IGF-II/M6P receptor immunoreactivity was detected in all tissues studied with the highest amount of receptor being expressed in heart, thymus, and kidney and the lowest receptor content being measured in brain and muscle. The receptor content in ovary, testis, lung, and spleen was intermediate. The apparent molecular weight of the IGF-II/M6P receptor (220,000 kilos without reduction of disulfide bonds) varied among the different tissues: in brain the receptor was of lower molecular weight than in other organs. Immunoquantitation experiments employing 125I-protein A and protein extracts from human kidney at different ages revealed a small, albeit not significant, difference of the receptor content between fetal and postnatal tissues: as in other species, larger amounts of receptor seemed to be present in fetal than in postnatal organs. In addition, no significant difference of the receptor content between human fetal liver and early postnatal liver was measured employing 125I-protein A-immunoquantitation in three fetal and five postnatal liver tissue samples. The distribution of IGF-binding protein (IGEBP) species, another abundant and major class of IGF binding principles, was also measured in human fetal and early postnatal lung, liver, kidney, muscle, and brain using Western ligand blotting with 125I-IGF-II: as with IGF-II/M6P receptor immunoreactivity there was differential expression of the different classes of IGFBPs in the various organs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relative expression of aromatase cytochrome P450 in human fetal tissues as determined by competitive polymerase chain reaction amplification.

The aromatase enzyme complex is responsible for the conversion of C19 steroids to estrogens. Aromatase activities ranging from moderate to very low have been measured in human fetal tissues. The inability to demonstrate aromatase cytochrome P450 (P450AROM) messenger RNA (mRNA) in several fetal tissues by northern blotting has been attributed to low levels of specific message. In order to identify and compare P450AROM mRNA levels in fetal tissues, we developed a specific competitive polymerase chain reaction amplification technique. This reaction uses coamplification of a rat P450AROM complementary RNA to normalize differences in amplification efficiencies. Using this technique, P450AROM mRNA was identified in all fetal tissues studied including; liver, lung, brain, skin, intestine, kidney, spleen, and heart. Fetal liver contained far more P450AROM mRNA per total RNA than any other tissues studied. Fetal brain and intestine also tended to have slightly higher levels than other tissues.     

Secretion of soluble insulin-like growth factor-II/mannose 6-phosphate receptor by rat tissues in culture.

The insulin-like growth factor-II/mannose 6 phosphate receptor, which binds both IGF-II and mannose 6-phosphate containing proteins, such as lysosomal enzymes, has been detected in the serum of several species. Neither the source nor the role of this soluble, truncated form of the membrane receptor has been determined. This study has examined the ability of a variety of rat tissues in culture to synthesize and release soluble receptor. Explants (1 mm3) of 21-day fetal, neonatal and adult rat tissues were cultured in serum-free medium and the conditioned medium analyzed for the presence of receptor. Using IGF-II binding and immunochemical techniques, receptor was detected in media from heart, kidney, liver, lung and muscle explants. [35S]cysteine labeling indicated that de novo synthesis of the soluble receptor occurs in the cultured explants and can be inhibited by cycloheximide. This rat tissue explant culture system demonstrates that soluble receptor is released by a number of tissues, and should provide a useful model for further investigations into its function and regulation.     

Expression of IGF-II in early experimental hepatocellular carcinomas and its significance in early diagnosis

AIM: To investigate the serum level and expression of insulin growth factor II (IGF-II) in liver tissues of rats with early experimental hepatocellular carcinomas (HCC) and its significance in early diagnosis. METHODS: Early experimental hepatocellular carcinomas were induced by diethylnitrosamine (DENA) in 180 male SD rats. Another 20 male SD rats served as control. The IGF-II serum level was measured by ELISA. Immunohistochemistry and electron microscopic immunohistochemistry were used to observe the expression of IGF-II in normal and tumor liver tissues and its ultrastructural location in malignant hepatocytes. The expressions of IGF-II in human hepatoma cell lines HepG2, SMMC7721 and human embryonic liver cell line L-02 were measured by immunocytochemistry. IGF-II mRNA level was studied by in situ hybridization. RESULTS: IGF-II was expressed in the cytoplasm of both sinusoidal cells in paracancerous cirrhotic liver tissue and malignant hepatocytes in early experimental HCC tissues. Gold particles were seen on the rough endoplasmic reticulum and the mitochondrion in malignant hepatocytes. IGF-II was expressed in the human hepatoma cell lines. The mRNA level of IGF-II was higher in rat liver tumor tissue than in normal rat liver tissue. The serum IGF-II level of the early experimental HCC group was 34.67+/-10.53 ng.ml(-1) and that of the control group was 11.75+/-5.84 ng.ml(-1). The rank sum test was used for statistical analysis. There was a significant difference between the two groups (P<0.01). CONCLUSION: During the induction of early experimental HCC by DENA, IGF-II may promote hepatocytic proliferation via a paracrine mechanism in the pre-cancerous stage. When hepatocytes are transformed into malignant cells, they may secrete IGF-II and promote malignant cell proliferation by an autocrine mechanism. IGF-II may be a possible biological marker in the early diagnosis of HCC.     

Insulin-like growth factor II (IGF-II) mRNA expression during hepatocarcinogenesis in transgenic mice.

Insulin-like growth factor II (IGF-II) mRNA expression is developmentally regulated in liver tissue. We previously observed the reexpression of fetal IGF-II mRNAs in human primary liver cancer and in surrounding cirrhotic tissue. In order to determine the steps of liver cancer progression where the activation of IGF-II fetal mRNAs occurs, we analyzed IGF-II mRNA expression during hepatocarinogenesis in transgenic mice carrying an antithrombin III-SV40 early region hybrid gene. The comparative analysis of mRNAs encoding IGF-II and other differentiation-associated proteins, as well as histological analysis, indicate that the reexpression of fetal IGF-II mRNAs takes place in specific steps of liver cancer progression, both in early pretumorous lesions and in well-differentiated hepatocellular carcinomas.     

Expression of the genes for insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding proteins-1 and -2 in fetal rat under conditions of intrauterine growth retardation caused by maternal fasting.

Evidence suggests that insulin-like growth factors-I and -II (IGF-I and II) play a role in regulating fetal growth and development. In the fetus, IGF-I and -II are complexed with two specific binding proteins (IGFBP-1 and -2), which are thought to modulate the actions of the IGFs in target tissues. We examined regulation of the genes for IGF-I, IGF-II, IGFBP-1, and IGFBP-2 in fetal rat liver in an experimental model for intrauterine growth retardation caused by maternal fasting on days 17-21 of gestation. The mean weight of fetuses from the fasted dams was 27-32% lower than the mean weight of fetuses from the fed dams. The concentration of immunoreactive IGF-I was decreased by 71% in serum of fetuses from the fasting dams. The concentration of immunoreactive IGF-II was slightly decreased (by 12%) in serum of fetuses from the fasting dams, whereas the concentration of immunoreactive pro-IGF-II E-domain peptide was decreased by 31%. The abundance of hepatic IGF-I mRNA was decreased by 55% in fetuses from the fasting dams. In contrast, the abundance of IGF-II mRNA in fetal liver was not significantly decreased by maternal fasting. Maternal fasting caused a 2-fold increase in the abundance of IGFBP-1 mRNA in fetal liver, whereas it did not change the abundance of IGFBP-2 mRNA. The induction of IGFBP-1 mRNA in liver of the growth-retarded fetuses is similar to the induction that occurs in liver of fasting adults, while the lack of regulation of IGFBP-2 mRNA differs from the strong induction of IGFBP-2 mRNA that occurs in liver of fasting adults. In summary, these results indicate that maternal fasting causes a decrease in fetal IGF-I gene expression, a decrease in fetal serum IGF-I, and a slight decrease in fetal serum IGF-II and pro-IGF-II E-domain peptide concentrations. Maternal fasting also causes an increase in fetal IGFBP-1 gene expression. Changes in fetal insulin and glucose may be related to changes in expression of the IGF-I and IGFBP-1 genes in the growth-retarded fetuses. The decreased expression of IGF-I and -II and increased expression of the IGFBP-1 gene may contribute to the fetal growth retardation observed in this model system.     

Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets.

The relative amount of apolipoprotein (apo-) E mRNA in 12 different tissues of the rat and marmoset was examined by dot blot hybridization using cloned cDNA probes. As expected, it was found to be most abundant in the liver. However, substantial amounts of apo-E mRNA were found in the brain and adrenals at relative levels about one-third of that found in the liver. Significant quantities of apo-E mRNA were detected in all of the other peripheral tissues as well. The apo-E mRNA levels in these tissues were 2-10% of that found in the liver of the rat and 10-30% of that found in the liver of the marmoset. Apo-E mRNA was also abundant in human brain and in each species examined; it was distributed throughout all major areas of this organ. In contrast, apo-A-I mRNA was detected in abundant amounts only in the small intestine and in the liver. Extrahepatic apo-E mRNA appears to be functional, generating a translation product similar or identical to that generated by the liver. During fetal and neonatal development, apo-E mRNA is rapidly induced from low levels to approximately equal to 60% of adult levels in liver at parturition. The fetal yolk sac contains more apo-E mRNA than the fetal liver, suggesting a significant role for the yolk sac as a source of apo-E during gestation.     

Cholesterol synthesis and accretion within various tissues of the fetal and neonatal rat

The rate of cholesterol synthesis is reported to be higher in fetal relative to adult rats. Along with the observation that maternal diets high in fat and cholesterol are unable to alter the rate of cholesterol synthesis in the fetus, this has been taken as indirect evidence that the fetal rat meets its cholesterol needs through de novo synthesis. This study quantified the rates of cholesterol synthesis and accumulation in the liver, brain, intestine, and carcass of the fetal and neonatal rat and the placenta to determine whether these developing tissues are able to support their own cholesterol needs without the uptake of plasma lipoprotein cholesterol. The rate of cholesterol synthesis was determined in vivo using [3H]water. The rate of cholesterol accumulation was determined by calculating the difference in tissue cholesterol content between 2 subsequent days of development. Total fetal body cholesterol synthesis was sufficient to support the rate of cholesterol accumulation. Fetal and neonatal liver synthesized cholesterol at a rate in excess of cholesterol accumulation, suggesting hepatic secretion of cholesterol into the plasma. Before the onset of suckling, the rates of de novo cholesterol synthesis in the intestine, brain, and carcass were also sufficient but not higher than the need for cholesterol accretion. After the establishment of suckling, the rate of cholesterol accumulation in the intestine and carcass was in excess of synthesis, suggesting that neonatal tissues derive some of their cholesterol from dietary milk or liver. These studies suggest that the perinatal rat does not require exogenous cholesterol to support tissue cholesterol accretion. However, the fetal liver may support cholesterol accretion in other tissues through rates of synthesis in excess of accumulation and secretion into plasma. The placenta may derive some cholesterol from the maternal and/or fetal plasma.     

Pituitary control of growth in the neonatal rat: effects of neonatal hypophysectomy on somatic and organ growth, serum insulin-like growth factors (IGF)-I and -II levels, and expression of IGF binding proteins.

The neonatal period is a time of transition between pituitary-independent fetal growth and the pituitary-dependent growth seen in older mammals. To evaluate pituitary-dependent neonatal growth, Wistar rats were hypophysectomized (Hx) on postnatal day 6. Nineteen days post-Hx, body weight and tail length were inhibited 48% and 34%, respectively, compared with sham-Hx controls. Organ weights determined on days 10, 15, 20, 25, and 30 revealed three patterns of pituitary-dependence: 1) pituitary-independent growth in the brain and lung; 2) moderate pituitary-dependent growth in the heart, liver, kidney, and intestine; and 3) marked pituitary-dependent growth in the adrenals, spleen, and testes. Both serum insulin-like growth factor (IGF)-I and -II levels fell significantly in Hx pups by 54 h after Hx (P = 0.0005), and Northern analysis on day 15 showed a significant decrease in liver messenger RNA (mRNA) for IGF-II. Analysis of the major IGF binding proteins (BPs) was performed by Western ligand blots. Hx performed on day 6 resulted in a linear decrease in the amount of the 22k BP from day 10 to day 30. In contrast, the major neonatal BP (IGFBP-2, a 29.5k molecule) showed a biphasic response to neonatal Hx. On postnatal day 10, 4 days after Hx, a significant decrease in IGFBP-2 occurred, which persisted through day 15; by postnatal day 20 and continuing through postnatal day 30, the amount of IGFBP-2 in the serum dramatically increased. The 40 to 50k fraction of IGFBP-3 first appeared in significant quantities by postnatal day 20, and after Hx dropped to 10% of sham-control values. Similarly, Northern analysis on day 15 demonstrated a significant decrease in liver, but not brain, mRNA for IGFBP-2 after Hx, whereas on postnatal day 25, liver mRNA for IGFBP-2 was increased in Hx pups compared with sham controls. We conclude that the pituitary gland exerts significant but selective effects on neonatal growth, with the notable exception of brain growth. Serum levels of both IGF-I and IGF-II, as well as their BPs, are pituitary dependent in the neonatal period. Pituitary-dependent neonatal growth thus appears to be mediated by IGF and modulated by IGF-binding proteins. On the other hand, that portion of the persistent growth in the neonatal Hx rat that is independent of the pituitary-IGF axis may be a good model for investigation of fetal growth.     

Effect of Ethanol on Insulin-Like Growth Factor-II Release from Fetal Organs

This study examines the effect of ethanol (ETOH) exposure and nutrient restriction on the release of insulin-like growth factor (IGF)-II from 18- and 20-day explanted fetal organs. Fetuses were exposed to ETOH (E) in utero by feeding dams a 36% (calories derived from ETOH: 6.6% v/v) ETOH liquid diet. Control fetuses were offsprings of dams either pair-fed (P) a control liquid diet or ad libitum (A) fed a standard pelleted lab chow. Brain, heart, kidney, liver, lung, muscle, and placenta of fetuses from the same litter were pooled and explanted, and IGF-II concentration in explanted media was analyzed by radioimmunoassay. Maternal and fetal weights were determined during pregnancy and at sacrifice, respectively, to evaluate the influence of ETOH on growth. Both maternal and fetal weights were substantially reduced by ETOH on 18 and 20 days of gestation compared with both A and P controls. At 18 days of gestation, E fetuses (1.33 ± 0.03 g) weighed less than either A (1.47 ± 0.03 g) or P (1.54 ± 0.04 g) fetuses. By 20 days, A mean fetal weight (4.19 ± 0.23 g) was significantly greater than both P (3.74 ± 0.06 g) and E (3.28 ± 0.06 g) fetuses. IGF-II concentration in media from 18-day fetal explants was highest from E (brain, heart, liver, and placenta) and P tissues (kidney, lung, and muscle). IGF-II in media from A tissues (except placenta) was lower than both E and P levels. A significant difference between treatments occurred in heart. By 20 days, IGF-II levels were highest in media from all A tissues (except placenta). IGF-II in media from E tissues (except lung) was lower than those from P tissues. A significant difference between treatments occurred in the brain. With regard to the developmental pattern, IGF-II release generally increased between 18 and 20 days of gestation, with the greatest increases occurring in A tissues. Increased secretion by P tissues was greater than that by corresponding E tissues, and tended to follow the A trend. On the other hand, E brain, kidney, and placenta released only slightly more IGF-II at 20 days compared to 18 days, whereas E heart, liver, lung, and muscle released slightly less hormone. This study suggests that even moderate nutrient deprivation influences the pattern of IGF-II release from fetal organs, even though there is only a small decrease in overall body size. At the same level of nutrient deprivation, ETOH more dramatically alters both fetal weight and the pattern of IGF-II release. Because IGFs are autocrine/ paracrine factors that influence growth, differentiation, and function, the reduced availability of IGF-II may be one of the factors contributing to ETOH-induced growth retardation and impaired functional capacity of some organ systems.