Fetal Liver Membranes Research Articles

A short periconceptional exposure to maternal type-1 diabetes is sufficient to disrupt the feto-placental phenotype in a rabbit model

Tight metabolic control of type-1 diabetes is essential during gestation, but it could be crucial during the periconception period. Feto-placental consequences of maternal type-1 diabetes around the time of conception need to be explored.Using a rabbit model, type-1 diabetes was induced by alloxan 7 days before mating. Glycemia was maintained at 15–20 mmol/L with exogenous insulin injections to prevent ketoacidosis. At 4 days post-conception (dpc), embryos were collected from diabetic (D) or normoglycemic control (C) dams, respectively, and transferred into non-diabetic recipients. At 28dpc, D- and C-feto-placental units were collected for biometry, placental analyses and lipid profiles.D-fetuses were growth-retarded, hyperglycemic and dyslipidemic compared to C-fetuses. The efficiency of D-placentas was associated with an increased gene expression related to nutrient supply and lipid metabolism whereas volume density of fetal vessels decreased.Fetal plasma, placental and fetal liver membranes had specific fatty acid signatures depending on embryonic origin. Tissues from D-fetuses contained more omega-6 polyunsaturated fatty acids. The concentrations of docosahexaenoic acid decreased while linoleic acid increased in the heart of D-fetuses.This study demonstrates that a short exposure to maternal type-1 diabetes in the periconception window, until the blastocyst stage, is able to irreversibly malprogram the feto-placental phenotype, through precocious and persistent structural and molecular adaptations of placenta.

119 A SHORT PERICONCEPTIONAL MATERNAL HYPERGLYCEMIA IS SUFFICIENT TO DISRUPT THE FETO-PLACENTAL PHENOTYPE IN A RABBIT MODEL

Pre-gestational type 1-diabetes (T1D) increases the risk of miscarriage and congenital malformations and programs the offspring to develop metabolic syndrome in adulthood. Management of maternal diabetes is essential during gestation but could be also highly important around the time of conception. Using a rabbit model, the effects of maternal T1D during the periconceptional period on pre-implantation blastocysts have been well documented, but the effects on feto-placental phenotype at 28 dpc (term = 31 days) has not been explored. Diabetes was induced by Alloxan in dams 7 days before mating. Glycemia was maintained at 15 to 20 mmol L–1 with exogenous insulin injections. At 4 dpc, embryos were collected and transferred into nondiabetic recipients. At 28 dpc, control (C) and diabetic (D) fetuses were collected for biometric records, placental analyses including stereology and gene expression, and lipid profiles of feto-placental tissues by gas chromatography. Lipid data were analysed by principal component analysis. D-fetuses were growth retarded, hyperglycemic, and dyslipidemic compared with C fetuses. Moreover, placental efficiency was much higher in D- than in C-fetuses. The volume density of fetal vessels was significantly decreased in D-placentas compared to C-placentas, whereas the volume density of trophoblast tended to increase (P = 0.051). This morphometric disruption was associated with a deregulation of the expression of genes related to nutrient supply and lipid metabolism. In fetal plasma, a specific fatty acid signature was observed in D- and C-groups. Moreover, the composition of placental and fetal liver membranes differed according to maternal status and fetal sex. Tissues from D-fetuses contained significantly more n-6 polyunsaturated fatty acids compared with C. Docosahexaenoic acid decreased whereas linoleic acid increased in the cardiac membranes of D-fetuses, indicating a higher risk of ischemia. This study demonstrates that exposure to high plasma glucose during the short periconceptional period is sufficient to adversely program fetal phenotype by reducing fetal growth, altering placental function and lipid profiles in all fetal tissues.

Uteroferrin induces lipid peroxidation in endometrial and conceptus microsomal membranes and is inhibited by apotransferrin, retinol binding protein, and the uteroferrin-associated proteins.

Iron-containing proteins catalyze lipid peroxidation when combined with either H2O2 or ascorbic acid (ASC). Microsomal membranes were prepared from Day 13 endometrial and conceptus tissues (5 pigs) and from Day 30 endometrial, placental, fetal liver, and fetus minus fetal liver tissues (5 pigs). Microsomal membranes were subjected to the following in vitro treatments: 1) no treatment, 2) 50 microM ASC, 3) 100 microM uteroferrin (UF), 4) 50 microM ASC + 100 microM UF, 5) 50 microM ASC + 100 microM UF + 10 microM apotransferrin (transferrin with no iron bound; ATF), and 6) 50 microM ASC + 100 microM UF + 10 microM holotransferrin (transferrin saturated with iron; HTF). For treatments 7 through 10, membranes were preincubated (0 degrees C, 3 h) with either 7) no treatment, 8) 50 microM fetuin, 9) 50 microM holoretinol binding protein (holoRBP: retinol binding protein [HoloRBP] with retinol bound), or 10) 50 microM apoRBP (RBP with no retinol bound) followed by incubation with 50 microM ASC + 100 microM UF. Lipid peroxidation was measured in the samples as thiobarbituric acid reactive substances (TBARS). Endogenous TBARS were greater (p < 0.05) in Day 13 conceptus than in Day 13 endometrium and were highest (p < 0.05) on Day 30 in fetal liver. Combined ASC and UF caused a large increase (p < 0.05) in TBARS in all membranes except Day 30 placental membranes. Addition of ATF, but not HTF, decreased TBARS production in all membrane preparations. HoloRBP, but not fetuin or apoRBP, decreased (p < 0.05) TBARS production in all but Day 30 endometrial membranes. In other experiments, when combined with ASC, UF/UF-associated protein complex induced less (p < 0.01) lipid peroxidation in fetal liver microsomal membranes than did free UF. Catalase and superoxide dismutase had no effect on UF-induced lipid peroxidation in fetal liver membranes. These results indicate that 1) UF combined with ASC induces lipid peroxidation in Day 13 endometrial and conceptus and Day 30 endometrial, fetal liver, and fetus minus liver microsomal membranes, and 2) ATF, holoRBP, and the UF-associated proteins, but not catalase or superoxide dismutase, inhibit this reaction.

The growth hormone receptor messenger ribonucleic acid present in ovine fetal liver is a variant form.

Specific binding of ovine GH (oGH) to microsomal membranes isolated from fetal sheep liver is slight to nonexistent. The complementary DNA sequence encoding the oGH receptor (oGHR) has been reported, and Northern blot analysis has indicated that oGHR messenger RNA (mRNA) is present in fetal liver and skeletal muscle from mid- to late gestation. In human tissues, the GHR mRNA exists in multiple forms, including the deletion of exon 3 and variable 5'-untranslated regions. In rodents, the GHR mRNA exists in two forms, one encoding the membrane-bound receptor and the other encoding the soluble GH-binding protein. To further characterize the oGHR mRNA transcript present in ovine fetal liver during gestation, we designed a series of primers to be used in reverse transcriptase-polymerase chain reactions (RT-PCR), which generate products that span from the 5'-untranslated region through the coding region of the oGHR mRNA. Nucleotide sequences of the resulting complementary DNAs revealed that an oGHR mRNA is present from mid- to late gestation (days 60-135) which contains the region analogous to exon 3 of the human GHR gene. However, the 5'-untranslated region previously reported in adult tissues was not present until day 135 of gestation in fetal liver, nor was it present in day 100 fetal skeletal muscle. Northern hybridization analysis indicates that the major oGHR transcript in day 105 fetal liver is 5.8 kilobases (kb) in size, with minor transcripts observed at 4.7 kb and three transcripts greater than 6.5 kb. By day 135 of gestation, the transcript size is the same as that observed in day 100 pregnant ewe liver (5.5 kb). We conclude that the oGHR mRNA present in midgestation fetal liver differs structurally from the transcript present in late gestation fetal liver and adult liver, and this difference may explain the lack of specific GH binding to ovine fetal liver membranes. Furthermore, our results suggest that there is a developmental switch in the structure of oGHR mRNA that occurs shortly before term, potentially preparing the fetus to respond to GH postnatally.

The growth hormone receptor messenger ribonucleic acid present in ovine fetal liver is a variant form

Specific binding of ovine GH (oGH) to microsomal membranes isolated from fetal sheep liver is slight to nonexistent. The complementary DNA sequence encoding the oGH receptor (oGHR) has been reported, and Northern blot analysis has indicated that oGHR messenger RNA (mRNA) is present in fetal liver and skeletal muscle from mid- to late gestation. In human tissues, the GHR mRNA exists in multiple forms, including the deletion of exon 3 and variable 5'-untranslated regions. In rodents, the GHR mRNA exists in two forms, one encoding the membrane-bound receptor and the other encoding the soluble GH-binding protein. To further characterize the oGHR mRNA transcript present in ovine fetal liver during gestation, we designed a series of primers to be used in reverse transcriptase-polymerase chain reactions (RT-PCR), which generate products that span from the 5'-untranslated region through the coding region of the oGHR mRNA. Nucleotide sequences of the resulting complementary DNAs revealed that an oGHR mRNA is present from mid- to late gestation (days 60-135) which contains the region analogous to exon 3 of the human GHR gene. However, the 5'-untranslated region previously reported in adult tissues was not present until day 135 of gestation in fetal liver, nor was it present in day 100 fetal skeletal muscle. Northern hybridization analysis indicates that the major oGHR transcript in day 105 fetal liver is 5.8 kilobases (kb) in size, with minor transcripts observed at 4.7 kb and three transcripts greater than 6.5 kb. By day 135 of gestation, the transcript size is the same as that observed in day 100 pregnant ewe liver (5.5 kb). We conclude that the oGHR mRNA present in midgestation fetal liver differs structurally from the transcript present in late gestation fetal liver and adult liver, and this difference may explain the lack of specific GH binding to ovine fetal liver membranes. Furthermore, our results suggest that there is a developmental switch in the structure of oGHR mRNA that occurs shortly before term, potentially preparing the fetus to respond to GH postnatally.

Localization of the growth hormone receptor, identified by immunocytochemistry, in second trimester human fetal tissues and in placenta throughout gestation.

Pituitary GH secretion appears largely unnecessary for the attainment of normal birth size in many species, including man. This is believed to be due to an immaturity and/or an absence of GH receptors in many fetal tissues. However, in vitro studies using late first trimester human fetal tissues have demonstrated mitogenic actions of GH on liver and stimulation of insulin biosynthesis in pancreas. To resolve this discrepancy, we have employed immunocytochemistry to identify the presence and distribution of GH receptors in various human fetal tissues. Fetuses of 14-16 weeks gestation were obtained after therapeutic abortion, tissues were fixed, and immunocytochemistry was performed using monoclonal antibodies against purified rat or rabbit GH receptor. The specificity of staining was confirmed by preabsorption of the antibodies with 1) adult rat liver membranes or 2) human fetal liver membranes, both of which possess specific GH-binding sites, or 3) human fetal skeletal muscle membranes, which do not specifically bind GH. Positive staining was seen in a subpopulation of liver parenchymal cells, in the ductal and endocrine tissue of pancreas, in the germinal layer of the epidermis and the deeper dermal layers of skin, and in the tubular epithelium of kidney. No immunopositive staining was seen in skeletal or cardiac muscle, epiphyseal growth plate, lung, intestine, or adrenal. Positive staining was present in the neuronal cell bodies of the cerebral cortex. GH receptor was also detectable as early as 8 weeks gestation in syncytial layers of the placenta and was maintained until term. Results demonstrate the presence of immunoreactive GH receptor/binding protein in some human fetal tissues early in development. In particular, these results would support a role for GH in the growth and function of liver and pancreas.

Evidence for the expression of non-HLA-A,-B,-C class I genes in the human fetal liver.

HLA class I Ag expression was investigated in the human fetal liver. By immunostaining, mAb to monomorphic class I Ag showed widespread reactivity with both epithelial and hemopoietic cells. By contrast, mAb to polymorphic determinants showed more restricted reactivity that was confined to a proportion of hemopoietic cells: the hepatic epithelium was essentially unreactive. This suggested that the developing liver might express nonclassical HLA class I. Class I Ag were examined in membrane and cytosol fractions of mid-trimester fetal liver. Because of its broad reactivity with HLA-A,-B, mAb Q1/28 was selected to identify classical class I Ag in these studies. Immunoprecipitations were carried out against radiolabeled glycoprotein extracts of fetal liver membranes. W6/32 detected a 40-kDa product characteristic of nonclassical class I proteins, as well as a 43-kDa product, in lysates immunodepleted with Q1/28. By immunoblotting, an anti-H chain antiserum (HC) identified a Q1/28- 40-kDa component and a 43-kDa Q1/28+ component in fetal liver membrane glycoproteins. The fetal liver cytosol fraction was found to contain a 42- to 43-kDa product by W6/32-chromatography. This component partitioned to the aqueous phase upon condensation in TritonX-114 detergent and by immunoblotting was reactive with monomorphic mAb HC10 but not with Q1/28. Total RNA and polymerase chain reaction amplified class I transcripts of fetal liver were probed using oligonucleotides specific for HLA-E, -F, and -G. HLA-F, was readily detected in total RNAs by Northern analysis. HLA-E, HLA-F, and HLA-G were all detected in fetal liver by polymerase chain reaction. Differential expression of these genes may occur between the first and second trimester of liver development. Overall therefore, the human fetal liver expresses multiple class I protein products and contains transcripts for non-classical class I genes; particularly HLA-F.

Ontogeny of Hepatic Type I Insulin-Like Growth Factor Receptors in the Rat

We studied the pre- and postnatal developmental regulation of the hepatic type I IGF receptor in the rat. Fetal rat liver membranes bound IGF-I throughout the latter part of gestation (d 17 to 21). After birth, binding diminished rapidly, reaching barely detectable levels by the 13th postnatal d. However, the presence of type I IGF receptors was readily demonstrated by affinity-labeling throughout the immediate postnatal period and in adult rats. Furthermore, IGF-I-dependent autophosphorylation of type I receptors could be seen in both fetal and adult liver membranes. Fasting for 48 h in adult rats led to a 2- to 3-fold increase in affinity-labeled type I IGF receptors. In contrast, nutrient deprivation to the fetus, via maternal fasting, did not alter fetal hepatic IGF-I binding or affinity-labeling of the type I receptor. These results support a role for the IGF and the type I IGF receptor in the autocrine/paracrine regulation of hepatic growth through the latter stages of gestation in the rat. The demonstration of enzymatically active type I IGF receptors in adult liver, and their increased expression in fasted adult rats is consistent with an autocrine/paracrine role for hepatic IGF-I in the adult.

Hepatic Protein Kinase-C and Protein Phosphatase Type-2A in the Fetal Rat

The Ca2(+)- and phospholipid-dependent protein kinase, protein kinase-C (PK-C), was studied in fetal rat liver between d 17 and 21 of gestation. Initial studies showed that rat liver, membrane-associated PK-C could be detected as a protein of Mr = 80,000 using a polyclonal rat brain PK-C antiserum. Fetal hepatic membrane-bound PK-C activity rose as gestation progressed with adult levels (21 +/- 3 pmol/min/mg protein) being attained by term. Although fasting for 48 h led to PK-C activation in adult livers, fetal hepatic PK-C was not activated by 48 h of maternal fasting. Membrane-associated protein phosphatase activity that might reverse PK-C action was also studied. PK-C sites in casein (an artificial PK-C substrate) were selectively dephosphorylated by a membrane-associated, poly-cation-stimulated protein phosphatase. This activity, thus classified as protein phosphatase type-2A, was constitutively expressed in fetal liver membranes from 17-21 d gestation. We have previously reported that the other major hepatic protein phosphatase, protein phosphatase type-1, also is constitutively expressed during the later stage of gestation. Taken together with the results of our present study, our data indicate that PK-C-dependent phosphorylation in fetal liver probably increases with advancing gestation.

Expression of Hepatic Transforming Growth Factor Receptors During Late Gestation in the Fetal Rat*

Transforming growth factor-alpha (TGF alpha) promotes DNA synthesis in adult rat hepatocytes, an effect opposed by picomolar concentrations of TGF beta. Recently, the presence of these growth factors in fetal rat liver has been demonstrated. Since a regulatory role for TGF alpha and TGF beta in fetal hepatic growth requires the presence of high affinity receptors, the receptors for these hormones were studied in membranes from normal fetuses at 17-21 days gestational age and growth-retarded fetuses of mothers fasted for 48 h. Fetal liver membranes bound [125I]epidermal growth factor ([125I]EGF) with high affinity (Kd = 1-2 nM). TGF alpha could compete with EGF for the same binding site, albeit at 4-fold lower affinity. EGF receptor number increased from nearly undetectable levels at 17 days to adult levels (0.15-0.3 nmol/mg membrane protein) by 21 days. Affinity labeling of fetal liver membranes with [125I]TGF alpha identified the 170,000 mol wt (Mr) EGF receptor. The intensity of labeling correlated with EGF receptor number based on binding analyses. TGF beta bound to fetal liver membranes with high affinity (Kd = 30 pM) and at a level (20-30 pmol/mg throughout late gestation) that was 3-fold higher than in adult liver. Affinity labeling of fetal hepatic membranes with [125I]TGF demonstrated high affinity 85,000 Mr TGF beta receptors and lower affinity 66,000 and 130,000 Mr receptors. Although TGF beta binding did not change with advancing gestation, affinity labeling of the 85,000 Mr protein doubled from day 18 to day 21 and was decreased by 50% in fetuses from fasted mothers. These data, demonstrating the presence and regulation of the receptors for TGF alpha and TGF beta, support roles for these hormones in the regulation of fetal hepatic growth.

Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors.

Proliferin is a prolactin-related glycoprotein secreted by proliferating mouse cell lines and by mouse placenta. In an attempt to identify target sites for proliferin action, we looked for proliferin receptors in murine fetal and maternal tissues during pregnancy using proliferin purified from the conditioned medium of a constructed Chinese hamster ovary cell line carrying amplified copies of proliferin cDNA. Purified proliferin bound to membrane preparations from fetal or maternal liver and from placenta with a Kd of 1 to 2 nM. The amount of proliferin bound per microgram of membrane protein varied markedly during pregnancy; maximal binding to day 16 fetal liver membranes was approximately 25 times that to liver membranes from adult animals. Binding to fetal and maternal receptors was specifically and completely inhibited by mannose 6-phosphate, with half-maximal inhibition at 10 microM. Furthermore, non-glycosylated proliferin did not inhibit the binding of the glycosylated protein. A approximately 300 Kd proliferin receptor was purified from the liver of pregnant mice using a proliferin affinity column and elution with mannose 6-phosphate. This receptor reacted with antibodies directed against the rat cation-independent mannose 6-phosphate receptor. We conclude that 1) proliferin secreted by cultured cell binds to cation-independent mannose 6-phosphate receptors and therefore may be a lysosomal protein or targeted to lysosomes, and 2) the concentration or activity of mannose 6-phosphate receptors in murine fetal and maternal liver and in placenta is regulated during pregnancy.

The ontogeny of serum GH binding protein in man: a possible indicator of hepatic GH receptor development.

Experiments in rabbits suggest that a serum GH binding protein (GH-BP) probably is derived largely from hepatic membrane bound GH receptors. Human serum contains a specific GH binding protein which can be easily measured by incubation with [125I] hGH and separation of [125I] hGH-BP complexes from free [125I] hGH by gel filtration through an Ultrogel AcA 44 minicolumn. In each assay GH-BP activity of a reference (normal young adult) serum is similarly run and the results are expressed as a percentage of the activity of the unknown serum divided by the GH-BP activity of the reference serum after correction for the expected inhibition of GH binding resulting from the GH content of the unknown serum. The mean relative specific GH binding protein (RSGH-BP) of cord serum from 11 premature infants was only 3.2 +/- 1.4% (SE) and of cord serum from 17 full term infants was 14.9 +/- 2.5%. During the first two decades of life there was a progressive rise of RSGH-BP with considerable individual variation. The mean serum RSGH-BP of 13 such subjects was 54.5 +/- 6.2%. More uniform RSGH-BP results were obtained in serum from 15 young adults, 91.7 +/- 7.4%. Lower RSGH-BP 77.2 +/- 5.4% was found in serum from 12 healthy older adults (age 60 to 70 years). The low levels of RSGH-BP in fetal serum are consistent with the reported low concentrations of GH receptors in sheep and rat fetal liver membranes. We suggest the measurement of GH-BP activity provides a simple, noninvasive measure of the ontogeny of GH receptors of human beings.

Insulin-receptor development in normal and diabetic pregnancies. Role of membrane fluidity.

In the hyperinsulinemic offspring of the diabetic mother, both significant macrosomia and postnatal hypoglycemia are thought to be due to increased insulin sensitivity. The purpose of this study is to characterize changes in insulin-receptor development in fetal offspring of an experimental model of diabetic pregnancy. Two groups of Sprague-Dawley female rats were studied after timed mating. Both groups received injections of either vehicle (controls) or streptozocin (diabetic), 40 mg/100 g body wt, on day 7 of pregnancy and were killed at either 17, 20, or 21 days of gestation. Maternal and fetal blood were assayed for glucose and insulin, and fetal liver membranes were prepared for 125I-labeled insulin binding, lipid composition, and fluorescence polarization studies with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Maternal and pooled fetal glucose levels were elevated in streptozocin-treated rats; however, pooled fetal insulin values were not elevated in the offspring of diabetic animals compared with controls (33 +/- 1 vs. 50 +/- 5 microU/ml). 125I-insulin binding was greater in fetal offspring of diabetic (FD) rat membranes at each gestational age studied [P less than .001 by analysis of variance (ANOVA)] due to significantly greater numbers of both high- and low-affinity receptors. The highest insulin-binding capacity was seen on membranes obtained from FD rats at day 21 (9.92 M X L-1 X 100 micrograms membranes-1 vs. 6.38 M X L-1 X 100 micrograms protein-1 in fetal control (FC) rats. At each gestational age, membranes from FDs had lower values for fluorescence polarization (using the probe DPH) than did gestational-age--matched controls.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

INSULIN RECEPTOR (IR) DEVELOPMENT IN NORMAL (N) AND DIABETIC (D) PREGNANCIES: DOES INSULIN (I) UP REGULATE THE RECEPTOR?

Offspring of D pregnancies have increased concentrations of IR on plasma membranes associated with differences in membrane lipid composition and physical structure. We have previously suggested that increased [I] occurring in human and animal offspring of D pregnancies caused up-regulation of IR. Since ambient [I] is the difference between its secretion by the pancreas and its degradation, we have studied these factors in rat fetuses from both N and D pregnancies. Twenty nine pregnant rats of timed gestation received either saline (N, n=13) or streptozotocin 60 mg/kg (D, n=16) on d 7 of pregnancy, and were sacrificed on d 21. Pooled fetal serum I levels were comparable (47 ± 8 vs. 34 ± 2μU/ml), whereas blood glucose levels were markedly elevated in offspring of D, (39 ± 13 vs. 321 ± 63 mg/dl, p < 0.001). Tracer I binding was also elevated on liver plasma membranes from D (5.3 ± 1.0 vs. 2.1 ± 0.3%/100μ g protein, p <0.02) due to increased high and low affinity receptors. I content of fetal pancreatic islets, quantitated by immunoperoxidase staining, was significantly lower in D offspring. I degradation was significantly higher in fetal liver homogenates from D offspring compared to N, (34 ± 1.7 vs. 26 ± 1.2% degradated/30 min/100μg protein, p<0.001).Conclusions: Elevation in IR concentrations on fetal liver membranes in D pregnancies appears to occur independently of I presented to the liver. The data support the concept that I degradation and IR regulation occur by separate pathways. A causal relationship between fetal hyperglycemia and/or hyperlipidemia and alterations of membrane structure increasing IR exposure cannot be excluded.

Influence of maternal diabetes on fetal rat development: alteration of insulin receptors in fetal liver and lung.

The influence of maternal diabetes on fetal development was studied in rats made diabetic by administration of streptozotocin on day 2 of gestation as well as in genetically diabetic BB Wistar rats. A dose of 65 mg streptozotocin/kg produced severe diabetes with plasma glucose levels of approximately 36 mmol/l, this was associated with fetal growth retardation but not fetal hyperinsulinaemia. In contrast, a smaller dose of streptozotocin (45 mg/kg) produced moderate diabetes with plasma glucose levels of approximately 20 mmol/l and was associated with fetal hyperinsulinaemia but only a marginal effect on fetal size. In both groups of diabetic animals, maternal body weight gain was decreased, maternal plasma insulin levels were low and fetal glucose levels were similar. In a small group of genetically diabetic BB rats on insulin therapy the fetuses were macrosomic and hyperinsulinaemic. The specific binding of 125I-labelled insulin to partially purified liver and lung membranes of fetuses of both groups of streptozotocin-induced diabetic rats was significantly lower than the binding to membranes from fetuses of control animals. The specific binding of 125I-labelled insulin to fetal liver and lung membranes from the diabetic BB Wistar rats also appeared to be reduced when compared to tissues from controls. Decreased insulin receptors in fetal lung and liver of diabetic rats suggest a role for insulin in the development of these organs during the fetal and neonatal period.

Ontogeny of the mammalian insulin receptor: Studies of human and rat fetal liver plasma membranes

Insulin binding to human fetal plasma liver membranes was studied in preparations segregated into three pools according to length of gestation: 15–18 weeks (Pool A), 19–25 weeks (Pool B), and 26–31 weeks (Pool C). Receptor numbers, calculated by extrapolation of Scatchard plots to the X axis, increased from 25 × 10 10 sites per 100 μg protein in the youngest group (Pool A) to 46 × 10 10 sites per 100 μg protein in Pool B. No further increase in receptor number was seen in Pool C. The affinity constant for insulin at tracer concentrations, K e (“empty site”), was 1.53 × 10 8 M −1 in Pool A and was only slightly higher than K f (“filled site”). K e was higher in Pool B, 1.75 × 10 8 M −1, and in Pool C reached a value of 5.63 × 10 8 M −1. In Pool C K f was 2.3 × 10 8 M −1. Insulin binding of liver plasma membranes from rat fetuses aged 14, 16, 18, and 21 (term) days and adults was also studied. Maximum binding capacity tended to increase with gestational age and was 130 × 10 10 sites per 100 μg protein at term, which was in excess of that found in adult rats (89–90 × 10 10). In addition, K e increased from 0.75 × 10 8 M −1 at 14 days to 3.02 × 10 8 M −1 at term, a value higher than that found in pregnant and nonpregnant adults. Dissociation of insulin in the presence of high concentrations of insulin was significantly enhanced in tissues from 18-day and term fetuses and adults, but not in membranes from fetal rats aged 14 and 16 days. These data appear to indicate that site-site interactions are not present in early fetal existence. These changes in insulin binding with increased length of gestation are not ascribable to changes in relative proportions of hematopoietic and parenchymal tissue. Human fetal plasma liver membranes demonstrated elevated insulin binding with increased gestational age, but comparison of fetal and adult liver could not be done. However, newborn human infants have been shown to have a higher capacity for binding insulin to circulating monocytes than adults. Also, human fetuses apparently lack the capability to diminish monocyte receptors in the presence of hyperinsulinemia. These experiments show that an increase in insulin receptor binding capacity and affinity also occurs in the liver of the rat fetus at term as compared to the adult rat. The reasons and mechanisms underlying enhanced capacity for insulin binding by fetal and newborn members of human and rodent species are not known.

Development of glucagon sensitivity in neonatal rat liver.

The ontogenesis of the hepatic glucagon-sensitive adenylate cyclase system has been studied in the rat. With a partially purified liver membrane preparation, fetal adenylate cyclase was less responsive to glucagon than the enzyme from neonatal or adult livers. Similar results were obtained in gently prepared liver homogenates, suggesting that destruction of essential components of the fetal liver membrane did not account for the relative unresponsiveness of the adenylate cyclase enzyme to glucagon. Investigation of other factors that might account for diminished fetal hepatic responsiveness to glucagon indicate (a) minimal glucagon degradation by fetal membranes relative to 8-day or adult tissue; and (b) available adenylate cyclase enzyme, as suggested by a 13-fold increase over basal cyclic AMP formation with NaF in fetal liver membranes. These results indicate that neither enhanced glucagon degradation nor adenylate cyclase enzyme deficiency accounts for the relative insensitivity of the fetal hepatic adenylate cyclase system to glucagon. In early neonatal life, hepatic adenylate cyclase responsiveness to glucagon rapidly developed and was maximal 6 days after birth. These changes were closely paralleled by a fivefold increase in glucagon binding and the kinetically determined Vmax for cyclic AMP formation. These observations suggest that (a) fetal hepatic unresponsiveness to glucagon may be explained by a limited number of glucagon receptor sites; (b) during the neonatal period, the development of glucagon binding is expressed primarily as an increase in adenylate cyclase Vmax; (c) the ontogenesis of hepatic responsiveness to glucagon may be important in the resolution of neonatal hypoglycemia.

Fetal monkey absorption of 55Fe from amniotic fluid.

A single dose of 0.5 mCi radioiron (55Fe) was injected directly into the amniotic cavity of each of four pregnant monkeys near term. At 6 or 24 hours after 55Fe adminstration, samples of fetal jejunum, ileum, blood, spleen, liver, lung and fetal membranes were taken for radioautography and scintillation counts. Counts were also made on corresponding maternal tissues. Intestinal radioiron levels and localization indicated that fetuses swallowed and absorbed 55Fe within 6 to 24 hours after injection. Silver grains over the meconium bodies of fetal ileum suggested that intestinal epithelium retained at least some of the absorbed radioiron. However, elevated 55Fe levels in fetal blood and hematopoietic organs indicated that transport of radioiron to the circulation had occurred, presumably through fetal intestine, extraembryonic membranes and/or fetal lungs. Transfer of 55Fe across the placenta from fetus to mother occurred at a slow rate, i.e., samples of maternal organs obtained at 24 hours gave low counts.