A low maternal protein diet during pregnancy and lactation has sex- and window of exposure-specific effects on offspring growth and food intake, glucose metabolism and serum leptin in the rat.

Extensive epidemiological and experimental evidence indicates that a sub-optimal environment during fetal and neonatal development in both humans and animals may programme offspring susceptibility to later development of chronic diseases including obesity and diabetes that are the result of altered carbohydrate metabolism. We determined the effects of protein restriction during pregnancy and/or lactation on growth, serum leptin, and glucose and insulin responses to a glucose tolerance test in male and female offspring at 110 days postnatal life. We fed Wistar rats a normal control 20% casein diet (C) or a restricted diet (R) of 10% casein during pregnancy. Female but not male R pups weighed less than C at birth. After delivery, mothers received the C or R diet during lactation to provide four offspring groups: CC (first letter maternal pregnancy diet and second maternal lactation diet), RR, CR and RC. All offspring were fed ad libitum with C diet after weaning. Relative food intake correlated inversely with weight. Offspring serum leptin correlated with body weight and relative, but not absolute, food intake in both male and female pups. Serum leptin was reduced in RR female pups compared with CC and increased in RC males compared with CC at 110 days of age. Offspring underwent a glucose tolerance test (GTT) at 110 days postnatal life. Female RR and CR offspring showed a lower insulin to glucose ratio than CC. At 110 days of age male RR and CR also showed some evidence of increased insulin sensitivity. Male but not female RC offspring showed evidence of insulin resistance compared with CC. Cholesterol was similar and triglycerides (TG) higher in male compared with female CC. Cholesterol and TG were higher in males than females in RR, CR and RC (P < 0.05). Cholesterol and TG did not differ between groups in females. Cholesterol and TG were elevated in RC compared with CC males. Nutrient restriction in lactation increased relative whole protein and decreased whole lipid in both males and females. RC females showed decreased relative levels of protein and increased fat. We conclude that maternal protein restriction during either pregnancy and/or lactation alters postnatal growth, appetitive behaviour, leptin physiology, TG and cholesterol concentrations and modifies glucose metabolism and insulin resistance in a sex- and time window of exposure-specific manner.

Sex differences in transgenerational alterations of growth and metabolism in progeny (F2) of female offspring (F1) of rats fed a low protein diet during pregnancy and lactation.

Compelling epidemiological and experimental evidence indicates that a suboptimal environment during fetal and neonatal development in both humans and animals may programme offspring susceptibility to later development of several chronic diseases including obesity and diabetes in which altered carbohydrate metabolism plays a central role. One of the most interesting and significant features of developmental programming is the evidence from several studies that the adverse consequences of altered intrauterine environments can be passed transgenerationally from mother (F0) to daughter (F1) to second generation offspring (F2). We determined whether when F0 female rats are exposed to protein restriction during pregnancy and/or lactation their F1 female pups deliver F2 offspring with in vivo evidence of altered glucose and insulin metabolism. We fed F0 virgin Wistar rats a normal control 20% casein diet (C) or a protein restricted isocaloric diet (R) containing 10% casein during pregnancy. F1 female R pups weighed less than C at birth. After delivery, mothers received C or R diet during lactation to provide four F1 offspring groups CC (first letter pregnancy diet and second lactation diet), RR, CR and RC. All F1 female offspring were fed ad libitum with C diet after weaning and during their first pregnancy and lactation. As they grew female offspring (F1) of RR and CR mothers exhibited low body weight and food intake with increased sensitivity to insulin during a glucose tolerance test at 110 days of postnatal life. Male F2 CR offspring showed evidence of insulin resistance. In contrast RC F2 females showed evidence of insulin resistance. Sex differences were also observed in F2 offspring in resting glucose and insulin and insulin: glucose ratios. These sex differences also showed differences specific to stage of development time window. We conclude that maternal protein restriction adversely affects glucose and insulin metabolism of male and female F2 offspring in a manner specific to sex and developmental time window during their mother's (the F1) fetal and neonatal development.

Sex hormone-binding globulin stimulates chorionic gonadotrophin secretion from human cytotrophoblasts in culture.

The effects of sex hormone-binding globulin (SHBG) on the secretion of human chorionic gonadotrophin (HCG) and cAMP by cultured human cytotrophoblasts were investigated. Cytotrophoblasts obtained from normal term placentae were cultured in serum-free medium with or without the addition of human SHBG. The presence of SHBG in the medium increased the release of HCG and the accumulation of cAMP. Ligand-free SHBG was able to raise both HCG and cAMP concentrations and the maximal response was observed with 1 nM of the steroid-binding globulin. Addition of either oestradiol or 5alpha-dihydro-testosterone (DHT) to cultures previously incubated with SHBG in a final molar ratio of 1:10 resulted in a further increase of HCG and cAMP concentrations. This effect was blocked when cultured placental cells were exposed to SHBG that was previously saturated with DHT or when incubated in the presence of steroids only. The results of the present study provide evidence for the in-vitro regulation of HCG secretion by SHBG and further support the concept that this steroid-binding protein may act as a mediator of steroid action at the cellular level. Finally, the increase in cAMP suggests that SHBG receptor located in the surface of syncytiotrophoblast membranes is coupled to adenylate cyclase as part of the G-protein receptor family. Our results may provide new insights into the biological implications of extracellular steroid-binding proteins as well as new perspectives on the endocrinology of pregnancy.

New benzoic acid derivatives with retinoid activity: lack of direct correlation between biological activity and binding to cellular retinoic acid binding protein.

In this paper the biological activity of several newly synthesized benzoic acid derivatives of the Am- and Ch- series, which are structurally different from retinoic acid and arotinoids, was examined. These compounds inhibit squamous cell differentiation of rabbit tracheal epithelial cells in vitro as indicated by the inhibition of transglutaminase Type I and cholesterol 3-sulfate levels. In contrast to the inhibition of differentiation in rabbit tracheal cells, these compounds induce differentiation of mouse embryonal carcinoma F9 and human promyelocytic leukemia HL60 cells. The Am- and Ch- series of compounds also affect several parameters of cell proliferation. These agents are very potent inhibitors of growth of melanoma S91 cells and inhibit the induction of ornithine decarboxylase activity by phorbol 12-myristate 13-acetate in 3T6 fibroblasts. These results show that the Am- and Ch- derivatives elicit in several cell systems the same cellular responses as retinoic acid. We propose, therefore, that they exhibit mechanism(s) of action similar to those of retinoids. Comparison of the biological response with the binding capacity to the cellular retinoic acid-binding protein shows a lack of a direct correlation.

Retinoic acid and substratum regulate the differentiation of rabbit tracheal epithelial cells into squamous and secretory phenotype. Morphological and biochemical characterization.

In this paper we show that the expression of the squamous differentiated phenotype and mucosecretory phenotype by cultured rabbit tracheal epithelial cells can be regulated by substratum and the presence of retinoic acid. Cells grown on a type I collagen gel matrix in the absence of retinoic acid stratify and undergo squamous differentiation as indicated by the appearance of squamous, cornified cells. Under these conditions cells are rich in desmosomes and heavy tonofilament bundles. These cells also express several biochemical markers for squamous differentiation such as high levels of type I transglutaminase and cholesterol sulfate. High levels of transglutaminase were also observed in areas of squamous metaplasia in tracheas of vitamin A-deficient hamsters. Treatment with retinoic acid not only blocked squamous differentiation as evidenced by the inhibition of the biochemical markers for squamous differentiation but induced the appearance of columnar, polarized cells many of which contained secretory granules. These granules stained positively with periodic acid thiocarbohydrazide and certain lectins indicating the presence of glycoconjugates. Analysis of radiolabeled glycoconjugates released into the medium indicated the synthesis of mucous glycoproteins. It appears that retinoic acid determines the pathway of differentiation whereas the collagen gel matrix is permissive for the expression of both phenotypes. The morphological and biochemical similarities between this in vitro cell system and the normal and metaplastic tracheal epithelium suggest that this rabbit tracheal epithelial cell system is a useful and relevant model to study the regulation of differentiation of the tracheobronchial epithelium.

Synthesis of mucous glycoproteins by rabbit tracheal cells in vitro. Modulation by substratum, retinoids and cyclic AMP.

One function of airway epithelium is the secretion of mucins, which comprise an important component of the mucous lining layer. We demonstrate that rabbit tracheal epithelial cells grown in primary culture incorporate [3H]glucosamine into material released into the medium which is characterized as mucin by the following criteria: high Mr, monosaccharide composition, ion-exchange behaviour different from that of glycosaminoglycans and oligosaccharides attached via N-acetylgalactosamine. The production of mucin by the cells requires growth on a substratum of collagen gel and is enhanced by retinoids in the extracellular medium. In the presence of retinoids, 8-bromo cyclic AMP and factors present in medium from 3T3 fibroblasts each further stimulate mucin production. These results indicate that an isolated epithelial-cell culture system, in the absence of nervous, mesenchymal or other tissue types, can be used to answer questions about the regulation of mucin production at the cellular level.

Mechanism of resistance of a variant of P388 leukemia to L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin).

Acivicin [L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; NSC 163501] is a fermentation-derived amino acid antibiotic antagonistic to L-glutamine which exhibits potent oncolytic properties. We have developed a variant of P388 leukemia resistant to acivicin (P388/ACIA) and compared its properties with those of the parent line (P388/S). An examination of the enzymes utilizing L-glutamine revealed that the basal specific activities of L-asparagine synthetase and L-glutaminase were 1-to 3-fold higher in the parent line. The activities of carbamoyl phosphate synthetase II, L-asparagine synthetase, formylglycinamide ribonucleotide amidotransferase, and guanosine monophosphate synthetase were about equally inhibited in the two cell lines, while there was a partial inhibition of 5-phosphoribosyl-1-pyrophosphate amidotransferase, fructose-6-phosphate amidotransferase, and L-glutaminase activities, found only in the sensitive line. Cytidine triphosphate synthetase activity was not inhibited in either line. There was no difference in the dose response or restitution of L-glutamine utilizing enzyme activities between the two lines. Acivicin treatment produced a 2- to 3-fold augmentation of the L-glutamine pools only in the sensitive line. Drug injection induced increased 5-phosphoribosyl-1-pyrophosphate levels in both lines. Acivicin perturbed guanosine nucleotide pools only in the sensitive line, indicating that the primary mechanism of action of acivicin in P388 leukemia may be directed at guanosine monophosphate synthetase. Transport studies demonstrated a restricted uptake of acivicin by the resistant cells. These studies suggest that the transport of acivicin and L-glutamine plays an important role in determining the sensitivity or resistance to acivicin in these tumors.