Survey on awareness of folic acid recognition and intake by female students.

To reduce the risk of neural tube defects, studies have been conducted on female students of medical services, nutritional science, and nursery education that investigated the awareness of folic acid by using questionnaires. Many investigators have suggested the need to provide detailed information about the awareness of folic acid and knowledge about folic acid intake and neural tube defect risk reduction. The dietary habits of female students showed a positive correlation with their estimated folic acid intake, suggesting that improvements in dietary habits are associated with the consumption of folic acid. The importance of folic acid intake must be more aggressively promoted among female students. Thus, many learning opportunities should be provided for such students to help increase their folic acid intake.

The role of leptin in the development of the cerebral cortex in mouse embryos.

Leptin is detected in the sera, and leptin receptors are expressed in the cerebrum of mouse embryos, suggesting that leptin plays a role in cerebral development. Compared with the wild type, leptin-deficient (ob/ob) mice had fewer cells at embryonic day (E) 16 and E18 and had fewer 5-bromo-2'-deoxyuridine(+) cells at E14 and E16 in the neuroepithelium. Intracerebroventricular leptin injection in E14 ob/ob embryos increased the number of neuroepithelium cells at E16. In cultured neurosphere cells, leptin treatment increased Hes1 mRNA expression and maintained neural progenitors. Astrocyte differentiation was induced by low-dose (0.1 microg/ml) but not high-dose (1 microg/ml) leptin. High-dose leptin decreased Id mRNA and increased Ngn1 mRNA in neurosphere cells. The neuropeptide Y mRNA level in the cortical plate was lower in ob/ob than the wild type at E16 and E18. These results suggest that leptin maintains neural progenitors and is related to glial and neuronal development in embryos.

Maternal factors in a model of type 1 diabetes differentially affect the development of insulitis and overt diabetes in offspring.

Type 1 diabetes, a multifactorial disease involving genetic and environmental factors, results from the destruction of pancreatic beta-cells. The maternal environment has been suggested to be important in the development of diabetes. To assess the role of maternal factors in the development of insulitis and overt diabetes, we transplanted pre-implantation stage embryos of nonobese diabetic (NOD) mice, a model of type 1 diabetes, into the uterus of each recipient. Recipients were ICR and DBA/2J mice without diabetic genetic predisposition and NOD mice not exhibiting overt diabetes during the experiment; offspring were designated as NOD/ICR, NOD/DBA, and NOD/NOD, respectively; unmanipulated NOD offspring were also examined. NOD/ICR and NOD/DBA offspring developed insulitis significantly earlier than NOD/NOD offspring. However, overt diabetes was significantly suppressed in NOD/ICR and NOD/DBA offspring in comparison with NOD/NOD offspring. Insulin autoantibodies (IAAs) were undetectable in ICR and DBA/2J surrogate mothers and in NOD/ICR and NOD/DBA offspring at the onset of insulitis, suggesting that maternal factors other than transmitted IAAs induced the earlier onset. The present study indicates that altered maternal factors modify the immune response to islets, which in turn might affect the pathogenic course from insulitis to overt diabetes.

Analysis of polyploid cells in mouse embryonic cells cultured under diabetic conditions.

To clarify the cytogenetic effects of glucose and ketone bodies on the pathogenesis of diabetes-associated congenital anomalies, we cultured cells from gestation-day-8 ICR mouse embryos under the diabetic condition. Cells were cultured in the medium with glucose (300 mg/dL) plus DL-2-hydroxybutyric acid (32 mM) (G + B group), glucose alone (G group), or neither of them (C group) for 5 days. At the end of the culture, cells were analyzed for the chromosomes. After 3-4 days culture, when the living cells grew into a mono-layered sheet, cells floating in the medium were observed and showed morphological features of apoptosis. Ratio of the floating cells was significantly higher in the G + B group than in the G or C group (P < 0.05), suggesting the deleterious effect of glucose and ketone body. Polyploidy was observed in the cultured cells more frequently in the G + B group (64.1%) than in the G group (49.0%), which was higher than the C group (20.5%) (G + B vs G: P < 0.05, G vs C: P < 0.001). The higher ratio of the polyploidy, but not of the aneuploidy, in the G + B and G groups suggested the specific effect of glucose and ketone body for inducing polyploidy. These results suggest that diabetic condition causes polyploidy in cultured embryonic cells.

Leptin deficiency causes pycnotic change in fetal cingulate cortical cells.

Leptin is an obese gene product, and leptin-deficient ob/ob mice develop hyperphagia and reduced locomotor activity. Leptin is thought to be related to brain development, because leptin receptors are widely expressed in the brain, and because brain weight as well as brain protein and DNA contents were reduced in adult ob/ob mice. In this study, we investigated the effect of leptin on the fetal cingulate cortex, since the leptin receptor is expressed in the neurons of the cingulate cortex, which is involved in emotion as well as in sensory, motor, and cognitive processes. The ob/ob fetuses had more pycnotic cells than wild-type fetuses in the cingulate cortex at embryonic day (E) 18. Many pycnotic cells were observed in the intermediate zone of the cingulate cortex. Most cells observed in this area were neuronal lineage cells, while few undifferentiated cells and oligodendrocyte precursor cells were found. At E18 there was no significant difference in the rostrocaudal length of the corpus callosum, which contains the neuronal projection from the cingulate cortex, between ob/ob and wild-type fetuses. We also showed that the length of the cerebrum was greater and the width of the cerebrum and cerebellum were lesser in ob/ob fetuses than in wild-type at E16. These results suggest an increased cell death in neuronal lineage cells in the intermediate zone of the cingulate cortex in leptin-deficient ob/ob mice. Leptin deficiency may also alter the gross morphology of the brain in development, but not the formation of the corpus callosum.

Maternal environment affects endogenous virus induction in the offspring of type 1 diabetes model non-obese diabetic mice.

Type 1 diabetes results from the destruction of pancreatic b-cells (insulitis). It is a multifactorial disease involving genetic and environmental factors, including the maternal environment. Viruses have also been implicated in the pathogenesis of human type 1 diabetes as well as in its model non-obese diabetic (NOD) mice during the perinatal period, as endogenous viruses and/or as infectious agents vertically transmitted from mothers. However, the role of virus as genetic or environmental factor and its interaction with other maternal factors remain unclear. In a series of experiments, we transplanted preimplantation-stage NOD embryos into the uterus of recipient Institute of Cancer Research (ICR) mice, which are without diabetic genetic predisposition, and NOD mice, which did not exhibit overt diabetes during the experiment, and designated offspring as NOD/ICR and NOD/NOD, respectively. We previously observed that NOD/ICR offspring developed insulitis significantly earlier than NOD/NOD offspring. To assess the role of viruses in the development of insulitis, we examined the appearance of viral particles and expression of retroviruses between NOD/ICR and NOD/NOD. NOD/ICR showed earlier expression of env region of the xenotropic type C retrovirus by polymerase chain reaction analysis than NOD/NOD, while the retrovirus-like particles were observed in the islet b-cells similarly in both groups by electron microscopy. Serum corticosterone level, which is suggested to enhance retroviral induction, was significantly higher in the ICR than in the NOD surrogate mothers. These findings suggest that the observed virus is endogenous and that maternal environmental factors, including hormone levels, affect the induction of endogenous viruses and cause the earlier onset of insulitis.

Role of nutritional factors at the early life stages in the pathogenesis and clinical course of type 1 diabetes.

Nutrition has been suggested as an important environmental factor other than viruses and chemicals in the pathogenesis of type 1 diabetes (T1D). Whereas various maternal dietary nutritional elements have been suggested and examined in T1D of both humans and experimental animals, the results largely remain controversial. In a series of studies using T1D model nonobese diabetic (NOD) mice, maternal dietary n-6/n-3 essential fatty acid ratio during pregnancy and lactation period, that is, early life stages of the offspring, has been shown to affect pathogenesis of insulitis and strongly prevent overt T1D of the offspring, which is consistent with its preventive effects on other allergic diseases.

PSK, a biological response modifier, modifies p53 expression, mitosis and apoptosis in X-ray irradiated mouse embryos: possible cellular mechanism of the anti-teratogenic effect.

We previously showed that PSK, a biological response modifier, suppressed X-ray irradiation induced ocular anomalies in mouse embryos. In the present study, in mouse embryos irradiated at E7.5, PSK, when administered immediately after irradiation, suppressed mitosis and increased apoptosis as compared with embryos not treated with PSK at 12 hrs after irradiation. In the irradiated embryos, p53, which is normally expressed at a high level in early embryos, increased at 6 hrs and decreased at 12 hrs after irradiation. In the irradiated and PSK-treated embryos, the p53 level did not change at 6 hrs, increased at 12 hrs and decreased at 24 hrs after irradiation. This timing of PSK-induced delayed increase of p53 coincided with that of the PSK-induced decrease in mitosis and increase in apoptosis. These results suggested that PSK modified the p53 level and affected cell proliferation and apoptosis, which might contribute to the suppression of teratogenesis.

Chromosome analysis of blastocysts cultured under the diabetic condition.

Chromosomes of Slc:ICR mouse blastocysts cultured under the diabetic condition were analyzed to clarify the effect of glucose and ketone body (DL-beta-hydroxybutyric acid). In the group exposed to glucose plus ketone body or glucose alone, blastocysts showed higher incidences of chromosome abnormalities, especially numerical abnormalities such as aneuploidy and polyploidy, than in the control group (p < 0.01). The association of nucleolus organizing regions was increased in the blastocysts exposed to glucose plus ketone body, which seems to be related to the increase in numerical abnormalities. Structural abnormalities such as break and fragment were also observed, but there was no significant difference between the diabetic and nondiabetic conditions. These results from chromosome analysis of the cultured blastocysts suggest that the diabetic condition may directly cause chromosome abnormalities in early embryos, especially aneuploidies, and may thus induce duplications or deletions of genes. These chromosomal damages may disrupt the developmental programs for organogenesis and may be involved in diabetes-induced teratogenesis.

Direct and indirect effects of neuropeptide Y and neurotrophin 3 on myelination in the neonatal brains.

Neuropeptide Y (NPY) is expressed in the developing central nervous system, however, its role in the brain development remains unclear. In this study, C57/B6 mice were intraperitoneally administered 1 nmol/capita/day of NPY, 10 nmol/capita/day of an NPY-receptor 1-specific antagonist (Y1R-A), or NPY and Y1R-A simultaneously (NPY+Y1R-A) from postnatal day (P) 7 to P14. Recombinant NPY reached the P14 cerebrum in 1 hour. These treatments didn't significantly affect body weight gain or P14 brain weight. The ratio of myelinated axons to total axons in the parietal cerebrum was significantly higher in the NPY group than in the control group. The expression of myelin basic protein (MBP)-mRNA in the cerebrum was significantly higher in the NPY group than in the control group and was significantly lower in the NPY+Y1R-A group than in the NPY group, while it was significantly higher in the NPY+Y1R-A group than in the control group. In cultured oligodendroglioma-derived B12 cells, NPY didn't influence the MBP-mRNA expression, while neurotrophin 3 (NT3) increased MBP mRNA via receptor-type tyrosine kinase type C (Trk C). NPY administration significantly increased NT3-mRNA expression in the P14 cerebrum as deduced by quantitative real-time PCR. The change in phosphorylated Trk C (P-Trk C) was proportional to that of the NT3-mRNA expression, and the proportion of P-Trk C was higher in the NPY group than in the control group. These results suggest that NPY, partially via Y1R, induces NT3 which, via Trk C phosphorylation, accelerates myelination by oligodendrocytes in the mouse brain during the neonatal period.

Diet with a low n-6/n-3 essential fatty acid ratio when started immediately after the onset of overt diabetes prolongs survival of type 1 diabetes model NOD mice.

Type 1 diabetes is a multifactorial disease involving genetic and environmental factors and results from the destruction of pancreatic islet ß cells, virtually the only source of insulin. When the majority of ß cells are lost, a 'honeymoon' period of variable length follows: namely, a fleeting phase of residual endogenous insulin production, during which glycemic control is achieved with modest or no doses of insulin. However, the remaining ß cells are eventually lost, causing the individual to become insulin-dependent and to require long-term insulin therapy or islet transplantation. Here we show that NOD mice, a type 1 diabetes model, survived significantly longer when their diet was changed from one chow with a high essential fatty acid (EFA) ratio (n-6/n-3, 14.5) to another with a low n-6/n-3 ratio (3.0) within 6 days after the onset of overt diabetes (i.e. the 'honeymoon' period), than mice that were continuously fed with the chow with the high n-6/n-3 ratio. This effect was not observed when the chow was changed later than 9 days after the onset. Significantly larger number of islets remained with suggestive islet neogenesis from the pancreatic duct and pathological changes in renal glomeruli were significantly milder in NOD mice fed the chow with the low n-6/n-3 ratio within 6 days after the onset of overt diabetes than those continuously fed with the high-n-6/n-3-ratio chow. These findings indicate that a diet with a low n-6/n-3 ratio prolongs the 'honeymoon' period by retaining the ß cell mass, suggesting its potential therapeutic merit.

Maternal dietary n-6/n-3 fatty acid ratio affects type 1 diabetes development in the offspring of non-obese diabetic mice.

Environment factors, including maternal or infant dietary nutrition have been reported to have an influence on the pathogenesis of type 1 diabetes. In the present study, to investigate the effect of maternal or post-weaning offspring's nutrition, in particular the essential fatty acid ratio (n-6/n-3) on the development of type 1 diabetes, we prepared two kinds of chows with n-6/n-3 ratios of 3.0 (L) and 14.5 (H), and provided them to mothers of non-obese diabetic (NOD) mice during gestation and lactation and to the offspring after weaning. The n-6/n-3 ratios in breast milk and erythrocyte membrane of NOD offspring became nearly the same with that of the maternal diet at 2 weeks after birth. In the L chow-fed offspring from L chow-fed mother (LLL), levels of insulitis were higher than those in the H chow-fed offspring from H chow-fed mother (HHH) at 4 weeks of age, while the levels in the LLL offspring became lower than those in the HHH after 6 weeks. Early insulin autoantibody expressions were found from 2 to 6 weeks in the HHH offspring, but not in the LLL. The LLL offspring exhibited strong suppression of overt diabetes development in regard to the onset and accumulated incidence of diabetes compared to the HHH. The study with combined L and H chows during gestation, lactation in mother and in post-weaning offspring revealed that only the LLH chow significantly suppressed the development of diabetes with similar kinetics to LLL chow, although the other combinations may delay the onset of diabetes. The present findings suggest that n-6/n-3 ratio of the maternal diet during gestation and lactation rather than that of offspring after weaning strongly affects the development of overt diabetes in NOD mice.

Individual variation in organ histogenesis as a causative factor in the developmental origins of health and disease: unnoticed congenital anomalies?

Morphological studies of congenital anomalies have mainly focused on abnormal shape (i.e. malformation) and thus on disturbed organogenesis. However, in regard to postnatal functions of organs that develop through branching mechanisms, organ size is another important morphological feature. These organs consist of a large number of structural and functional units, such as nephrons in the kidney, and the total number of these units, that is approximately proportional to the organ size, has been shown to vary widely among individuals. Organ-specific cells are differentiated and organized to form structural units and realize organ-specific functions during the histogenetic period (i.e. from mid-gestation to the early postnatal period). The total number of units is attained at the end of histogenesis and determines the total functional capacity, including the functional reserve of the organ, and thus may be related to predispositions to postnatal organ-based diseases, because the functional reserve decreases during the course of life and eventually become short of the minimum requirement of each organ. Therefore, it may be hypothesized that a smaller number of units of organs at the end of histogenesis is one of the predisposing factors for postnatal diseases (i.e. a form of unnoticed but late-manifested congenital anomalies), in this era of extended longevity. However, the mechanisms that control the total number of units in each organ during histogenesis and the possible relationship among the numbers of units in different organs remain unknown. Here, we review our trials based on the above hypothesis in order to (1) mathematically analyze the morphometric data of the different organs in fetuses to elucidate relationship among developing organs, (2) analyze the developing neuro-immuno-endocrine network as a series of mechanisms to systemically correlate the histogenesis of multiple organs, and (3) examine the maternal environment, including dietary fat, as a factor to influence histogenesis and thus the predisposition to type 1 diabetes.