Toshio Harigaya

Bisphenol A affects glucose transport in mouse 3T3‐F442A adipocytes

Recently, environmental chemicals have appeared in daily human life, and these chemicals have been incidentally taken in by humans. The serum concentrations of some of these chemicals have been found to be associated with the onset and incidence rate of diabetes mellitus. It has been suggested that one of the environmental chemicals, bisphenol A (BPA), has hormone‐like activity. It has also been demonstrated that some hormones affect insulin resistance and fat distribution in the body. To study the effects of these environmental chemicals on glucose metabolism, the effect of BPA on glucose transport in mouse 3T3‐F442A adipocytes was investigated. The 3T3‐F442A adipocytes were incubated with various concentrations of BPA in a medium. Deoxyglucose uptake assay was performed with and without insulin. Immunoblot analysis was performed with a glucose transporter (GLUT) 4‐specific antibody and antiphosphotyrosine antibody. The BPA treatment enhanced basal and insulin‐stimulated glucose uptake, and caused an increased amount of GLUT4 protein. Thus, the enhanced glucose uptake resulting from the BPA treatment was at least partially due to the increased amount of GLUT4. Tyrosine phosphorylation of insulin receptor substrate‐1 with insulin stimulation was not significantly affected. In conclusion, it was demonstrated that BPA, one of the chemicals that we intake incidentally, affects the glucose transport in adipocytes, and also that the environmental chemicals may be identified as one of the environmental factors that affect diabetes and obesity.

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.

Hormonal Regulation of Prolactin Cell Development in the Fetal Pituitary Gland of the Mouse

The developmental process of prolactin (PRL) cells in the fetal pituitary gland was studied in mice. Although PRL cells were hardly detectable in the pituitary gland of intact fetuses, a treatment with 17beta-estradiol (E(2)) in vitro induced a number of PRL cells that varied drastically in number depending on the stage of gestation with a peak at embryonic d 15. This effect was specific to E(2), with epidermal growth factor, insulin, and forskolin failing to induce PRL cells. Although both estrogen receptor (ER)alpha and ERbeta were expressed in the fetal pituitary gland, the results from ER knockout models showed that only ERalpha mediates E(2) action on PRL cells. A few PRL cells were observed in ERalpha-deficient mice as well as in their control littermates, suggesting that estrogen is not required for the phenotype determination of PRL cells. Unexpectedly, the effect of E(2) on the induction of PRL cells in vitro was diminished after embryonic d 15. Present results suggest that the exposure of fetal PRL cells to glucocorticoids (GCs) results in a reduction of sensitivity to E(2). The mechanism underlying the down-regulation of estrogen sensitivity by GCs was found not to be down-regulation of ER levels, induction of annexin 1, a GC-inducible inhibitor of PRL secretion, or a decrease in the number of PRL precursors by apoptosis. The effect of GCs appeared within 2 h and did not require a de novo protein synthesis. GCs are considered to be involved in the mechanisms of silencing pituitary PRL in gestation possibly through a novel mechanism.

Cause of Failure of Lactation in Mouse Mammary Tumor Virus/Human Transforming Growth Factor α Transgenic Mice

Abstract Transgenic female mice bearing human transforming growth factor-α (TGFα) cDNA under the control of the mouse mammary tumor virus enhancer/promoter became pregnant but failed lactation. TGFα mRNA was detected in the mammary glands of these mice by the reverse transcriptase-polymerase chain reaction. By the use of collagenase-dissociated mammary epithelial cells, the binding of prolactin to its receptor was determined before and after parturition. At the end of pregnancy, the binding in TGFα transgenic (TGFα[+]) mice was small and its amount was comparable to that in the TGFα negative (TGFα[-]) mice. On the day of parturition, prolactin binding in TGFα(+) mice increased approximately 1.9-fold (insignificant), while that in TGFα(-) mice elevated over 5.3-fold (P < 0.01). The binding sites per cell were also higher in TGFα(-) mice. Radioimmunoassay of prolactin suggested that in TGFα(+) mice the low level of prolactin binding after parturition was not due to masking effect of serum prolactin. Among six TGFα(+) mice assayed, one mother with the highest prolactin binding activity (3.7-fold increase) initiated lactation, but the others did not. As there was little difference between groups in the growth and synthesis in the mammary glands, it was concluded that the failure of lactation in TGFα(+) mice is principally due to the lack of elevation of mammary prolactin receptor after parturition. At present, the role of TGFα in this process is obscure; however, TGFα was revealed not to interfere with the binding of prolactin to the receptor.

Elevated vasoinhibin derived from prolactin and cathepsin D activities in sera of patients with preeclampsia.

Elevated vasoinhibin derived from prolactin and cathepsin D activities in sera of patients with preeclampsia

Vasoinhibins, N-terminal mouse prolactin fragments, participate in mammary gland involution

Vasoinhibins are a family of peptides that act on endothelial cells to suppress angiogenesis and promote apoptosis-mediated vascular regression. Vasoinhibins include the N-terminal fragments from prolactin (PRL), GH, and placental lactogen. One of the vasoinhibins, the N-terminal PRL fragment of 16 kDa, is generated by the lysosomal representative protease cathepsin D (Cath D). Because the normal growth and involution of the mammary gland (MG) are profoundly affected by the expansion and regression of blood vessels and also because PRL stimulates the growth and differentiation of MG, we proposed that intact PRL produced during lactation contributes to MG angiogenesis and increased blood flow, whereas during involution, the N-terminal PRL fragment would have proapoptotic effects on mammary epithelial cells (MECs). Therefore, we investigated the production of the N-terminal PRL fragment and its direct effect on the MG. Mouse PRL (mPRL) was proteolytically cleaved by Cath D between amino acids 148 and 149. N-terminal PRL fragment and Cath D expression increased during MG involution. Furthermore, incubation of MG fragments and MCF7 with recombinant 16 kDa mPRL revealed a proapoptotic effect in MECs. Ectopic mPRL in MECs was cleaved to 16 kDa PRL by Cath D in the MG lysosomal fraction. The majority of PRL derived from pituitary gland was cleaved to 16 kDa PRL in culture medium. Therefore, N-terminal PRL fragment increases during the involution period, has a proapoptotic effect on MECs, and is mainly generated by secreted Cath D in the extracellular space of MG.

The mRNA Expression of Neurocan, a Brain-Specific Chondroitin Sulfate Proteoglycan, in Neoplastic Mammary Glands in Mice

Abstract In the experiment of mouse transforming growth factor alpha (TGF&agr;) gene expression in mammary tumors, various sizes of amplified products by reverse transcriptase-polymerase chain reaction (RT-PCR) using mouse TGF&agr; primers were detected in addition to a predicted size in four strains of mice. During the further analysis of these RT-PCR products in mouse mammary tumors, the transcript of neurocan gene was detected in the mammary tumor from SHN mice by the cloning and nucleotide sequence analysis after RT-PCR reaction using mouse TGF&agr; primers. The 5′-nucleotide sequence of sequential 246bp in the amplified cDNA of 527bp was completely identical to a middle part of mouse neurocan cDNA sequence, one of the chondroitin-sulfate proteoglycan expressed in the nervous tissue.

Sexual dimorphism in amino acid compositions of mouse prolactin

Sexual dimorphism was found in amino acid compositions and immunogenecity of variant types of prolactin (PRL) purified from the pituitary gland of normal adult C57BL mice by a high performance liquid chromatography. From the pituitary gland of female mice, three female variant types of PRL were isolated, whereas from the pituitary gland of male mice, two male variant types of PRL (M1-PRL and M3-PRL) and a female variant type of PRL (M2-PRL) were obtained. The amino acid composition of M3-PRL was different from any of female variant types which were very similar to each other and contained less varine, isoleucine, leucine, tyrosine and lysine but more alanine than the latter. Immunoreactivity of any of female variant types of PRL against anti-PRL serum was 100%, whereas that of M1-PRL was as much as 85% and that of M3-PRL was nearly undetectable.

Variable expression of human transgenes in SHN mice

The mouse mammary tumour virus/human transforming growth factor α (MMTV/hTGFα) gene or the mouse whey acidic protein/human growth hormone (mWAP/hGH) gene was introduced to a high mammary tumour strain of SHN virgin females by mating with males bearing each gene. We maintained transgenic mice by backcrossing males with the hTGFα transgene or high serum hGH levels (>50 ng/ml) to SHN virgins in the subsequent generations. Expression of the transgenes was examined at each generation. At the first and the second generations of backcrossing (N1 and N2), females showed hTGFα mRNA in the mammary glands associated with a marked outgrowth of the glands. Both females and males had high hGH levels in the circulation, but these characteristics disappeared completely after the third generation (N3). All findings indicate that SHN mice are specific in the resistance to the expression of some human transgenes.

Vasoinhibin, an N-terminal Prolactin Fragment, Directly Inhibits Cardiac Angiogenesis in Three-dimensional Heart Culture

Vasoinhibins (Vi) are fragments of the growth hormone/prolactin (PRL) family and have antiangiogenic functions in many species. It is considered that Vi derived from PRL are involved in the pathogenesis of peripartum cardiomyopathy (PPCM). However, the pathogenic mechanism of PPCM, as well as heart angiogenesis, is not yet clear. Therefore, the aim of the present study is to clarify whether Vi act directly on angiogenesis inhibition in heart blood vessels. Endothelial cell viability was decreased by Vi treatment in a culture experiment. Furthermore, expression of proangiogenic genes, such as vascular endothelial growth factor, endothelial nitric oxide synthase, and VE-cadherin, were decreased. On the other hand, apoptotic factor gene, caspase 3, and inflammatory factor genes, tumor necrosis factor α and interleukin 6, were increased by Vi treatment. In three-dimensional left ventricular wall angiogenesis assay in mice, Vi treatment also inhibited cell migration, neovessel sprouting, and growth toward collagen gel. These data demonstrate that Vi treatment directly suppresses angiogenesis of the heart and support the hypothesis that Vi induce PPCM.