RESUMEN
Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.
Asunto(s)
Lactancia , Glándulas Mamarias Animales , Factores de Transcripción , Destete , Animales , Femenino , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Embarazo , Lactancia/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Claudina-4/genética , Claudina-4/metabolismo , CadherinasRESUMEN
Anoctamin 1 (encoded by the Ano1 gene) is a Ca2+-activated Cl- channel critical to many physiological functions. It has been speculated that Ano1 expression is regulated in a tissue-dependent manner via alternative promoters. However, variation in the 5'-end sequence of mouse Ano1 (mAno1) and its tissue-dependent regulation are poorly understood. We identified a novel 5'-terminal exon (designated exon 1a) of mAno1 instead of the known 5'-terminal exon (exon 0) using 5'-rapid amplification of cDNA ends (RACE) analysis. Unexpectedly, the novel 5'-end variant mAno1Ex1a was abundantly expressed in many tissues including the salivary and mammary glands, rectum, lung, trachea and prostate. In contrast, the known variant mAno1Ex0 predominated only in male reproductive tissues such as the epididymis and testis. In a heterologous expression system, mAno1Ex0 encoded a longer protein than mAno1Ex1a, and this long isoform was abolished by a mutation in the exon 0 start codon. Moreover, the mAno1Ex0-specific N-terminal sequence was immunohistochemically detected in epididymis but not in salivary gland. Our data suggest that mAno1 expression is regulated via alternative promoters, and its transcriptional variation results in variation of the N-terminal sequence of the Ano1 protein due to the alternative translation initiation sites. These tissue-specific variations might contribute to the regulation of mAno1 expression and activity according to the physiological function of each tissue.
Asunto(s)
Anoctamina-1/química , Anoctamina-1/genética , Exones/genética , Variación Genética/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Animales , Anoctamina-1/análisis , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos/genéticaRESUMEN
It is well known that retinoic acid (RA) suppresses adipogenesis, although there are some contradicting reports. In this study, we examined the effect of extracellular glucose on RA-induced suppression of adipogenesis in 3T3L1 cell culture. When the cells were cultured in normal glucose medium (NG), the addition of RA suppressed lipid accumulation. However, when cultured in high glucose medium (HG), addition of RA to the cells enhanced lipid accumulation. These changes were accompanied by parallel alterations in fatty acid synthase (FAS) and sterol regulatory element-binding protein (SREBP)-1 gene expression. Transfection of SREBP-1 siRNA suppressed RA-induced enhancement of lipid accumulation and FAS expression in the cells cultured with HG. Transfection of the nuclear form of SREBP-1a cDNA into the cells cultured with NG inhibited RA-induced suppression of lipid accumulation and FAS expression. Moreover, RA- and HG-induced SREBP-1a expression occurred at the early phase of adipogenesis and was dependent on glucocorticoid to induce liver X receptor (LXR) ß, peroxisomal proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR), the key nuclear factors influencing the SREBP-1a gene expression. These results suggest that RA suppresses and enhances lipid accumulation through extracellular glucose concentration-dependent modulation of SREBP-1 expression.
Asunto(s)
Adipocitos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Tretinoina/farmacología , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Humanos , Queratolíticos/farmacología , Ratones , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
Glycoconjugates in the olfactory system play critical roles in neuronal formation, and α1-2 fucose (α1-2Fuc) glycan mediates neurite outgrowth and synaptic plasticity. Histochemical findings of α1-2Fuc glycan in the mouse olfactory system detected using Ulex europaeus agglutinin-I (UEA-I) vary. This study histochemically assessed the main olfactory and vomeronasal pathways in male and female ICR and C57BL/6J mice aged 3-4 months using UEA-I. Ulex europaeus agglutinin-I reacted with most receptor cells arranged mainly at the basal region of the olfactory epithelium. The olfactory nerve layer and glomerular layer of the main olfactory bulb were speckled with positive UEA-I staining, and positive fibers were scattered from the glomerular to the internal plexiform layer. The lateral olfactory tract and rostral migratory stream were also positive for UEA-I. We identified superficial short-axon cells, interneurons of the external plexiform layer, external, middle and internal tufted cells, mitral cells and granule cells as the origins of the UEA-I-positive fibers in the main olfactory bulb. The anterior olfactory nucleus, anterior piriform cortex and olfactory tubercle were negative for UEA-I. Most receptor cells in the vomeronasal epithelium and most glomeruli of the accessory olfactory bulb were positive for UEA-I. Our findings indicated that α1-2Fuc glycan is located within the primary and secondary, but not the ternary, pathways of the main olfactory system, in local circuits of the main olfactory bulb and within the primary, but not secondary, pathway of the vomeronasal system.
Asunto(s)
Fucosa/metabolismo , Vías Olfatorias/metabolismo , Lectinas de Plantas/metabolismo , Polisacáridos/metabolismo , Animales , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Bulbo Olfatorio/metabolismo , Mucosa Olfatoria/metabolismo , Órgano Vomeronasal/metabolismoRESUMEN
It is well documented that estrogen is predominant inducer of hepatocyte growth factor (HGF) in a variety of cell types. However, the effect of progesterone (P) remains to be elusive. Thus, in the present study, we examined the effect of P and combined effect of P and 17ß-estradiol (E2) on HGF expression and production in 3T3-L1 fibroblastic preadipocytes and mature adipocytes, as a model of stromal cells. Northern blot analysis showed that hgf mRNA expressed in preadipocytes was notably higher than that of mature adipocytes, and increased by treatment of preadipocytes with E2 or 10 nM P, but not with 1,000 nM P. The E2-induced hgf mRNA expression was enhanced by 10 nM P, but suppressed by 1,000 nM P. Western blot analysis revealed that biological active forms of HGF protein was found in the preadipocyte culture medium, while the lesser amount of HGF precursor protein was detected in the mature adipocyte culture medium. The amounts of HGF were changed dependently on the hgf mRNA expression levels. These results indicate that HGF production is intricately regulated by E2 and P at the transcriptional levels in 3T3-L1 cells, and may explain the changes in the HGF production during the mammary gland development, especially decrease in HGF expression during pregnancy when P concentration is high.
Asunto(s)
Adipocitos/efectos de los fármacos , Factor de Crecimiento de Hepatocito/metabolismo , Progesterona/administración & dosificación , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estradiol/administración & dosificación , Expresión Génica/efectos de los fármacos , RatonesRESUMEN
The Cl- secretion via Ca2+-activated Cl- channel (CaCC) is critical for fluid secretion in exocrine glands like the salivary gland. Also in the mammary gland, it has been hypothesized that CaCC plays an important role in the secretion of Cl- and aqueous phase of milk. However, there has been no evidence for the functional expression of CaCC in native mammary secretory (MS) cells of lactating animals. We therefore assessed membrane current in MS cells that were freshly isolated from lactating mice using whole cell patch-clamp techniques. In MS cells, we detected CaCC current that exhibited the following characteristics: 1) Ca2+-dependent activation at the concentrations of submicromolar range; 2) voltage-dependent activation; 3) slow kinetics for activation and deactivation; 4) outward rectification of the steady-state current; 5) anion permeability in the sequence of I- > NO3- > Br- > Cl- >> glutamate; 6) inhibition by Cl- channel blockers (niflumic acid, DIDS, and CaCCinh-A01). These characteristics of native CaCC current were similar to reported characteristics of heterologously expressed TMEM16A. RT-PCR analyses showed the expression of multiple CaCC channels including TMEM16A, Best1, and Best3 in the mammary glands of lactating mice. Immunohistochemical staining revealed the localization of TMEM16A protein at the apical membrane of the MS cells. Collectively, our data strongly suggest that MS cells functionally express CaCC, which is at least partly constituted by TMEM16A. The CaCC such as TMEM16A at the apical membrane of the MS cells may influence the quantity and/or quality of milk.
Asunto(s)
Calcio/metabolismo , Canales de Cloruro/metabolismo , Lactancia/metabolismo , Glándulas Mamarias Humanas/metabolismo , Animales , Aniones/metabolismo , Anoctamina-1 , Bestrofinas , Proteínas del Ojo/metabolismo , Femenino , Humanos , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , PermeabilidadRESUMEN
K(+) channels in mammary secretory (MS) cells are believed to play a role in transcellular electrolyte transport and thus determining ionic composition of the aqueous phase of milk. However, direct evidence for specific K(+) channel activity in native MS cells is lacking at the single-cell level. Here, we show for the first time that an inwardly rectifying K(+) (Kir) channel is functionally expressed in fully differentiated MS cells that were freshly isolated from the mammary gland of lactating mice. Using the standard whole cell patch-clamp technique, we found that mouse MS cells consistently displayed a K(+) current, whose electrophysiological properties are similar to those previously reported for Kir2.x channels, particularly Kir2.1: 1) current-voltage relationship with strong inward rectification, 2) slope conductance approximately proportional to the square root of external K(+) concentration, 3) voltage- and time-dependent and high-affinity block by external Ba(2+), and 4) voltage-dependent inhibition by external Cs(+). Accordingly, RT-PCR analysis revealed the gene expression of Kir2.1, but not Kir2.2, Kir2.3, and Kir2.4, in lactating mouse mammary gland, and immunohistochemical staining showed Kir2.1 protein expression in the secretory cells. Cell-attached patch recordings from MS cells revealed that a 31-pS K(+) channel with strong inward rectification was likely active at the resting membrane potential. Collectively, the present work demonstrates that a functional Kir2.1-like channel is expressed in lactating mouse MS cells. We propose that the channel might be involved, at least in part, in secretion and/or preservation of ionic components of milk stored into the lumen of these cells.
Asunto(s)
Lactancia/metabolismo , Glándulas Mamarias Animales/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/metabolismo , Animales , Bario/metabolismo , Transporte Biológico , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/biosíntesis , Canales de Potasio de Rectificación Interna/genética , ARN Mensajero/biosíntesisRESUMEN
Lactation is a characteristic physiological function of mammals and is important for nourishing infants and the dairy industry; however, the molecular mechanisms underlying the function remain to be elucidated. A technique to directly evaluate the quantity and quality of milk in mice is necessary for the study of the lactation mechanism in vivo. By measuring the changes in milk amount after different durations of milk accumulation (0-24 h) using a ductal cannulation technique and oxytocin supplementation, we estimated the milk production rate at a single mammary gland level. In addition, collected milk was available to assess milk quality, including creamatocrit, osmolarity, and concentrations of ions, lactose, and total protein. Moreover, as a proof of principle, the effects of intraductal administration of a hypertonic solution to the abdominal mammary gland were examined. This stimulation increased milk amount, possibly by osmosis, compared with the contralateral control gland. These results demonstrated that this method is useful for examining the lactation ability and mechanisms in vivo. Studies using this method will contribute to the further understanding of lactation mechanisms in mammals.
Asunto(s)
Glándulas Mamarias Humanas , Leche , Humanos , Femenino , Ratones , Animales , Leche/metabolismo , Lactancia/fisiología , Mamíferos , Glándulas Mamarias Animales/metabolismoRESUMEN
Grainyhead-like 2 (Grhl2) is a transcription factor regulating cell adhesion genes. Grhl2 acts as an epithelial-mesenchymal transition suppressor, and it is a proto-oncogene involved in estrogen-stimulated breast cancer proliferation. However, its expression during ovarian hormone-dependent mammary ductal development remains obscure. We here examined Grhl2 expression in the mammary gland of normal and steroid-replaced ovariectomized mice. Grhl2 protein signals were detected in both the mammary luminal epithelial and myoepithelial nuclei. The ratio and density of Grhl2-positive nuclei increased after the onset of puberty and progressed with age, whereas Grhl2-negative epithelial cells were detected in mature ducts. Claudin 3, claudin 4, claudin 7, and E-cadherin gene expression in the mammary gland was upregulated, and their expression was highly correlated with Grhl2 gene expression. Furthermore, Grhl2 mRNA expression and ductal lumen width were significantly increased by the combined treatment of estrogen and progesterone compared with estrogen alone. These results suggest that Grhl2 expressed in the luminal epithelial and myoepithelial cells from the early phase of ductal development, controlling the expression of cell adhesion molecules to establish functional ducts.
Asunto(s)
Glándulas Mamarias Animales/crecimiento & desarrollo , Factores de Transcripción/genética , Animales , Estrógenos/metabolismo , Femenino , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/ultraestructura , Ratones Endogámicos C57BL , Progesterona/metabolismo , ARN Mensajero/genéticaRESUMEN
AIMS/HYPOTHESIS: C-peptide reduces renal damage in diabetic patients and experimental animal models. In vitro studies suggest that the renal effects of C-peptide may, in part, be explained by stimulation of Na(+)/K(+)-ATPase activity. However, the responses of Na(+)/K(+)-ATPase expression in the kidney of diabetic animals to C-peptide administration remain unclear. The aim of this study was to clarify the responses. METHODS: Type 1 diabetic rats were produced by injecting streptozotocin (STZ), and some of the rats were treated with either C-peptide or insulin by the aid of an osmotic pump for 1 week. The mRNA expression and immunohistochemical localization of Na(+)/K(+)-ATPase alpha1-, alpha2- and beta3-subunits were investigated in the kidney of these rats. RESULTS: Na(+)/K(+)-ATPase alpha1-subunit was abundantly expressed in the medullary collecting ducts of control animals, but the expression was markedly decreased in the diabetic state with concomitant decrease in its mRNA expression. Similar decreases were observed in the insulin-treated diabetic rats, whereas in the C-peptide-treated diabetic rats, there was no reduction in the alpha1-expression. The beta3-subunit was expressed in podocytes and parietal cells in the glomeruli, vascular endothelial cells, and cortical collecting ducts, but lesser signals were observed in the proximal and distal tubules. However, the beta3-subunit did not appear to be affected by the diabetic state. CONCLUSIONS: Diabetes selectively reduced Na(+)/K(+)-ATPase alpha1-subunit expression and abundance. Chronic administration of C-peptide prevented this decrease. This implies a role for C-peptide in the long-term regulation of Na(+)/K(+)-ATPase function.
Asunto(s)
Péptido C/farmacología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Riñón/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Análisis de Varianza , Animales , Glucemia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Ensayo de Inmunoadsorción Enzimática , Hipoglucemiantes/uso terapéutico , Inmunohistoquímica , Insulina/sangre , Insulina/uso terapéutico , Riñón/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Retinol-binding protein 4 (RBP4) is a plasma protein involved in retinol transportation, and recent evidence in rodents suggests that RBP4 is also a metabolic regulator that modifies insulin sensitivity. To assess how RBP4 levels are regulated in ruminants, we determined the RBP4 concentrations in bovine plasma and milk using Western blot analysis. Plasma RBP4 levels in non-pregnant non-lactating (control) cows were around 45 microg/ml, which were sustained during 60-h fasting, but decreased significantly 4 h after lipopolysaccharide (LPS) administration. Basal plasma retinol concentration was around 30 microg/dl, but this decreased to approximately one-third and one-half of these values during fasting and 8 h after LPS challenge, respectively. Plasma RBP4 and retinol levels in cows 3-6 d before parturition were comparable to those of the controls. However, on the day of parturition both were significantly decreased and had returned to basal levels by two weeks after calving. Interestingly, RBP4 was clearly detected in colostrum (16.4+/-5.6 microg/ml) but was only faintly detected in milk from cows at 7 d and 15 d after calving. Retinol concentrations in colostrum were almost 10-fold higher than those in plasma, while those in milk were comparable to those in plasma. These results suggest that RBP4 and retinol levels are independently regulated under physiological and pathophysiological conditions and that RBP4, like retinol, is transferred from maternal stores to calves through colostrum.
Asunto(s)
Bovinos/metabolismo , Calostro/química , Ayuno/sangre , Lipopolisacáridos/administración & dosificación , Parto/sangre , Proteínas Plasmáticas de Unión al Retinol/análisis , Animales , Enfermedades de los Bovinos/sangre , Femenino , Inflamación/sangre , Inflamación/inducido químicamente , Inflamación/veterinaria , Embarazo , Proteínas Plasmáticas de Unión al Retinol/metabolismoRESUMEN
Oxytocin, a posterior pituitary hormone, causes the contraction of the mammary myoepithelial cells that surround the acini. This ejects milk from the acini into the primary mammary ducts. The milk ejection responses by oxytocin have not yet been exactly evaluated in mice. Thus, we present a novel method for quantitatively evaluating oxytocin-induced milk ejection in anesthetized lactating mice. We cannulated the mammary duct, administered oxytocin intraperitoneally or intravenously, and collected and measured the ejected milk. Intraperitoneal oxytocin administration (150 mU) induced continuous but oscillatory milk ejection. Repeated intravenous administration of 1.5 mU of oxytocin elicited repeated transient milk ejection. The volume of the ejected milk as a proportion of the stored volume just before each ejection (rather than ejection volume itself) was an expedient and reliable parameter representing the potency of ejection. The oxytocin sensitivity of mice at day 18 of lactation was determined from a sigmoidal dose-response curve as ED50 ≈ 2.69 mU. Based on this dose-response relationship, the specific activity of the oxytocin receptor agonists (Thr4, Gly7)-oxytocin and WAY 267464 were estimated as 976 and 6.87 U/mg, respectively. The assay presented here could be useful for physiological and pharmacological investigations of oxytocin-induced milk ejection.
Asunto(s)
Lactancia/efectos de los fármacos , Oxitocina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Ratones Endogámicos C57BL , Oxitocina/administración & dosificaciónRESUMEN
To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK gene transcription.
Asunto(s)
Péptido C/farmacología , Células Endoteliales/metabolismo , Pulmón/irrigación sanguínea , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavonoides/farmacología , Regulación Enzimológica de la Expresión Génica , Ratones , ARN Mensajero/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Previously, we demonstrated that hepatocyte growth factor (HGF) potently stimulates endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production through a calcium- and Akt-mediated phosphorylation at Ser-1179 (Ser-1177 human) in bovine aortic endothelial cells. The regulation of eNOS, however, also involves interaction with chaperone proteins such as heat shock protein (HSP) 90, which can be enhanced by agonist stimulation of the enzyme. In the present work, the role of HSP90 in HGF stimulation of eNOS was examined in an endothelial cell culture system. Treatment of endothelial cells with geldanamycin, a commonly used HSP90 inhibitor, augmented HGF-stimulated eNOS phosphorylation at Ser-1179, while it did not alter eNOS phosphorylation at Thr-497. However, other HSP90 inhibitors, namely 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and radicicol, did not possess similar effects. Neither HGF nor geldanamycin treatment, independently or in combination, altered HSP90/eNOS interaction in endothelial cells. In addition, geldanamycin treatment did not enhance the HGF-induced phosphorylation of Akt, ERK1/2 and p38MAPK. Src kinase inhibition by PP2 also failed to block the geldanamycin effects. These results suggest that geldanamycin, but neither 17-AAG nor radicicol, may enhance HGF-mediated eNOS Ser-1179 phosphorylation by some as yet unknown mechanisms independently of HSP90 inhibition.
Asunto(s)
Benzoquinonas/farmacología , Células Endoteliales/enzimología , Proteínas HSP90 de Choque Térmico/fisiología , Factor de Crecimiento de Hepatocito/fisiología , Lactamas Macrocíclicas/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Bovinos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Macrólidos/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-met/fisiología , Transducción de SeñalRESUMEN
We examined the effect of stroma-derived factors, hepatocyte growth factor (HGF) and leptin, on morphological differentiation of bovine mammary epithelial cells (BMEC) in collagen gel three-dimensional culture in vitro. BMEC treated with HGF, but not leptin, formed duct-like organoids. The formation of organoids by HGF was enhanced by treatment with a mixture of insulin, cortisol and prolactin, while BMEC treated with the mixture alone did not produce the organoid. In contrast, the formation of organoids by HGF was dose-dependently inhibited by simultaneous addition of leptin, regardless of the presence or absence of the hormone mixture. These results suggest that stroma-derived factors intricately regulate mammary epithelial morphogenesis.
Asunto(s)
Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Leptina/farmacología , Glándulas Mamarias Animales/efectos de los fármacos , Animales , Bovinos , Diferenciación Celular/efectos de los fármacos , Interacciones Farmacológicas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Factor de Crecimiento de Hepatocito/farmacología , Técnicas In Vitro , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Morfogénesis/efectos de los fármacos , Morfogénesis/fisiologíaRESUMEN
Mammary glands are physiologically active in female mammals only during nursing. Immediately after weaning, most lactation-related genes are downregulated and milk production ceases. In our previous study, we have detected an inwardly rectifying potassium channel (Kir) 2.1-like current in mammary secretory (MS) cells freshly isolated from lactating mice. This current is highly sensitive to external Ba2+. The potassium permeability of the Kir channels may contribute to the secretion and/or preservation of ions in milk. We hypothesized that the functions of the Kir channels in MS cells are regulated after weaning. To test this hypothesis, we examined the effect of forced weaning on the Ba2+-sensitive Kir current and Kir2.1 expression in the mouse mammary glands. Twenty-four hours after weaning, the lumina of mammary acini were histologically enlarged by milk accumulation. The whole-cell patch-clamp analyses showed that the Ba2+-sensitive Kir current in the post-weaning MS cells was smaller than in the lactating MS cells. The inward conductances of the current in the lactating and post-weaning cells were 4.25 ± 0.77 and 0.93 ± 0.34 nS, respectively. Furthermore, real-time PCR and Western blot analyses showed that Kir2.1 mRNA and protein expression decreased in the post-weaning mammary gland (mRNA, 90% reduction; protein, 47% reduction). Moreover, the local milk accumulation caused by teat sealing decreased Kir conductance in MS cells (2.74 ± 0.45 and 0.36 ± 0.27 nS for control and sealed mammary glands, respectively). This was concomitant with the reduction in the Kir2.1 mRNA expression. Our results suggest that milk stasis after weaning immediately decreases the Kir conductance in MS cells. This decrease in the Kir conductance may be partly caused by the reduction in the Kir2.1 mRNA and protein expression. These alterations during the post-weaning period may be involved in the cessation of ion secretion and/or preservation in the milk.
Asunto(s)
Lactancia/fisiología , Glándulas Mamarias Animales/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potasio/metabolismo , Destete , Animales , Western Blotting , Células Cultivadas , Femenino , Técnicas para Inmunoenzimas , Transporte Iónico , Masculino , Glándulas Mamarias Animales/citología , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp/métodos , Canales de Potasio de Rectificación Interna/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K(m) for 2-phosphoglycerate without affecting V(max). The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo.
Asunto(s)
Péptido C/metabolismo , Membrana Celular/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células Cultivadas , Células HL-60 , Humanos , Datos de Secuencia MolecularRESUMEN
The thermogenic function of brown adipose tissue (BAT) is increased by norepinephrine (NE) released from sympathetic nerve endings, but the roles of NPY released along with NE are poorly elucidated. Here, we examined effect of NPY on basal and NE-enhanced thermogenesis in isolated brown adipocytes that express Y1 and Y5 receptor mRNA. Treatment of cells with NPY did not influence the basal and NE-enhanced rates of oxygen consumption and cAMP accumulation. Treatment with NPY also failed to induce ERK (Thr202/Tyr204) phosphorylation in the brown adipocytes. In contrast, treatment with NPY increased ERK phosphorylation in cultured stromal vascular cells from the BAT that express Y1 receptor mRNA. In the latter treatment with NPY also increased STAT3 (Ser727) phosphorylation. These results suggest that NPY mainly acts on stromal vascular cells in BAT and plays roles in the regulation of their gene transcription through ERK and STAT3 pathways, while NPY does not affect the thermogenic function of brown adipocytes.
Asunto(s)
Adipocitos Marrones/efectos de los fármacos , Tejido Adiposo Pardo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neuropéptido Y/farmacología , Factor de Transcripción STAT3/metabolismo , Células del Estroma/efectos de los fármacos , Adipocitos Marrones/citología , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Animales , AMP Cíclico/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Norepinefrina/farmacología , Consumo de Oxígeno/efectos de los fármacos , Fosforilación/efectos de los fármacos , ARN Mensajero/biosíntesis , Receptores de Neuropéptido Y , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/metabolismo , Termogénesis/efectos de los fármacos , Termogénesis/fisiologíaRESUMEN
Mammary glands develop postnatally in response to the hypothalamic-pituitary-gonadal axis. Obesity-induced changes in the local environment, however, retard mammary gland development during late pregnancy and lactation. To clarify the effects of obesity on fundamental duct development, we compared the mammary glands of nulliparous nonpregnant obese mice fed a high-fat diet with those of lean mice fed a normal diet. Obese mice had enlarged mammary glands, reflecting fat pad size, whereas the ducts in obese mice showed a less dense distribution with less frequent branching. Additionally, the ducts were surrounded by thick collagen layers, and were incompletely lined with myoepithelium. Because leptin receptors were localized in the epithelium region and leptin that was highly expressed in the obese glands suppressed mammary epithelial cell proliferation in vitro, the present results suggest that obesity disrupts mammary ductal development, possibly by remodeling the mammary microenvironment and promoting the expression of such paracrine factors as leptin.
Asunto(s)
Grasas de la Dieta/efectos adversos , Leptina/metabolismo , Glándulas Mamarias Animales/anomalías , Glándulas Mamarias Animales/fisiopatología , Obesidad/fisiopatología , Receptores de Leptina/metabolismo , Adiponectina/sangre , Animales , Dieta , Grasas de la Dieta/administración & dosificación , Ácidos Grasos no Esterificados/sangre , Femenino , Insulina/sangre , Leptina/sangre , Glándulas Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/patologíaRESUMEN
BACKGROUND: Proinsulin C-peptide shows ameliorative effects on diabetic complications, possibly through the production of nitric oxide (NO). On the contrary, increased local availability of NO and expression of endothelial NO synthase (eNOS) in the renal endothelium are shown to be involved in the progression of diabetic nephropathy. The aim of this study was to elucidate the effect of C-peptide and insulin as a reference on the eNOS expression in the early phase of type 1 diabetic rat kidney. METHODS: Type 1 diabetes in rats was produced by streptozotocin injection and some of the rats were treated with either C-peptide or insulin by the aid of an osmotic pump for 1 week. Conventional biochemical and histological analyses were performed on tissue samples. RESULTS: The diabetic rats showed hyperglycemia with over 90% reduction of endogenous insulin and C-peptide. Replacement with C-peptide or insulin resulted in recovery of weight lost, but only insulin infusion lowered plasma-glucose concentration. The eNOS protein was localized in glomeruli and endothelial cells of arterioles, and its amounts in the kidneys, but not in the lungs, of diabetic rats was increased. Replacement with C-peptide or insulin-abrogated diabetes-induced increase of renal eNOS protein. CONCLUSION: The results indicate that C-peptide suppresses diabetes-induced abnormal renal eNOS expression, by which C-peptide may exert beneficial effects on diabetic nephropathy.