RESUMO
Lymphaticovenular anastomosis (LVA) using supermicrosurgical techniques is effective for treating and preventing progression of lymphedema. We analyzed the influence of pregnancy on LVA in five patients from a total 2179 LVA cases. Previous studies offer conflicting reports on whether pregnancy worsens pre-existing lymphedema. This is the first report on the influence of pregnancy on lower limb lymphedema previously treated by multisite LVA (mLVA). Five patients with primary (n=4) and secondary (n=1) lower leg lymphedema were analyzed for this study. Patient age ranged from 18 to 31 (average 22.6) years old with 4 right and 1 left extremities involved. Duration of symptoms ranged from one to 19 (average 7.4) years and the periods of compression therapy were from 1 to 19 years (6.6 years). Four patients had single pregnancies and one patient was multiparous with 3 pregnancies. Final follow-up ranged from 5.8 to 18 years (average 8.9 years) after the primary mLVA. All patients had normal pregnancy, birth, and no serious complications after surgeries. Following pregnancy three patients had complete functional recovery (limb volume reduction and no compression requirement), one with functional improvement (limb volume reduction but required compression), and one with no change in symptoms (not worse and continued need for compression). There were no occurrences of infection following pregnancy. Based on this case series, it is suggested that pregnancy does not worsen the pre-existing lymphedema in patients who had previously undergone mLVA. Further studies with larger number of patients are needed to confirm these results.
Assuntos
Anastomose Cirúrgica , Extremidade Inferior/patologia , Linfedema/cirurgia , Microcirurgia , Adulto , Anastomose Cirúrgica/métodos , Feminino , Seguimentos , Humanos , Extremidade Inferior/cirurgia , Vasos Linfáticos/cirurgia , Linfedema/diagnóstico , Linfedema/etiologia , Microcirurgia/métodos , Gravidez , Complicações na Gravidez , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
OBJECTIVE: Hepatic artery (HA) reconstruction is an important part of resective surgery for advanced hepatobiliary and pancreatic malignancies, but few reports have been published. To identify indications for HA reconstruction, we retrospectively analyzed our surgical procedures and outcomes. METHODS: En-bloc resection of advanced hepatobiliary and pancreatic malignancies followed by HA reconstruction was performed in 35 patients. Patients ranged in age from 27 to 81 years and included 18 men and 17 women. The primary site of cancer included the bile duct in 22 patients, the pancreas in 7, and others in 6. Reconstruction of the HA was necessitated by HA resection due to direct cancer invasion in 29 patients and by accidental arterial injury during surgical procedure in 6 patients. RESULTS: The HA was reconstructed with end-to-end anastomosis between hepatic arteries in 17 patients. Transposition of an intra-abdominal artery, such as the gastroepiploic artery, was required in 14 patients, and arterial grafting was required in 4 patients. Although the HA patency was achieved in 30 patients, 4 cases of arterial thrombosis and 1 case of arterial rupture developed postoperatively. The overall RFS time was analyzed in all patients, and mean and median RFS times were 18 and 9 months, respectively. CONCLUSION: Although oncologic outcomes remain poor, HA resection and reconstruction can be performed in selected patients. We believe that the method of first choice for HA reconstruction is end-to-end anastomosis between HAs. A vascular autograft should be used only in selected cases.
Assuntos
Neoplasias do Sistema Biliar/cirurgia , Procedimentos Cirúrgicos do Sistema Biliar/efeitos adversos , Hepatectomia/efeitos adversos , Artéria Hepática/lesões , Artéria Hepática/cirurgia , Neoplasias Hepáticas/cirurgia , Pancreatectomia/efeitos adversos , Neoplasias Pancreáticas/cirurgia , Procedimentos Cirúrgicos Vasculares/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anastomose Cirúrgica , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos de Cirurgia Plástica/métodos , Resultado do TratamentoRESUMO
AIMS/HYPOTHESIS: A decrease in plasma adiponectin levels has been shown to contribute to the development of diabetes. However, it remains uncertain whether adiponectin plays a role in the regulation of insulin secretion. In this study, we investigated whether adiponectin may be involved in the regulation of insulin secretion in vivo and in vitro. METHODS: The effect of adiponectin on insulin secretion was measured in vitro and in vivo, along with the effects of adiponectin on ATP generation, membrane potentials, Ca2+ currents, cytosolic calcium concentration and state of 5'-AMP-activated protein kinase (AMPK). In addition, insulin granule transport was measured by membrane capacitance and total internal reflection fluorescence (TIRF) analysis. RESULTS: Adiponectin significantly stimulated insulin secretion from pancreatic islets to approximately 2.3-fold the baseline value in the presence of a glucose concentration of 5.6 mmol/l. Although adiponectin had no effect on ATP generation, membrane potentials, Ca2+ currents, cytosolic calcium concentrations or activation status of AMPK, it caused a significant increase of membrane capacitance to approximately 2.3-fold the baseline value. TIRF analysis revealed that adiponectin induced a significant increase in the number of fusion events in mouse pancreatic beta cells under 5.6 mmol/l glucose loading, without affecting the status of previously docked granules. Moreover, intravenous injection of adiponectin significantly increased insulin secretion to approximately 1.6-fold of baseline in C57BL/6 mice. CONCLUSIONS/INTERPRETATION: The above results indicate that adiponectin induces insulin secretion in vitro and in vivo.
Assuntos
Adiponectina/farmacologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , AMP Cíclico/metabolismo , Eletrofisiologia , Glicólise , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Ácido Palmítico/metabolismoRESUMO
The incidence and number of deaths from soft tissue sarcoma (STS) have been reported to increase in many countries. However, those in Japan have not been analysed over a long span of time. The objective of this study was to analyse the changing trends in the number of deaths from STS in Japan. We analysed the annual trends in the number of deaths from STS from 1955 to 2002 in Japan using the data from the Vital Statistics of Japan, Statistics and Information Department, Minister's Secretariat, Ministry of Health, Labour and Welfare. Until 2000, the number of deaths from STS had increased. The recent value of the annual increased ratio of deaths from STS was 0.5%[95% confidence interval (CI): -1.2-2.2%]. Men continued to have a higher number of deaths than women. The general trends in age-standardized death rates were roughly upward before 1995, although the death rates tended to decrease thereafter. The number and proportion of deaths at or after 60 years of age were increasing. The peak age group of deaths was roughly the sixties before 1982, and the seventies after 1983. The individuals in their sixties and seventies should be the focus of health promotion activities.
Assuntos
Sarcoma/mortalidade , Neoplasias de Tecidos Moles/mortalidade , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Distribuição por SexoRESUMO
AIMS/HYPOTHESIS: The interaction of syntaxin-1 and SNAP-25 with insulin exocytosis was examined using the diabetic Goto-Kakizaki (GK) rat and a total internal reflection fluorescence (TIRF) imaging system. METHODS: Primary rat pancreatic beta cells were immunostained with anti-syntaxin-1A, anti-SNAP-25 and anti-insulin antibodies, and then observed by TIRF microscopy. The real-time image of GFP-labelled insulin granules motion was monitored by TIRF. RESULTS: The number of syntaxin-1A and SNAP-25 clusters, and the number of docked insulin granules on the plasma membrane were reduced in GK beta cells. When GK rats were treated with daily insulin injection for 2 weeks, the number of syntaxin-1 and SNAP-25 clusters was restored, along with the number of docked insulin granules. The infection of GK beta cells with Adex1CA SNAP-25 increased the number of docked insulin granules. TIRF imaging analysis demonstrated that the decreased number of fusion events from previously docked insulin granules in GK beta cells was restored when the number of docked insulin granules increased by insulin treatment or Adex1CA SNAP-25 infection. CONCLUSIONS/INTERPRETATION: There was a close correlation between the number of syntaxin-1 and SNAP-25 clusters and the number of docked insulin granules, which is associated with the fusion of insulin granules.
Assuntos
Antígenos de Superfície/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Insulina/análise , Ilhotas Pancreáticas/patologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Insulina/química , Insulina/farmacologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Fusão de Membrana , Ratos , Ratos Endogâmicos , Ratos Wistar , Proteínas SNARE , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , Proteínas de Transporte Vesicular/metabolismoRESUMO
Magnetic X-ray absorption fine-structure (XAFS) spectra have been measured for Ni-Mn alloys. The magnetic XAFS in the near-edge region (X-ray absorption near-edge structure, XANES) and X-ray magnetic circular dichroism (XMCD) of the Mn and Ni K-edge for Ni(1-x)Mn(x) (x = 0.25, 0.24 and 0.20) show that (i) the local magnetic structure around the Mn atom is quite different from that around the Ni atom, and (ii) the peak intensity in the magnetic XANES of the Mn K-edge depends on the magnetization of the sample in contrast to the Ni K-edge. The Mn K-edge magnetic EXAFS (extended XAFS) for Ni(0.76)Mn(0.24) is also measured. The second and fourth peaks in the Fourier transform are observed to be enhanced in comparison with the non-magnetic EXAFS, indicating that the second- and fourth-shell Ni atoms are replaced by Mn atoms due to heat treatment (atomic ordering). Semi-relativistic theoretical calculation explains the observed magnetic EXAFS.
RESUMO
We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.
Assuntos
Proteínas de Membrana/fisiologia , Proteínas de Transporte Vesicular , Venenos de Vespas/farmacologia , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Linhagem Celular , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Insulina/metabolismo , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intercelular , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Proteínas de Membrana/genética , Camundongos , Mutação , Peptídeos , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Sintaxina 1RESUMO
We investigated whether adenovirus-mediated preproinsulin gene transfer into insulin target tissues (adipocytes) ameliorates hyperglycemia in diabetic mice. KKA(y) mice, a genetically obese type 2 diabetic animal model, were treated with a single subcutaneous injection of recombinant adenovirus, Adex1CA-human preproinsulin (Adex1CA-pchi), into the epididymal fat pads. pchi mRNA was expressed only in adipose tissue in which mature insulin was produced. Three days after virus injection these mice showed a marked decrease of blood glucose levels (from about 400 to 200 mg/dl), and an intraperitoneal glucose tolerance test revealed the markedly improved glucose tolerance. There was no significant difference in serum insulin levels between control and recombinant adenovirus-treated KKA(y) mice. The normalized glucose levels in diabetic mice were maintained for at least 2 weeks after the virus injection. This strategy could provide a novel and, most importantly, a simple and convenient gene therapy for obese type 2 diabetes patients.
Assuntos
Adenoviridae/genética , Tecido Adiposo/metabolismo , Diabetes Mellitus Experimental/terapia , Técnicas de Transferência de Genes , Hiperglicemia/terapia , Proinsulina/genética , Precursores de Proteínas/genética , Animais , Southern Blotting , Ensaio de Imunoadsorção Enzimática , Teste de Tolerância a Glucose , Immunoblotting , Imuno-Histoquímica , Insulina/sangue , Camundongos , Camundongos Obesos , Modelos Biológicos , Proinsulina/metabolismo , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Biologically active insulin consists of two polypeptide chains, the A chain(21 amino acids) and the B chain(30 amino acids). Several regions of invariability closely related to the biological activity, such as (a) the position of cysteines that form the disulfide bridges, (b) the N- and C-terminal regions of the A-chain, and (c) hydrophobic residues at the C-terminus of the B chain. The functional insulin receptor is a heterotetrameric protein composed of two alpha and two beta subunits. The alpha subunits contain the insulin binding site and the binding causes conformational changes in the receptor molecule. The quaternary structure of the beta subunit then changes to allow for stimulating autophosphorylation of its tyrosine residues. Those in the catalytic domain(tyrosines 1146, 1150, 1151) are essential to promote the kinase activity of the receptor toward other protein substrates in insulin signalling system. The elucidation of detailed mechanisms of insulin binding to the receptor will be useful for the development of a novel hypoglycemic agent 'insulin receptor agonist', which directly acts on the insulin site and can be orally administered for the treatment of diabetes mellitus.
Assuntos
Insulina , Transdução de Sinais , Sítios de Ligação , Humanos , Insulina/química , Insulina/fisiologia , Fosforilação , Conformação Proteica , Sinais Direcionadores de Proteínas , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismoRESUMO
We discuss the angular dependent K-edge X-ray Magnetic Circular Dichroism (XMCD) spectra based on the semi-relativistic full multiple scattering theory, where 2-spinor formalism is used to describe spin-orbit coupling. So far most of theoretical approaches have been limited to the simplest case where the circularly polarized X-ray propagation coincides with the direction of the magnetic field. Here we discuss more general cases, using the above theoretical approaches. We separately discuss atomic, single and full multiple scattering XMCD spectra; in particular anisotropic features of them are studied in detail.
RESUMO
X-ray magnetic circular dichroism (XMCD) has been measured at Mn and Cu K-edge in Cu2MnX (X=Al, In, and Sn) Heusler alloy. The Mn K-edge spectrum shows a dispersion-type profile and the Cu K-edge resembles the Mn spectrum, which suggests that polarization of the p unoccupied bands originates commonly in Mn 3d states. To reproduce the observed spectrum by full multiple scattering calculations. Madelung potential has been taken into account. Charge redistribution is an important factor for the electronic structure in Cu2MnX Heusler alloy.
RESUMO
alpha'-NaV2O5 transforms at Tc = 34K, and the origin of the phase transition seems to be caused by the charge order and spin-Peierls mechanism. However the experiments which reveal the existence of charge order are very little. We measured XANES of V atoms which relates to the valence of V. It was found that the valences of V atoms in NaV2O5 were V4.5+ in the high temperature phase by comparing pre-edge and XANES of NaV2O5 with that of LiV2O5 CaV2O5 and V2O5. The analysis of EXAFS also shows the environment of the V4.5+ state.
RESUMO
The effects of fatty acids on pancreatic beta cell are still controversial. Here, in order to determine whether free fatty acids acutely affect beta cell functions, we studied the effect of palmitic acid (PA) on proinsulin biosynthesis and insulin secretion using rat islets in vitro. Exposure of islets to PA for 1 h reduced glucose-stimulated proinsulin biosynthesis in a dose-dependent manner; in contrast, no change in insulin secretion was observed after 1 h incubation with PA. Furthermore, PA treatment did not cause any change of preproinsulin mRNA level during 1-h incubation period. Thus, our data indicate that PA primarily suppresses glucose-induced proinsulin biosynthesis within 1 h at the translational level.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ácido Palmítico/farmacologia , Proinsulina/biossíntese , Proinsulina/genética , Animais , Glucose/farmacologia , Técnicas In Vitro , Insulina , Masculino , Proinsulina/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Cellubrevins are integral membrane proteins expressed in a wide variety of tissues and usually localized in recycling vesicles. Here, we investigated the cellular localization of a cellubrevin-related peptide, endobrevin, in pancreatic (beta) cells and its implication in the exo-endocytosis of insulin and (gamma)-amino butyric acid (GABA). Immunocytochemistry showed that endobrevin is associated with tubulo-vesicular structures, which are colocalized with early endosomes labeled by early endosome antigen (EEA)-1 in insulinoma MIN6 cells. To determine the cellular localization of endobrevin, we appended the green fluorescent protein (GFP) to endobrevin and the fusion protein was introduced into MIN6 cells. The subcellular localization of GFP-endobrevin was visualized by confocal laser microscopy. Colocalization study based on the expressed GFP-endobrevin and endocytosed Texas-Red(Tx-R) labeled transferrin receptor and immunocytochemistry with anti-EEA1 antibody revealed that endobrevin was preferentially localized in the early endosome. Then, we examined the functional role of endobrevin in the exocytosis of insulin and GABA from pancreatic (beta) cells. Endobrevin overexpression increased the amount of GABA released from MIN6 cells; in contrast, it decreased the glucose-stimulated insulin release from rat islets, MIN6 and INS1-D cells to approximately 50% of the control levels. Both in vitro and in vivo binding studies showed that endobrevin binds to syntaxin 1. Finally, using the fluorescent probe FM4-64, it was revealed that endobrevin overexpression accelerates vesicle recycling. We conclude that (1) endobrevin is localized in the early endosome in pancreatic (beta) cells and (2) endobrevin plays a physiological role in the exo-endocytosis of insulin and GABA from pancreatic (beta) cells, probably via an interaction between endocytic vesicles and the endosome.
Assuntos
Endocitose/fisiologia , Endossomos/fisiologia , Exocitose/fisiologia , Ilhotas Pancreáticas/fisiologia , Proteínas de Membrana/fisiologia , Vesículas Secretórias/fisiologia , Animais , Endossomos/metabolismo , Corantes Fluorescentes/metabolismo , Expressão Gênica , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Compostos de Piridínio/metabolismo , Proteínas Qa-SNARE , Compostos de Amônio Quaternário/metabolismo , Proteínas R-SNARE , Ratos , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Vesículas Secretórias/metabolismo , Sintaxina 1 , Proteína 3 Associada à Membrana da Vesícula , Xantenos/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The Rho family of small GTPases occupies a key position in the control of cell motility and morphology in response to extracellular stimuli. Rho proteins trigger the formation of contractile stress fibers, resulting in regulation of cell motility. We explored the expression and function of RhoA in human endometrium and decidua. RhoA immunoreactivity had a predominantly glandular epithelial distribution in the proliferative phase and midsecretory phase. In decidua, the expression of RhoA was more pronounced in the stromal cells as well as in the glandular epithelium. RhoA protein levels in proliferative phase and midsecretory phase endometrium as well as decidua were evaluated by immunoblotting; a single band of RhoA protein with a molecular mass of 21 kDa was detected in all cell lysates. Cultured human decidual cells were found to have few actin stress fibers. Decidual cells lost their actin stress fibers by the treatment with C3, an exoenzyme produced by Clostridium botulinum, whereas new actin stress fibers appeared in human decidual cells stimulated with lysophosphatidic acid (LPA). Mouse blastocysts became attached to cultured human decidual cells after embryos hatched from the zona pellucida. The majority of hatched blastocysts attached to human decidual cells within 24 h. Blastocysts attached to decidual cells exhibited extensive outgrowth after 48 h in culture. Treatment of decidual cells with C3 exoenzyme or LPA did not affect the rates of hatching and attachment of blastocysts, but outgrowth of embryos on decidual cells was inhibited by C3 exoenzyme treatment in a dose-dependent manner. Contrariwise, addition of LPA to decidual cells dose dependently increased the outgrowth of embryos on decidual cells. These findings suggest that RhoA in decidual cells is important for embryonic development and differentiation after attachment.
Assuntos
Toxinas Botulínicas , Implantação do Embrião/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , ADP Ribose Transferases/metabolismo , Actinas/metabolismo , Adulto , Animais , Blastocisto/metabolismo , Western Blotting , Adesão Celular , Células Cultivadas , Decídua/metabolismo , Endométrio/citologia , Endométrio/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Indicadores e Reagentes , Lisofosfolipídeos/farmacologia , Camundongos , GravidezRESUMO
The physiological role of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in insulin exocytosis has been reported in pancreatic beta-cells. To determine whether the beta-cells of GK rats, a nonobese rodent model of type 2 diabetes, exhibit abnormalities in their SNARE proteins, we studied the expression and function of target (t)-SNAREs, syntaxin 1A, and synaptosomal-associated protein of 25 kDa (SNAP-25) in GK rat islets. Although insulin release and insulin content of islets isolated from 12-week-old GK rats were reduced, the proinsulin biosynthetic rate was about twofold higher than that in control rat islets, and no change in the preproinsulin mRNA level was observed. Pulse-chase experiments suggested the increased degradation of insulin in GK rat islets. Immunoblot analysis revealed that protein levels of syntaxin 1A and SNAP-25 in GK rat islets decreased to approximately 60% of the levels in control rat islets. We then examined whether the restoration of the decreased expression of t-SNAREs to the normal level in GK rat islets affected insulin secretion. Restoration was achieved by the overexpression of syntaxin 1A and SNAP-25 via the recombinant adenovirus-mediated gene transduction system, which recovered levels of these proteins to almost control levels. Glucose-stimulated insulin release from AdexlCA syntaxin 1A and Adex1CA SNAP-25-infected GK rat islets increased up to approximately 135 and 200%, respectively, of those from uninfected GK rat islets, although no difference in basal (2.2 mmol/l glucose) insulin release was evident between them. We conclude that decreased expression of t-SNAREs in GK rat islets is in part the defect responsible for impaired insulin secretion.
Assuntos
Antígenos de Superfície/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Insulina/genética , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Transporte Vesicular , Animais , Antígenos de Superfície/metabolismo , Glicemia/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Glucose/farmacologia , Insulina/biossíntese , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Valores de Referência , Proteínas SNARE , Proteína 25 Associada a Sinaptossoma , Sintaxina 1RESUMO
We previously demonstrated that GLUT2, a facilitated-diffusion glucose transporter isoform known to play critical roles in the regulation of systemic blood glucose level, is present at the apical ends of Müller cells in the rat retina. As a means of elucidating the ontogeny and possible role(s) of GLUT2 in the developing retina, this study examined its expression at various stages of retinal development by immunofluorescence staining using GLUT2-specific antibody. Evidence of GLUT2 expression first appeared at embryonic day 14 (E14) as linear staining along the boundary between the inner and outer layers of the optic cup, with this staining pattern being present throughout subsequent embryonic and neonatal stages. After the development of photoreceptor cell inner and outer segments (i.e., photoreceptor layer), GLUT2 immunoreactivity was localized along the boundary between the outer nuclear layer and photoreceptor layer. Localization of GLUT2 expression and the timing of its appearance, which coincided with the formation of choriocapillaries, together suggest that GLUT2 is involved in the anterior transport of glucose supplied by choroidal circulation from the early stages of retinal development.
Assuntos
Proteínas de Transporte de Monossacarídeos/análise , Retina/química , Retina/embriologia , Animais , Capilares/citologia , Células Epiteliais/citologia , Imunofluorescência , Transportador de Glucose Tipo 2 , Epitélio Pigmentado Ocular/embriologia , Epitélio Pigmentado Ocular/ultraestrutura , Ratos , Ratos Sprague-Dawley , Retina/ultraestrutura , Vasos Retinianos/citologiaRESUMO
To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells.
Assuntos
Proteínas de Transporte/fisiologia , Grânulos Citoplasmáticos/metabolismo , Exocitose , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Transporte Vesicular , Adenoviridae/genética , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Glucose/farmacologia , Ilhotas Pancreáticas/ultraestrutura , Masculino , Ratos , Ratos Wistar , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaRESUMO
Amino acid residues 140-164 of integrin beta1 comprise an Arg-Gly-Asp (RGD) cross-linking region. The present study was undertaken to study the role of the RGD cross-linking region of integrin beta1 subunit in embryo implantation. Decidual cells attached to fibronectin (FN)-coated dishes. A peptide corresponding to integrin beta1[140-164] (DDL; DYPIDLYYLMDLSYSMKDDLENVKS) inhibited decidual cell attachment to FN-coated dishes in a dose-dependent manner. A variant integrin peptide in which Asp 157 and Asp 158 were replaced by Ala (AAL; DYPIDLYYLMDLSYSMKAALENVKS) did not affect decidual cell attachment to FN. Inhibition by DDL peptide was reversed by prior treatment with an RGD-containing peptide but not by prior treatment with an RGE-containing peptide. Mouse blastocysts became attached to cultured human decidual cells after embryos hatched from the zona pellucida. The majority of hatched blastocysts attached to human decidual cells within 24 h of culture. Blastocysts that attached to decidual cells exhibited extensive outgrowth after 48 h. Treatment of decidual cells with synthetic peptides did not affect the rates of hatching and attachment of blastocysts. The outgrowth of embryos on decidual cells was inhibited by DDL peptide in a dose-dependent manner, but not by AAL peptide. These findings suggest that integrin beta1[140-164] on decidual cells may be important in embryonic development and differentiation following attachment.
Assuntos
Blastocisto/fisiologia , Decídua/fisiologia , Implantação do Embrião , Integrina beta1/química , Integrina beta1/fisiologia , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Decídua/citologia , Desenvolvimento Embrionário e Fetal , Feminino , Fibronectinas , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Gravidez , Trofoblastos/fisiologiaRESUMO
The function of soluble N-ethylmaleimide-sensitive attachment protein-alpha (alpha-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of alpha-SNAP in the secretion of insulin and gamma-aminobutryric acid (GABA) from pancreatic beta cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed alpha-SNAP, but not beta-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that alpha-SNAP was present predominantly in the cytoplasm of the islets of Langerhans. alpha-SNAP overexpression in rat islets enhanced insulin release relative to the control levels. An in vitro binding study showed that both wild-type alpha-SNAP and C-terminal-deleted alpha-SNAP mutant (1-285) can bind to syntaxin 1A. alpha-SNAP mutant (1-285) was overexpressed to evaluate its activity as dominant-negative effector on insulin release. Overexpression of alpha-SNAP mutant (1-285) in rat islets and MIN6 cells decreased glucose-stimulated insulin release to about 50% of the control levels. Suppression of endogeneous alpha-SNAP in MIN6 cells by treatment with an antisense phosphorothioate oligonucleotide resulted in inhibition of insulin release. In order to examine if alpha-SNAP functions in exocytosis from synaptic-like microvesicles in pancreatic beta cells, the functional role of alpha-SNAP in GABA release from MIN6 cells was studied. The data showed no effect of alpha-SNAP mutant (1-285) overexpression on GABA release. We conclude that 1) alpha-SNAP plays a crucial role in insulin exocytosis via large dense core vesicles, but not GABA released via synaptic-like microvesicles, in pancreatic beta cells; and 2) the interaction of alpha-SNAP and syntaxin 1A may play an important role in the insulin exocytotic process.