RESUMEN
Mucins are high molecular weight epithelial proteins, strongly glycosylated, and are the main component of the mucus. Since mucus secretion can be altered in diseases, colon mucins can be regarded as a biomarker of chronic inflammatory bowel diseases or preneoplastic changes. Conventional histochemistry and lectin histochemistry combined with chemical treatment and enzymatic digestion were carried out to analyze the colon mucins in mice fed a high-fat diet for 25 weeks, a period sufficient to induce simple liver steatosis, to check whether the carbohydrate features of mucus can be altered by an inadequate diet. An increase in the sialo/sulfomucins ratio with respect to control mice, assessed by computerized image analysis, was observed in the colon, although differences in sialic acid acetylation between control and mice fed a high-fat diet were not found. High-fat diet was also associated with altered lectin-binding pattern of the mucus, with a probable shortening of oligosaccharide chains of glycoproteins. This pattern was leading to over-expression of Galß1,3GalNAc terminal dimers (TF antigen) and GalNAc terminal residues (Tn antigen). This altered composition of mucins can be related to a defect in the process of glycosylation, or to incomplete maturation of goblet cells, and may be an early indication of preneoplastic and neoplastic changes. In conclusion, our findings confirm that a fatty-rich diet (Western-style diet) induces alteration of mucins and may be associated with colon diseases. Our investigation corroborates the usefulness of lectins histochemistry in the early diagnosis of prepathological states of the colon.
Asunto(s)
Colon/química , Dieta Alta en Grasa/efectos adversos , Mucinas/química , Oligosacáridos/análisis , Oligosacáridos/química , Animales , Colon/patología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
AIM: To investigate the effectiveness of antioxidant compounds in modulating mitochondrial oxidative alterations and lipids accumulation in fatty hepatocytes. METHODS: Silybin-phospholipid complex containing vitamin E (Realsil(®)) was daily administered by gavage (one pouch diluted in 3 mL of water and containing 15 mg vitamin E and 47 mg silybin complexed with phospholipids) to rats fed a choline-deprived (CD) or a high fat diet [20% fat, containing 71% total calories as fat, 11% as carbohydrate, and 18% as protein, high fat diet (HFD)] for 30 d and 60 d, respectively. The control group was fed a normal semi-purified diet containing adequate levels of choline (35% total calories as fat, 47% as carbohydrate, and 18% as protein). Circulating and hepatic redox active and nitrogen regulating molecules (thioredoxin, glutathione, glutathione peroxidase), NO metabolites (nitrosothiols, nitrotyrosine), lipid peroxides [malondialdehyde-thiobarbituric (MDA-TBA)], and pro-inflammatory keratins (K-18) were measured on days 0, 7, 14, 30, and 60. Mitochondrial respiratory chain proteins and the extent of hepatic fatty infiltration were evaluated. RESULTS: Both diet regimens produced liver steatosis (50% and 25% of liver slices with CD and HFD, respectively) with no signs of necro-inflammation: fat infiltration ranged from large droplets at day 14 to disseminated and confluent vacuoles resulting in microvesicular steatosis at day 30 (CD) and day 60 (HFD). In plasma, thioredoxin and nitrosothiols were not significantly changed, while MDA-TBA, nitrotyrosine (from 6 ± 1 nmol/L to 14 ± 3 nmol/L day 30 CD, P < 0.001, and 12 ± 2 nmol/L day 60 HFD, P < 0.001), and K-18 (from 198 ± 20 to 289 ± 21 U/L day 30 CD, P < 0.001, and 242 ± 23 U/L day 60 HFD, P < 0.001) levels increased significantly with ongoing steatosis. In the liver, glutathione was decreased (from 34.0 ± 1.3 to 25.3 ± 1.2 nmol/mg prot day 30 CD, P < 0.001, and 22.4 ± 2.4 nmol/mg prot day 60 HFD, P < 0.001), while thioredoxin and glutathione peroxidase were initially increased and then decreased. Nitrosothiols were constantly increased. MDA-TBA levels were five-fold increased from 9.1 ± 1.2 nmol/g to 75.6 ± 5.4 nmol/g on day 30, P < 0.001 (CD) and doubled with HFD on day 60. Realsil administration significantly lowered the extent of fat infiltration, maintained liver glutathione levels during the first half period, and halved its decrease during the second half. Also, Realsil modulated thioredoxin changes and the production of NO derivatives and significantly lowered MDA-TBA levels both in liver (from 73.6 ± 5.4 to 57.2 ± 6.3 nmol/g day 30 CD, P < 0.01 and from 27.3 ± 2.1 nmol/g to 20.5 ± 2.2 nmol/g day 60 HFD, P < 0.01) and in plasma. Changes in mitochondrial respiratory complexes were also attenuated by Realsil in HFD rats with a major protective effect on Complex II subunit CII-30. CONCLUSION: Realsil administration effectively contrasts hepatocyte fat deposition, NO derivatives formation, and mitochondrial alterations, allowing the liver to maintain a better glutathione and thioredoxin antioxidant activity.
Asunto(s)
Hígado Graso/prevención & control , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fosfolípidos/farmacología , Silimarina/farmacología , Animales , Biomarcadores/sangre , Deficiencia de Colina/complicaciones , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hígado Graso/sangre , Hígado Graso/etiología , Hígado Graso/patología , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Queratina-18/sangre , Hígado/metabolismo , Hígado/patología , Masculino , Malondialdehído/sangre , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Proteínas Mitocondriales/metabolismo , Compuestos Nitrosos/sangre , Fosfolípidos/administración & dosificación , Ratas , Ratas Wistar , Silibina , Silimarina/administración & dosificación , Compuestos de Sulfhidrilo/sangre , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Tiorredoxinas/sangre , Factores de Tiempo , Tirosina/análogos & derivados , Tirosina/sangre , Vitamina E/farmacologíaRESUMEN
The mucins of colonic murine mucus are highly O-glycosilated sulfosialoglycoproteins. We have characterized the sialylation pattern of oligosaccharide chains of colonic murine mucins by conventional histochemical methods and by lectin histochemistry combined with chemical pretreatments and sialidase digestion. Oligosaccharide chains are strongly sulphated, with an increase of sulfation from the proximal toward the distal colon and a decrease of sialic acid expression and acetylation toward the distal colon. In the goblet cells of proximal colon, sialic acid bound α2,3 to Galß1,3GalNAc subterminal dimers is diacetylated at C7,C8;C7,C9;C8,C9 or triacetylated at C7,8,9. In the distal colon, sialic acid-linked α2,3 to Galß1,3GalNAc subterminal dimers shows reduced O-acetylation at C7 and/or C8, while acetyl substituents at C9 and at C4 are almost absent. Sialic acid is involved in different essential physiological functions; thus, alterations of its expression and acetylation in oligosaccharide chains of intestinal mucins are generally associated with diseases, such as ulcerative colitis and cancer. Mice may represent a suitable animal model to study alterations of oligosaccharidic chains in colonic mucins and lectin histochemistry combined with chemical pretreatments, and enzyme digestion may be a valuable tool for this study. Our present work may represent a landmark for further lectin histochemical studies to evaluate alterations of mouse colon mucins under different physiological, pathological, or experimental conditions, with possible translational value in humans.
Asunto(s)
Colon/química , Mucinas/química , Ácido N-Acetilneuramínico/análisis , Animales , Histocitoquímica/métodos , Lectinas/metabolismo , RatonesRESUMEN
The liver has a remarkable ability to regenerate after partial hepatectomy (PH), although the factors governing such ability are still poorly understood. During the prereplicative phase of the regeneration, ultrastructural alterations of periportal hepatocytes were seen, including mitochondrial swelling, abnormal accumulation of lipids, and myelin figures which could lead to the formation of lipid droplets. As it has been hypothesized that caveolin-1 is involved in lipidogenesis and in mitochondrial homeostasis, we aimed to study the subcellular distribution of caveolin-1 in hepatocytes at an early stage following PH. Liver samples were processed for light and electron microscopy at 0 h, 24 h, and 96 h after PH. The expression and subcellular distribution of caveolin-1 was assessed by immunohistochemical and immunocytochemical techniques. Following PH, at 24 h, membranes of altered mitochondria of periportal hepatocytes exhibited significant decrease of caveolin-1 expression compared with control. Myelin figures showing high expression of caveolin-1 were also seen. At 96 h, hepatocytes became ultrastructurally similar to the control liver, and the expression of caveolin-1 on mitochondria showed a moderate increase compared with 24 h after PH. Decrease of expression of caveolin-1 in the altered liver mitochondrial membranes at 24 h following PH, and the high expression of caveolin-1 observed on myelin figures, suggests involvement of caveolin-1 is in both mitochondrial homeostasis and lipidogenesis. Addressing the role played by caveolin-1 during liver regeneration might disclose additional features of mitochondrial homeostasis and lipidogenesis during frequent metabolic liver diseases.
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Caveolina 1/metabolismo , Hepatocitos/metabolismo , Regeneración Hepática , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Animales , Hepatectomía , Hepatocitos/ultraestructura , Homeostasis , Inmunohistoquímica , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Metabolismo de los Lípidos , Hígado/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Microscopía Inmunoelectrónica , Mitocondrias Hepáticas/ultraestructura , Dilatación Mitocondrial , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
BACKGROUND INFORMATION: Lipolytic glycerol, released from adipocytes, flows through the bloodstream to the liver, where its utilisation in supplying hepatocyte gluconeogenesis is rate-limited by the permeation step. An aquaglyceroporin expressed in hepatocytes, aquaporin-9 (AQP9), has been often linked to liver uptake of glycerol. However, the truthfulness of this postulation and the potential existence of additional pathways of glycerol import by hepatocytes have never been assessed directly. Here, we define the identity and extent of liver glycerol transport and evaluate the correlation between hepatic AQP9 expression and glycerol permeability (P(gly) ) in AQP9(+/+) wild-type mice in different nutritional states and circulating insulin levels. The liver P(gly) of AQP9 null mice is also assessed. RESULTS: By stopped-flow light scattering, facilitated diffusion of glycerol into hepatocytes was indicated by the low Arrhenius activation energy (3.5 kcal/mol) and strong inhibition by phloretin, an AQP9 blocker, that characterised the transport. Although fasting markedly increased hepatic AQP9, a straight parallelism was seen both in quantitative and time-space terms between P(gly) and AQP9 protein in AQP9(+/+) mice kept in fed or fasted/refed states. In line with these findings, the highest P(gly) (P(gly) ≈ 14.0 × 10(-6) cm/s at 20°C) at 18-h fasting coincided with the highest percent of phloretin inhibition (63%). Besides being markedly lower than that in AQP9(+/+) mice, the liver P(gly) of the AQP9 null mice did not increase during fasting. Reverse-transcription PCR analysis showed lack of compensation by AQP3 and AQP7, the other known murine glycerol facilitators, in AQP9 null mice. CONCLUSIONS: Overall, these results experimentally prove major functional significance for AQP9 in maximising liver glycerol import during states requiring increased glucose production. If any, alternative facilitated pathways would be of minor importance in transporting glucogenetic glycerol into hepatocytes during starvation. Refining the understanding of liver AQP9 in metabolic and energy homeostasis may reveal helpful for therapeutic purposes.
Asunto(s)
Acuaporinas/metabolismo , Glicerol/metabolismo , Hígado/metabolismo , Animales , Acuaporinas/análisis , Acuaporinas/genética , Difusión Facilitada , Ayuno , Eliminación de Gen , Gluconeogénesis , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , PermeabilidadRESUMEN
The characterization of mucus O-linked glycans in the proximal and distal mouse colon was performed by conventional histochemical methods and by lectin histochemistry in combination with enzymatic treatment (PNGase, α1,2 fucosidase, sialidase digestion), with and without prior desulfation. We demonstrated the presence of sialo- and sulfomucins in both the proximal and distal colon of the mouse. In the distal colon the sulfomucins were clearly prevalent, although there were always sialomucins with sialyl residues linked α2,6 to the subterminal galactose. Sialic acid was poorly O-acetylated, especially in the distal colon. The lectin binding pattern indicates a massive presence of fucose α1,2 linked to galactose in O-glycans and smaller quantities of fucose linked α1,6 to N-acetylglucosamine in the core of N-linked glycans. Lectin histochemistry also demonstrated the presence of glycosidic residues of N-acetylglucosamine, N-acetylgalactosamine, and galactose in oligosaccharide chains of highly sulfated mucins.
Asunto(s)
Colon/metabolismo , Ratones/anatomía & histología , Mucina 2/metabolismo , Polisacáridos/metabolismo , Acetilglucosamina/metabolismo , Animales , Conformación de Carbohidratos , Colon/citología , Fucosa/metabolismo , Galactosa/metabolismo , Glucosa/metabolismo , Células Caliciformes/metabolismo , Lectinas/química , Masculino , Manosa/metabolismo , Ratones/metabolismo , Ratones Endogámicos C57BL , Ácidos Siálicos/metabolismo , Coloración y EtiquetadoRESUMEN
BACKGROUND: Caveolin-1, the main structural protein of caveolae, is involved in cholesterol homoeostasis, transcytosis, endocytosis and signal transduction and thought to play an important role in lipidogenesis. Little is known about the pathophysiological role of caveolin-1 in nonalcoholic fatty liver disease (NAFLD), a condition frequently associated with the metabolic syndrome and characterized by abnormal accumulation of intrahepatic triglycerides with a potentially harmful risk of evolution to liver fibrosis, cirrhosis and hepatocellular carcinoma. MATERIALS AND METHODS: Liver steatosis (micro/macrovesicular) was induced in adult rats fed a choline-deficient diet for 14days and compared with a control normal diet. The expression and subcellular distribution of caveolin-1 was assessed using light and electron microscopy by immunohistochemical and immunocytochemical techniques and by Western blotting. RESULTS: Caveolin-1 was mainly associated with the hepatocyte basolateral plasma membrane. Fatty hepatocytes were characterized by a significant increase in the expression of caveolin-1 around and within the lipid droplets as well as in the inner membrane of mitochondria. CONCLUSIONS: Our data suggest the involvement of caveolin-1 in the case of abnormal lipogenesis and mitochondrial function typical of steatotic hepatocytes in NAFLD. Addressing the role played by caveolin-1 in liver membranes in NAFLD may help future therapeutic choices in a frequent metabolic liver disease.
Asunto(s)
Caveolina 1/metabolismo , Hígado Graso/metabolismo , Animales , Colina/metabolismo , Dieta , Hígado Graso/patología , Immunoblotting , Masculino , Ratas , Ratas Wistar , Estadística como AsuntoRESUMEN
Histochemical, lectin-histochemical, and immunohistochemical analyses were performed on parietal cells of the greater horseshoe bat, Rhinolophus ferrumequinum, to clarify the composition and distribution of oligosaccharide chains in the beta-subunit of the protonic pump H(+),K(+)-ATPase. PAS, Alcian Blue (pH 2.5) and Alcian Blue (pH 1.0) stainings detected only neutral glycoconjugates. Lectin-binding analyses included LTA, UEA-I, ConA, SBA, BSI-B4, AAA, DBA, PNA, and WGA. WGA-and PNA-bindings were also tested after beta-elimination to detect O-linked glycans. Parietal cells were negative for binding to LTA and UEA-I, and to PNA and WGA after beta-elimination, indicating the lack of (1,2) fucosylated residues and of N-linked glycans, respectively. Immunohistochemical tests with anti-alpha- and anti-beta-H(+),K(+)-ATPase were positive. Two alternative patterns of glycoconjugate distribution were found, i.e. a perinuclear and a diffuse one, indicating localization in the intracellular canaliculus and in the tubulovesicular system of the parietal cells, respectively. Both the subunits of the H(+),K(+)-ATPase and the galactosyl/galactosaminyl residues were co-distributed in both the perinuclear and the diffuse patterns, suggesting that the residues are part of the protonic pump. Glycosyl/glycosaminyl and mannosyl groups were concentrated in the tubulovesicular system, and fucosylated residues were found almost exclusively in the intracellular canaliculi; thus they are probably not included in the oligosaccharide chains of beta-H(+),K(+)-ATPase. These findings indicate that the oligosaccharide chains linked to the beta-H(+),K(+)-ATPase subunit in R. ferrumequinum have distinct features compared to the other mammals studied and confirms the taxon specificity of the chains in the proton pump.
Asunto(s)
Quirópteros/fisiología , Glicoconjugados/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Células Parietales Gástricas/metabolismo , Transporte de Proteínas/fisiología , Animales , Femenino , Masculino , Células Parietales Gástricas/citologíaRESUMEN
BACKGROUND: Hepatocytes are polarized epithelial cells with three morphologically and functionally distinct membrane surfaces: the sinusoidal, lateral and canalicular surface domains. These domains differ from each other in the expression of integral proteins, which concur to their polarized functions. We hypothesize that the cholestasis-induced alterations led to partial loss of hepatocyte polarity. An altered expression of membrane proteins may be indicative of functional disorders. Alkaline liver phosphatase (ALP), one of the most representative plasma membrane glycoproteins in hepatocytes, is expressed at the apical (canalicular) pole of the cell. Because the release of ALP protein in the bloodstream is significantly increased in cholestasis, the enzymatic levels of plasma ALP have major relevance in the diagnosis of cholestatic diseases. Here we assess the cholestasis-induced redistribution of membrane glycoproteins to investigate the ALP release. MATERIALS AND METHODS: We performed enzymatic histochemistry, immunohistochemistry, lectin histochemistry, immunogold and lectin-and immunoblotting studies. Experimental cholestasis was induced in rats by ligation of common bile duct (BDL). RESULTS: The BDL led to altered membrane sialoglycoprotein targeting as well as to ultrastructural and functional disorders. Disarrangement of the microtubular system, thickening of the microfilamentous pericanalicular ectoplasm and disturbance of the vectorial trafficking of membrane glycoprotein containing vesicles were found. CONCLUSIONS: Altogether, results indicate that the cholestasis-induced partial loss of hepatocyte cell polarity leads to mistranslocation of ALP to the sinusoidal plasma membrane from where the enzyme is then massively released into the bloodstream.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Colestasis/metabolismo , Hepatocitos/metabolismo , Hígado/patología , Glicoproteínas de Membrana/metabolismo , Animales , Colestasis/patología , Hepatocitos/patología , Histocitoquímica , Ratas , Ratas WistarRESUMEN
A multidisciplinary study involving lectin histochemistry, IHC, immuno-lectin blotting, and immunogold was carried out to determine the distribution of sugar residues in the glycoproteins of Rana esculenta oxynticopeptic cells. We considered animals in two experimental conditions, fasting and fed. It is known that, in mammals, the tubulovesicular membranes are rich in proteins with several functions. The proton pump H(+),K(+)-ATPase, a heterodimeric complex with a catalytic alpha-subunit and a heavily glycosylated beta-subunit, responsible for acid secretion, is the most abundant. No data have been published regarding the localization and the structures of H(+),K(+)-ATPase in amphibians. In the water frog, the luminal membrane and tubulovesicular system of oxynticopeptic cells, which differ in morphology according to their functional stage, reacted with the primary gold-conjugated antibody against the H(+),K(+)-ATPase alpha-subunit. By lectin histochemistry and immunoblotting, in the oxynticopeptic cells of R. esculenta we detected the presence of N-linked glycans having fucosylated (poly)lactosamine chains, which could correspond to the oligosaccharide chains of the beta subunit. The latter are somewhat different from those described in mammals, and this is probably because of an adaptation to the different microenvironmental conditions in which the oxynticopeptic cells find themselves, in terms of their different habits and phylogeny.
Asunto(s)
Mucosa Gástrica/metabolismo , Glicoproteínas/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/biosíntesis , Rana esculenta/metabolismo , Animales , Anticuerpos Monoclonales , Ingestión de Alimentos , Células Endocrinas/enzimología , Ayuno , Mucosa Gástrica/ultraestructura , Glicoproteínas/química , ATPasa Intercambiadora de Hidrógeno-Potásio/inmunología , Histocitoquímica , Microscopía Inmunoelectrónica , Oligosacáridos/análisis , Células Parietales Gástricas/enzimología , Lectinas de Plantas , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/inmunologíaRESUMEN
Rat hepatocytes express aquaporin-9 (AQP9), a basolateral channel permeable to water, glycerol, and other small neutral solutes. Although liver AQP9 is known for mediating the uptake of sinusoidal blood glycerol, its relevance in bile secretion physiology and pathophysiology remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, 1-day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL since at 7 days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the AQP9 immunoreactivity of the total liver membranes from day 7 of BDL rats was reduced by 49% compared with the sham counterpart. Results were confirmed by immunofluorescence and immunogold electron microscopy and consistent with biophysical studies showing considerable decrease of the basolateral membrane water and glycerol permeabilities of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL, indicating that the dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to negatively regulate AQP9 at a transcriptional level, since insulinemia was unchanged in 7-day BDL rats. Overall, these results suggest that extrahepatic cholestasis leads to downregulation of AQP9 in the hepatocyte basolateral plasma membrane and dysregulated aquaporin channels contribute to bile flow dysfunction of cholestatic hepatocyte.
Asunto(s)
Acuaporinas/metabolismo , Colestasis Extrahepática/fisiopatología , Hígado/metabolismo , Animales , Permeabilidad de la Membrana Celular/fisiología , Conducto Colédoco , Regulación hacia Abajo , Glicerol/metabolismo , Hepatocitos/metabolismo , Inmunohistoquímica , Ligadura , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
The morphofunctional organization of the exocrine cells in the foregut of the red-eared slider turtle, Trachemys scripta, was investigated by histochemistry (PAS, AB pH1.0 and pH 2.5, HID-AB, Bowie), lectin-histochemistry (WGA, SBA, UEA, ConA, PNA, DBA, sialidase-SBA, sialidase-PNA, Paradoxical ConA), and immunohistochemistry (antipepsin, anti-alpha-H+,K+ ATPase) to detect regional differences and verify the existence of an oro-aboral gradient in gastric juice secretion. Observations showed that pharyngeal goblet cells have mucins with terminal residuals of GalNAc and sialic acid. In the oesophagus, sulphomucins in the goblet cells are progressively substituted by sialomucins and no glands are found. Gastric surface cells secrete mostly sialomucins with residuals of GlcNAc and GalNAc. The cardias presents glands with cells secreting mostly sialomucins, differing from the surface ones in having GalNAcalpha1,3GalNAc sequences. The fundus presents complex glands with main and lesser tubules. Cells in the main tubules secrete stable, class-III mucins with sialylated residuals of glucose and/or mannose, GalNAcalpha1, 3GalNAc, and Galbeta1,3GalNAc sequences. In the lesser tubules, the oxynticopeptic cells are found, presenting pepsinogen granules and an affinity to the anti-alpha-H+,K+ ATPase, without any oro-aboral variation. The pyloric glands have a secretion similar to that of the neck cells of the fundic glands, consisting of stable, class-III mucins mostly sialylated with a high heterogeneity of residuals such as glucose and/or mannose, fucose, GlcNAc, and GalNAc. Mucins in the foregut are probably involved in several functions such as lubrication, protection against gastric juice, osmotic regulation to increase intestinal absorption, and protection against microbial injuries.
Asunto(s)
Sistema Digestivo/citología , Glándulas Exocrinas/citología , Tortugas/metabolismo , Animales , Sistema Digestivo/metabolismo , Glándulas Exocrinas/metabolismo , Inmunohistoquímica , Lectinas/metabolismo , Especificidad de Órganos , Unión ProteicaRESUMEN
Mitochondrial dysfunction and oxidative stress play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). This study aimed to elucidate the mechanism(s) responsible for mitochondrial dysfunction in nonalcoholic fatty liver. Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days. We examined the effect of CD diet on various parameters related to mitochondrial function such as complex I activity, oxygen consumption, reactive oxygen species (ROS) generation and cardiolipin content and oxidation. The activity of complex I was reduced by 35% in mitochondria isolated from CD livers compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. Hydrogen peroxide (H(2)O(2)) generation was significantly increased in mitochondria isolated from CD livers. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 38% as function of CD diet, while there was a significantly increase in the level of peroxidized cardiolipin. The lower complex I activity in mitochondria from CD livers could be completely restored to the level of control livers by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD.
Asunto(s)
Cardiolipinas/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Hígado Graso/metabolismo , Mitocondrias Hepáticas/metabolismo , Enfermedades Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Alcoholes , Animales , Respiración de la Célula/efectos de los fármacos , Cloro/deficiencia , Cloro/farmacología , Peróxido de Hidrógeno/metabolismo , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/patología , Enfermedades Mitocondriales/patología , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
Mucins in the gastrointestinal tract of Rhinolophus ferrumequinum were investigated by histochemistry and lectin histochemistry to evaluate morphofunctional variations of different regions and their possible physiological and evolutionary implications. Histochemical methods included periodic acid-Schiff (PAS), Alcian blue (AB) at pH 2.5 and 1.0 and high-iron-diamine AB pH 2.5. Binding of lectins Con A, DBA, WGA, LTA, LFA, PNA and SBA; LFA, PNA and SBA with prior sialidase treatment; and paradoxical Con A were evaluated. The oesophagus lacked glands. The stomach was divided into a short cardias, a wide fundus and a brief pylorus. The surface muciparous cells secreted sulpho- and sialomucins with N-acetylgalactosamine (GalNAc) residues, N-acetyllactosamine and (beta1,4 N-acetylglucosamine)(n) chains. Towards the pylorus, N-acetylgalactosamine residues disappeared and acidity decreased. Cardiac glands, neck cells in the fundic glands, pyloric and duodenal Brunner's glands all shared neutral, stable class-III mucins, mainly with N-acetylgalactosamine sequences. The intestine was divided into a duodenum, a jejuno-ileum and a short rectum. The goblet cells produced sulpho- and sialomucins with sialylated N-acetylgalactosamine sequences, (beta1,4 N-acetylglucosamine)(n) and N-acetyllactosamine, whose sialylation increased towards the rectum. The main features of the mucins are probably associated with the requirements of fast absorption and food passage and in protection against mechanical and pathogenic injuries.
Asunto(s)
Quirópteros/metabolismo , Tracto Gastrointestinal/metabolismo , Glicoconjugados/metabolismo , Lectinas/metabolismo , Animales , Femenino , MasculinoRESUMEN
In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal beta1,3 GalNAc and (GlcNAc beta1,4)(n) oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal beta1-3 GalNAc, Gal-alphaGal, and (GlcNAc beta1,4)(n) oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal beta1,3 GalNAc, (GlcNAc beta1,4)(n)oligomers and fucose linked alpha1,6 or terminal alpha1,3 or alpha1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-alphaGal, Gal beta1-3 GalNAc and (GlcNAc beta1,4)(n)oligomers; Fuc linked alpha1,2 to Gal, alpha1,3 to GlcNAc in (poly)lactosamine chains and alpha1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H(+)K(+)-ATPase beta-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc beta1,4)(n)oligomers and fucose linked alpha1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.
Asunto(s)
Tracto Gastrointestinal/química , Glicoconjugados/química , Lectinas/metabolismo , Triturus , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/metabolismo , Glicoconjugados/metabolismo , Histocitoquímica , Triturus/anatomía & histología , Triturus/metabolismoRESUMEN
The recent identification of aquaporin-8 (AQP8), an aquaporin (AQP) channel permeable to water and ammonia, in the inner membrane (IMM) of rat liver mitochondria suggested a role for such AQP in the hydration state and the metabolic function of mitochondria. Since thyroid hormone triiodothyronine (T3) is known to modulate both the shape and the metabolic activities of liver mitochondria, it was interesting to investigate the expression and distribution of AQP8 as well as the osmotic water permeability of the IMM in liver mitochondria from rats in different thyroid states. By semi-quantitative reverse transcriptase (RT)-PCR, when compared with the euthyroid counterpart, the levels of hepatic AQP8 mRNA significantly increased in the hypothyroid state, whereas they were strongly decreased after administration of T3. A similar pattern was seen at the protein level by immunoblotting mitochondrial membranes. The upregulation of mitochondrial AQP8 in the hypothyroid liver was confirmed by immunogold electron microscopy. Stopped-flow light scattering with IMM vesicles showed no significant differences in terms of osmotic water permeability among the IMMs in the various thyroid states. Overall, our data indicate that the T3 modulation of the AQP8 gene is a rapid downregulation of transcription. Modulation of hepatic AQP8 expression may be relevant to the regulation of mitochondrial metabolism by thyroid hormones.
Asunto(s)
Acuaporinas/metabolismo , Regulación hacia Abajo , Hipotiroidismo/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias Hepáticas/metabolismo , Triyodotironina/farmacología , Animales , Acuaporinas/genética , Expresión Génica , Immunoblotting/métodos , Membranas Intracelulares/efectos de los fármacos , Masculino , Microscopía Inmunoelectrónica , Mitocondrias Hepáticas/efectos de los fármacos , Consumo de Oxígeno , ARN Mensajero/análisis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacosRESUMEN
Our previous work supports a role for aquaporin-8 (AQP8) water channels in rat hepatocyte bile formation mainly by facilitating the osmotically driven canalicular secretion of water. In this study, we tested whether a condition with compromised canalicular bile secretion, i.e., the estrogen-induced intrahepatic cholestasis, displays defective hepatocyte AQP8 functional expression. After 17alpha-ethinylestradiol administration (5 mg x kg body wt(-1).day(-1) for 5 days) to rats, the bile flow was reduced by 58% (P < 0.05). By subcellular fractionation and immunoblotting analysis, we found that 34 kDa AQP8 was significantly decreased by approximately 70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, 17alpha-ethinylestradiol-induced cholestasis did not significantly affect the protein level or the subcellular localization of sinusoidal AQP9. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability (P(f)) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (73 +/- 1 vs. 57 +/- 2 microm/s) in cholestasis, consistent with defective canalicular AQP8 functional expression. By Northern blotting, we found that AQP8 mRNA expression was increased by 115% in cholestasis, suggesting a posttranscriptional mechanism of protein level reduction. Accordingly, studies in primary cultured rat hepatocytes indicated that the lysosomal protease inhibitor leupeptin prevented the estrogen-induced AQP8 downregulation. In conclusion, hepatocyte AQP8 protein expression is downregulated in estrogen-induced intrahepatic cholestasis, presumably by lysosomal-mediated degradation. Reduced canalicular membrane AQP8 expression is associated with impaired osmotic membrane water permeability. Our data support the novel notion that a defective expression of canalicular AQP8 contributes as a mechanism for bile secretory dysfunction of cholestatic hepatocytes.
Asunto(s)
Acuaporinas/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Colestasis/metabolismo , Hepatocitos/metabolismo , Agua/metabolismo , Animales , Acuaporinas/análisis , Acuaporinas/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colestasis/inducido químicamente , Colestasis/fisiopatología , Inhibidores de Cisteína Proteinasa/farmacología , Regulación hacia Abajo/efectos de los fármacos , Estradiol/análogos & derivados , Estradiol/farmacología , Estrógenos , Etinilestradiol/farmacología , Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Leupeptinas/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Lisosomas/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
The movement of water accompanying solutes between the cytoplasm and the mitochondrial spaces is central for mitochondrial volume homeostasis, an important function for mitochondrial activities and for preventing the deleterious effects of excess matrix swelling or contraction. While the discovery of aquaporin water channels in the inner mitochondrial membrane provided valuable insights into the basis of mitochondrial plasticity, questions regarding the identity of mitochondrial water permeability and its regulatory mechanism remain open. Here, we use a stopped flow light scattering approach to define the water permeability and Arrhenius activation energy of the rat liver whole intact mitochondrion and its membrane subcompartments. The water permeabilities of whole brain and testis mitochondria as well as liposome models of the lipid bilayer composing the liver inner mitochondrial membrane are also characterized. Besides finding remarkably high water permeabilities for both mitochondria and their membrane subcompartments, the existence of additional pathways of water movement other than aquaporins are suggested.
Asunto(s)
Mitocondrias Hepáticas/metabolismo , Agua/metabolismo , Animales , Transporte Biológico Activo , Fenómenos Biofísicos , Biofisica , Encéfalo/metabolismo , Encéfalo/ultraestructura , Técnicas In Vitro , Luz , Liposomas/química , Masculino , Microscopía Electrónica de Transmisión , Mitocondrias/metabolismo , Mitocondrias Hepáticas/ultraestructura , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Permeabilidad , Ratas , Ratas Wistar , Dispersión de Radiación , Testículo/metabolismo , Testículo/ultraestructura , Agua/químicaRESUMEN
C57L mice are susceptible and AKR mice are resistant to gallstone formation. We studied in male mice of both strains gallbladder histopathology, cholecystokinin-induced emptying, and concentrating function at 0, 14, 28, and 56 days on a lithogenic diet. Gallbladder wall thickness increased on the diet, with stromal granulocyte infiltration, progressive fibrosis, edema, and epithelial cell indentation, particularly in C57L. Strong basal cholecystokinin octapeptide-induced gallbladder emptying (70% of fasting volumes) occurred in both strains, but fasting gallbladder volumes were significantly larger in C57L (14.8 +/- 2.2 microl vs. 8.8 +/- 1.0 microl). On the diet, fasting volumes increased exclusively in C57L (28.6 +/- 2.9 microl on day 56), with progressively decreased emptying (27% of fasting volumes on day 56). Gallbladder emptying remained normal in AKR. Gallbladder concentrating function decreased on the lithogenic diet (especially in C57L), coinciding with decreased aquaporin-1 (AQP1) and AQP8 expression at the mRNA and protein levels. In additional experiments, similar downregulation of AQP1 and AQP8 mRNA expression occurred in farnesoid X receptor (FXR)-deficient mice after 1 week on the lithogenic diet, without any difference from corresponding wild-type mice. In conclusion, during murine lithogenesis, altered gallbladder histology is associated with impaired motility, reduced concentrating function, and decreased AQP1 and AQP8 expression, the latter without the involvement of the FXR.
Asunto(s)
Vesícula Biliar/patología , Cálculos Biliares/etiología , Cálculos Biliares/patología , Animales , Acuaporina 1/genética , Acuaporinas/genética , Secuencia de Bases , Bilis/metabolismo , Grasas de la Dieta/efectos adversos , Vesícula Biliar/efectos de los fármacos , Vesícula Biliar/fisiopatología , Vaciamiento Vesicular/efectos de los fármacos , Cálculos Biliares/genética , Cálculos Biliares/fisiopatología , Expresión Génica , Canales Iónicos/genética , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sincalida/farmacologíaRESUMEN
Formation of bile and generation of bile flow are driven by the active secretion of bile salts (BS), lipids and electrolytes into the canalicular and bile duct lumens followed by the osmotic movement of water. Although the transporting proteins involved in solute secretion have been cloned and their coordinated interplay defined both in health and disease, boosted by the discovery of the aquaporin water channels, only recently has considerable attention been addressed to the mechanism by which water, the major component of bile (> 95%), moves across the hepatobiliary epithelia. This review summarizes the novel acquisitions in liver membrane water transport and functional participation of aquaporin water channels in multiple aspects of hepatobiliary fluid balance. Emerging evidences suggesting involvement of aquaporins in the metabolic homeostasis of the hepatobiliary tract are also discussed.