High-fat diet alters the oligosaccharide chains of colon mucins in mice.

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.

Biophysical assessment of aquaporin-9 as principal facilitative pathway in mouse liver import of glucogenetic glycerol.

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.

Altered distribution of caveolin-1 in early liver steatosis.

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.

Altered membrane glycoprotein targeting in cholestatic hepatocytes.

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.

Co-distribution of glycoconjugates and H(+), K(+)-ATPase in the parietal cells of the greater horseshoe bat, Rhinolophus ferrumequinum (Schreber, 1774).

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.

Expression of H(+),K(+)-ATPase and glycopattern analysis in the gastric glands of Rana esculenta.

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.

Mitochondrial dysfunction in rat with nonalcoholic fatty liver Involvement of complex I, reactive oxygen species and cardiolipin.

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.

Water permeability of rat liver mitochondria: A biophysical study.

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.

Triiodothyronine modulates the expression of aquaporin-8 in rat liver mitochondria.

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.

Glycoconjugate histochemistry of the digestive tract of Triturus carnifex (Amphibia, Caudata).

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.

Lectin histochemistry of gastrointestinal glycoconjugates in the greater horseshoe bat, Rhinolophus ferrumequinum (Schreber, 1774).

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.

Water transport into bile and role in bile formation.

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.

Pepsinogen and H,K-ATPase mediate acid secretion in gastric glands of Triturus carnifex (Amphibia, Caudata).

The gastric glands of Triturus carnifex (Amphibia, Caudata) have been examined by histochemical and immunohistochemical methods with particular regard to hydrochloric acid and pepsinogen secretion. Fundic glands consist of mucous neck cells, endocrine cells and oxynticopeptic cells producing both pepsinogen and hydrochloric acid. The neck cells showed an unexpected distribution pattern which was only observed in the oral fundus, and produced neutral mucins with glycosidic residues of GalNAc and Gal beta1,3GalNAc, and in this respect they differ from the neck cells of anuran amphibians. The secretion of pepsinogen and hydrochloric acid as demonstrated by immunolabelling with anti-H,K-ATPase and with anti-pepsinogen, respectively, seems not to vary significantly along the longitudinal axis of the stomach. The mechanism of gastric acid secretion seems to be mediated by an ATPase, having similar features to the mammalian gastric H,K-ATPase, and is localised in the luminal membrane and in the subapical cytoplasm of the oxynticopeptic cells. Unusually, the same cytoplasmic areas revealed binding specificity for the winged pea lectin (WPA) from Lotus tetragonolobus, even after beta elimination, indicating the presence of fucosyl residues in N-linked oligosaccharidic chains in glycoproteins of beta-H,K-ATPase subunits.

Co-secretion of soluble caveolin-1 and pepsinogen in the oesophagus of the red-legged frog Rana aurora aurora.

Caveolin-1, an integral membrane protein, is the principal component of caveolae, which are specialised vesicular microdomains of the plasma membrane. Caveolae are found in most cell types, but they are most abundant in adipocytes, endothelial cells, fibroblasts, and muscle cells. Functionally, they have been implicated in endothelial transcytosis, potocytosis, and signal transduction. Recently, caveolin-1 has been found unexpectedly in the cytoplasm, mitochondria and elements of the secretory pathways of exocrine secretory cells. We have co-localised caveolin-1 and pepsinogen immunohistochemically in serous cells of oesophageal glands of the red-legged frog, Rana aurora aurora. Thus, according to its intracellular localisation pattern, caveolin-1 may be either a soluble protein, located in secretory droplets, or a protein that is inserted in caveolar membranes. Soluble caveolin-1, which is probably embedded in a lipid particle surrounded by a phospholipid shell, may be involved in intracellular and extracellular lipid transport. In the gut, caveolin-1-rich lipid particles can act as donor particles to facilitate (protein-mediated) intestinal uptake of cholesterol and phospholipids. Our findings strengthen the hypothesis that caveolin-1 has a physiological autocrine/paracrine function and demonstrate that secretion of this protein also occurs in vertebrates other than mammals, such as amphibians, which may be a useful alternative animal model to study caveolin-1.

Fine structure of the external nasal gland of two lizards (Podarcis sicula campestris and Chalcides chalcides).

In this study, we describe the ultrastructural features of the external nasal gland in two lizards: ruin lizard (Podarcis sicula campestris) and seps (Chalcides chalcides). Two secretory cell types, which differ interspecifically, have been found in the secretory endpieces of the glandular tubules in both species examined. An unusual morphological observation was the presence of paracrystalline structures in the secretory granules of the seromucous cells of the external nasal gland of the seps. These structures may be related to the packaging mechanism of glycoproteins or to their macromolecular structure. They may also reflect segregation of heterogeneous subcomponents within the same secretory granule. The striated cells are located in the distal segment of the glandular tubules, and have the typical ultrastructural features of the cells which in some species of reptiles, but not in these two lizards, are known to be capable of elaborating a hyperosmotic saline solution.

Histochemical characterization of the sialic acid residues in mouse colon mucins.

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.

A silybin-phospholipids complex counteracts rat fatty liver degeneration and mitochondrial oxidative changes.

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.

In situ characterization of O-linked glycans of Muc2 in mouse colon.

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.

Caveolin-1 and mitochondrial alterations in regenerating rat liver.

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.

Histochemical and immunohistochemical characterization of exocrine cells in the foregut of the red-eared slider turtle, Trachemys scripta (Emydidae).

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.