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High-fat diet (HFD) alters the glycosylation patterns of intestinal mucins leading to several health problems. We studied by histochemical and lectin-binding methods mucin alterations in the duodenum of mice fed a HFD for 25 weeks. Histochemical methods included periodic acid–Schiff, alcian blue pH 2.5, and high-iron diamine. Lectin-binding experiments were performed with SBA, PNA, WGA, MAA-II, SNA, ConA, UEA-I, LTA, and AAA. SBA, PNA, WGA, MAA-II, and SNA were tested also after desulfation and ConA after periodate-sodium borohydrate treatments (paradoxical ConA). Duodenal mucins are secreted by Brunner’s glands and goblet cells in the villi. Brunner’s glands of HFD mice showed increased secreting activity and a general reduction of glycosylated residuals, such as fucose and terminal α1,4-linked GlcNAc. Moreover, a general reduction of glycosylated residuals in the goblet cells of villi such as the fucosylated and sulfated ones was observed. Since the cited residuals are involved in cytoprotective and cytostatic functions, as well as in interactions with the intestinal microbiota and protection against parasites and inflammatory disorders, we conclude that HFD can predispose duodenum to several possible health disorders.

Urothelium is a multilayer epithelium covering the inner surface of the urinary bladder that acts as a blood-urine barrier and is involved in maintaining the wellbeing of the whole organism. Glycans serve in the maturation and differentiation of cells and thus play a key role in the morphology and function of the multilayered epithelium. The aim of the present study was to examine the glycoprotein pattern of the horse urinary bladder urothelium by lectin histochemistry.The study involved urinary bladders from four horse stallions. Tissue sections were stained with a panel of eleven lectins, in combination with saponification and sialidase digestion (Ks).Basal cells displayed high-mannose N-glycans (Con A), α2,6-linked sialic acid (SNA), and O-linked sialoglycans with sialic acids linked to Galβl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA). The young intermediate cells expressed terminal N-acetylglucosamine (GlcNAc) (GSA II), galactose (GSA I-BThis study showed that the glycan pattern becomes more complex from the basal to surface layer of the urothelium and that surface cells could modify the composition of urine via the secretion of glycoproteins.

Skin filaments are present on the heads of several Hippocampus species. Their clipping is a useful, non-lethal technique for marking individuals and sampling tissue. Little is known about the consequences of clipping on behaviour and details of regeneration. We present the results of a study on the effects of filament clipping on activity patterns and the regeneration of this structure at the macroscopic and microscopic level in Hippocampus guttulatus Cuvier, 1801 from the Gulf of Taranto (Apulia, Ionian Sea). Twelve individuals of both sexes underwent filament clipping, and their behaviour sequences (expressed as percent of total time spent swimming, resting, food searching, and swinging) were monitored for 10 wk in water tanks. In the first week, individuals spent significantly more time food searching and swimming compared to controls, whereas in the following weeks, no differences between groups were observed. Regeneration was observed in 12 other individuals reclipped after 3, 5, and 7 wk. Sections of paraffin-embedded filaments were stained with Mallory’s trichrome, alcian blue pH 2.5, periodic acid-Schiff (PAS), and PAS with diastase. Epidermal cells and basal membranes were the first to recover fully. Basal epidermal cells were filled with glycogen. In the dermis, the arrangement of bundles of fibres surrounded by melanocytes took longer to reconstitute. After 10 wk, the filaments grew to about 36.7% of their original length, whereas at the microscopic level, the tissues recovered fully. It is concluded that filament clipping does not significantly affect the general behavioural patterns of H. guttulatus, and regeneration at the microscopic level is fully accomplished.

Seasonal variation of liver glycogen, lipids and melanomacrophages were investigated in a non-hibernating population of Pelophylax kl. esculentus from Calabria by histochemical methods and computer-assisted image analysis. Twenty individuals of both sexes were sampled in a tank in Roseto Capo Spulico (Cosenza, Calabria) in four periods of the year 2016 (February, May, July, October). Portions of liver from each individual were included in paraffin for glycogen and melanomacrophages, and epoxydic resin-araldite for lipid analysis. Sections were stained with periodic acid-Schiff (PAS) for glycogen (with diastase-PAS as control) or osmium-tetroxide for lipids, or left unstained for melanomacrophages (appearing naturally black due to melanin). Image analyses were performed on 9–12 grayscale converted pictures per individual. Total areas per µm2 of glycogen, lipids and melanomacrophages, as well as counts of lipid droplets and melanomacrophages and mean area of single lipid droplets and melanomacrophages, were measured. Statistical analyses were performed by analysis of variance (ANOVA) with bootstrap resampling. Significant variation among sampling periods was found for each variable. Glycogen and lipids co-vary, with higher values observed in October–February and lower values in May–July, whereas melanomacrophages reach a peak in May and have much lower values in the other months. It is concluded that, in the absence of a hibernating period, reproduction is the main force regulating the annual cycles of reserve storing and melanin production.

Intracellular glycans in the urothelium of urinary bladder of 10 adult male Landrace pigs were characterized in situ by immunohistochemical detection of Muc1 mucin by anti MUC1 from rabbit, conventional histochemical techniques (Periodic-Acid Schiff, Alcian Blue pH 2.5, High-Iron Diamine), and binding with 13 lectins (PNA, DBA, RCA-I, WGA, SBA, BSI-B4, ConA, AAA, UEA-I, LTA, LFA, MAA-II, SNA) combined with chemical and enzymatic pre-treatments (β-elimination, desulfation and neuraminidase) to gather reference data for this model animal. Muc1 mucin was detected in the secreting granules of superficial cells and the underlying layer of intermediate cells. The secreting granules in both intermediate cells and superficial cells were rich in carbohydrates, with the oligosaccharidic chains mostly O-linked to proteins. Glycoproteins were prevailing over glycosaminoglycans (GAGs). In both superficial and intermediate cells sulfated and/or sialylated glycans were present, sulfation decreasing in the deeper layers. Lectin-binding detected presence of terminal sialic acid linked mostly in α2,6 to GalNAc, Gal terminal or subterminal to sulfates, GalNAc, GlcNAc, and Fuc, mostly linked in α1,6, α1,3 α1,4 and α1,2 to GlcNAc or Gal, but not to lactosamine chains. Except for fucosylation, the oligosaccharidic chains in the glycoproteins of the urothelium of pig urinary bladder were similar to those linked to human MUC1, which is fundamental in cell adhesion and immunological processes in the urothelium. The co-distribution of Muc1 and saccharidic residues suggests that many of them are linked to the glycoprotein.

The localization of intestinal glycoconjugates of the blunthead pufferfish Sphoeroides pachygaster and the grey triggerfish Balistes capriscus from the north-western Ionian Sea was analysed by histochemical methods (PAS, AB pH 2.5, HID) and lectin binding experiments (WGA, LFA, SBA, sialidase-SBA, PNA, sialidase-PNA, ConA, AAA, UEA-I, LTA) to assess how evolutionary loss of a functional stomach in S. pachygaster affects intestinal secretions relative to the B. capriscus, which retains the plesiomorphic gastric condition. Sphoeroides pachygaster had a lower content of acid mucins but more complex sialylation patterns than B. capriscus. GalNAc and GlcNAc residuals were present in both, but GalNAc residuals in S. pachygaster were subterminal to sialic acid. Balistes capriscus lacked galactosylated residuals and its enterocytes had a glycocalyx that differed in composition between the small intestine and the rectum and was missing from S. pachygaster. Functional and ecological implications of these findings are discussed.

The Octopus vulgaris farming is impaired by the high mortality of the paralarvae during the first month of life. Several factors have been investigated in this regard, but no data exist on the body surface mucus, which represents the interface with the outside environment. This study included morphometric analysis and glycoconjugates characterization of skin mucus in reared Octopus vulgaris paralarvae during the first month of life. Four types of mucous cells were distinguished: mucous 1 (m1) and mucous 2 (m2) cells were scattered in the mantle epidermis, mucous 3 (m3) and mucous 4 (m4) in the epithelium surrounding the sucker. Except for the presence of fucosylated and neutral glycoconjugates in all mucous cells, each cell type expressed a characteristic glycopattern. m2 and m4 contained also suphate and acid non-sulphate glycans, m3 lacked suphate glycoproteins. Lectin histochemistry showed that mantle mucous cells (m1, m2) expressed GlcNAc and lactosamine terminating glycans. m2 also contained GalNAc terminal or penultimate to sialic acid. m3 was distinguished by mannosylated glycans terminating with lactosamine and m4 by α2,6 sialoglycans. Glycoproteins terminating with lactosamine, Galβ1,3GalNAc, and α1,6-linked fucose were a common feature of paralarvae surface layer. Morphometry revealed a significant decrease of m1 and m2 abundance during the first month of life, afterwards the reared paralarvae died. Since the glycopattern did not change during the investigated period, the mantle mucous cells abundance could be related to the Octopus vulgaris paralarvae survival.

The extra-cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta-elimination PAS) and lectin-histochemical (Con A, WGA, Succinyl-WGA, PNA, RCA-1, DBA, SBA, AAA, UEA-I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J1 and J2). The fibers of J2 are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N-linked. In B. bufo there are also residuals of mannose and/or glucose and N-acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J1 layer of both species presents sialosulfoglycoproteins, mostly O-linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo, whereas the opposite is seen in the amount of fucosylated residuals. The J2 layer is similar in composition to J1 but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J2 . Several microorganisms, in particular blue algae, are observed in the J2 layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types.

Intracellular glycans in the urothelium of urinary bladder of 10 adult male Landrace pigs were characterized in situ by immunohistochemical detection of Muc1 mucin by anti MUC1 from rabbit, conventional histochemical techniques (Periodic-Acid Schiff, Alcian Blue pH 2.5, High-Iron Diamine), and binding with 13 lectins (PNA, DBA, RCA-I, WGA, SBA, BSI-B4, ConA, AAA, UEA-I, LTA, LFA, MAA-II, SNA) combined with chemical and enzymatic pre-treatments (β-elimination, desulfation and neuraminidase) to gather reference data for this model animal. Muc1 mucin was detected in the secreting granules of superficial cells and the underlying layer of intermediate cells. The secreting granules in both intermediate cells and superficial cells were rich in carbohydrates, with the oligosaccharidic chains mostly O-linked to proteins. Glycoproteins were prevailing over glycosaminoglycans (GAGs). In both superficial and intermediate cells sulfated and/or sialylated glycans were present, sulfation decreasing in the deeper layers. Lectin-binding detected presence of terminal sialic acid linked mostly in α2,6 to GalNAc, Gal terminal or subterminal to sulfates, GalNAc, GlcNAc, and Fuc, mostly linked in α1,6, α1,3 α1,4 and α1,2 to GlcNAc or Gal, but not to lactosamine chains. Except for fucosylation, the oligosaccharidic chains in the glycoproteins of the urothelium of pig urinary bladder were similar to those linked to human MUC1, which is fundamental in cell adhesion and immunological processes in the urothelium. The co-distribution of Muc1 and saccharidic residues suggests that many of them are linked to the glycoprotein.

Purpose: Chitosan microparticles containing celecoxib (CB), were developed as chemoprevention of bladder cancer. Furthermore two inclusion complexes of CB with methyl-?-cyclodextrin (C1 and C2) were prepared to improve the solubility of the drug. Methods: C1 and C2 were obtained by freeze-drying and characterized in the solid state and in solution. Microparticles loaded with CB or C1 or C2 were prepared by spray drying and fully characterized. Results: The yield and encapsulation efficiencies of microparticles depended by both the viscosity and the presence of the inclusion complex in the feed medium nebulised. Generally, the microparticles exhibited a spherical shape with mean diameter of approximately 2 ?m which was compatible with local intravesical administration using a catheter. The CB release studies from the microparticles allowed us to identify both immediate release systems (microparticles including the complexes) and prolonged release systems (microparticles including CB alone). The latter exhibited good adhesion to the bladder mucosa, as highlighted by a mucoadhesion study. Histological studies revealed a desquamation of the superficial cells when the bladder mucosa was treated with microparticles loaded with CB, while the morphology of the urothelium did not change when it was treated with microparticles loaded with the inclusion complex. Conclusion: A new CB intravesical formulation than can easily be administered with a catheter and is able to release the drug at the target site for several hours was realized. This new delivery system could be a good alternative to classic oral CB administration.

Characterization of mucins in the alimentary tract of the grass snake, Natrix natrix was performed by histochemical (PAS, Alcian Blue, pH 2.5 and pH 1.0, sialidase-Alcian Blue, pH 2.5, HID-AB pH 2.5) and lectin-histochemical (WGA, SWGA, PNA, sialidase-PNA, SBA, sialidase-SBA, DBA, sialidase-DBA, ConA, BSI-B4, AAA, UEA-1, LTA) techniques. Oesophageal lining epithelium consisted of ciliated and goblet cells, with no pluricellular glands. Mannosylated sialosulfomucins were observed. Fundic mucosa of stomach presented surface cells producing sialomucins with terminal sialic acid linked to galactose. In gastric glands neck and oxynticopeptic cells were found. Neck cells had sialomucins with mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine and fucose-α-(1,2)-linked residues. Cytoplasm of oxynticopeptic cells showed N-acetylgalactosamine and fucose residues. Secretion of surface cells in pyloric mucosa was similar to that of fundic ones, differing in having fucose. Goblet cells in the small intestine of N. natrix produced sulfo- and sialomucins, with sialic acid linked to galactose and N-acetylgalactosamine residues. Mucins also presented residues of mannose. Goblet cells in the large intestine presented sulfomucins only, with terminal N-acetylgalactosamine, galactose and N-acetylglucosamine. The glycosylation patterns found are probably related to protection against injuries, gastric juice and microorganisms, both pathogenic and decomposers, as well as to dietary adaptations.

Eur J Clin Invest 2011; 41 (6): 642–651 Abstract 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 14 days 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.

The mucins secreted by the Brunner's glands and the duodenal goblet cells of the Guinea-pig and the house mouse were compared by conventional and FITC-conjugated lectin histochemistry. Methylation/saponification and sialidase digestion were performed prior to lectin binding to detect the residues subterminal to sulfated groups and sialic acid, respectively. In the Guinea-pig the Brunner's glands produce class-III stable sulfosialomucins. Sialic acid is mostly 2,6-linked to galactose or to N-acetylgalactosamine and is in part O-acetylated in C7, C8, and C9. Sulfated groups are probably linked to sialic acid and N-acetylgalactosamine. Terminal residuals of N-acetylglucosamine, galactose, N-acetylgalactosamine and fucose linked in α1,2, α1,3, and α1,4 are also present. Duodenal goblet cells of the Guinea-pig present a lower number of residuals in respect to the Brunner's glandular ones, with sialic acid and N-acetylgalactosamine subterminal to sulfated groups. In the house mouse the Brunner's glands produce class-III stable neutral mucins, binding to same lectins as in the Guinea-pig except for those specific to sialic acid. A diversity of fucosylated residuals higher than in the Guinea-pig is observed. The mouse duodenal goblet cells lack stable class-III mucins, have little sialic acid and present a lower number of residuals in respect to the correspondent Brunner's glands. Regulation of the acidic intestinal microenvironment, prevention of pathologies and hosting of microflora can explain the observed results and the differences observed between the two rodents.

The extra-cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta-elimination PAS) and lectin-histochemical (Con A, WGA, Succinyl-WGA, PNA, RCA-1, DBA, SBA, AAA, UEA-I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J1 and J2). The fibers of J2 are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N-linked. In B. bufo there are also residuals of mannose and/or glucose and N-acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J1 layer of both species presents sialosulfoglycoproteins, mostly O-linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo, whereas the opposite is seen in the amount of fucosylated residuals. The J2 layer is similar in composition to J1 but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J2 . Several microorganisms, in particular blue algae, are observed in the J2 layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types.

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 Galβ1,3GalNAc 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.

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 Galβ1,3GalNAc 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 Galβ1,3GalNAc 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.

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, includ- ing 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 lipido- genesis and in mitochondrial homeostasis, we aimed to study the subcellular distribution of caveo- lin-1 in hepatocytes at an early stage following PH. Liver samples were processed for light and elec- tron microscopy at 0 h, 24 h, and 96 h after PH. The expression and subcellular distribution of cav- eolin-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 fol- lowing 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 homeosta- sis and lipidogenesis during frequent metabolic liver diseases. Microsc. Res. Tech. 00:000-000, 2012. V C 2012 Wiley Periodicals, Inc.

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, 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.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.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.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.

One form of liver steatosis, namely Non-Alcoholic Fatty Liver Disease (NAFLD), is a worrisome health problem worldwidecharacterized by intrahepatic triacylglycerol (TG) overaccumulation. NAFLD is a common feature of metabolic syndromebeing often associated with obesity, dyslipidemia and diabetes and mostly closely linked to insulin resistance. Themechanism of NAFLD pathogenesis is object of intense investigation especially regarding complex systems ultimatelyresulting in excessive TG deposition in hepatocytes. However, scarce is the attention about the relevance of hepatic importof glycerol, the other primary source (as glycerol-3-phosphate) of increased TG in hepatocytes. Obese leptin-deficient (ob/ob) mice, an animal model of NAFLD, were used to evaluate the functional involvement of Aquaporin-9 (AQP9), the majorpathway of liver glycerol entry, in hepatosteatosis. By RT-PCR and qPCR, the level of Aqp9 mRNA in the liver of starved obesemice was comparable with the corresponding control lean littermates. By immunoblotting, the AQP9 protein at thehepatocyte sinusoidal plasma membrane of obese mice was markedly lower (33%) than lean mice, a finding fully confirmedby immunohistochemistry. By stopped-flow light scattering, the liver glycerol permeability of ob/ob mice was significantlylower (53%) than lean mice, a finding consistent with both the observed down-regulation of AQP9 protein and increasedlevel of plasma glycerol characterizing obese mice. In summary, our results suggest implication of AQP9 in liver steatosis.The reduction of hepatocyte AQP9 and, consequently, glycerol permeability might be a defensive mechanism to counteractfurther fat infiltration in liver parenchyma. Fil: Gena, Patrizia. Department of Biosciences. Biotechnologies and Biopharmaceutics. University of Bari Aldo Moro; Italy; Fil: Mastrodonato, María. Department of Biology. University of Bari Aldo Moro; Italy; Fil: Portincasa, Piero. Department of Biomedical Sciences and Human Oncology. University of Bari Aldo Moro; Italy; Fil: Fanelli, Elena. Department of Biosciences. Biotechnologies and Biopharmaceutics. University of Bari Aldo Moro; Italy; Fil: Mentino, Donatella. Department of Biology. University of Bari Aldo Moro; Italy; Fil: Rodríguez, Amaia. Metabolic Research Laboratory. Clıínica Universidad de Navarra. The Spanish Biomedical Research Centre in Physiopathology of Obesity and Nutrition; Spain; Fil: Marinelli, Raul Alberto. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Fisiologia Experimental (i); Fil: Brenner, Catherine. Institut National de la Sante et de la Recherche Medicale U769. LabEx LERMIT. Universite Paris-Sud. France; Fil: Frühbeck, Gema. Metabolic Research Laboratory. Clıínica Universidad de Navarra. The Spanish Biomedical Research Centre in Physiopathology of Obesity and Nutrition; Spain; Fil: Svelto, María. Department of Biosciences. Biotechnologies and Biopharmaceutics. University of Bari Aldo Moro; Italy; Centro di Eccellenza di Genomica in campo Biomedico ed Agrario; Italy.; Fil: Calamita, Giuseppe. Network of Apulian Public Research Laboratories ‘‘WAFITECH’’; Italy.