Donatella Mentino

Liver Glycerol Permeability and Aquaporin-9 Are Dysregulated in a Murine Model of Non-Alcoholic Fatty Liver Disease

One form of liver steatosis, namely Non-Alcoholic Fatty Liver Disease (NAFLD), is a worrisome health problem worldwide characterized by intrahepatic triacylglycerol (TG) overaccumulation. NAFLD is a common feature of metabolic syndrome being often associated with obesity, dyslipidemia and diabetes and mostly closely linked to insulin resistance. The mechanism of NAFLD pathogenesis is object of intense investigation especially regarding complex systems ultimately resulting in excessive TG deposition in hepatocytes. However, scarce is the attention about the relevance of hepatic import of 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 major pathway of liver glycerol entry, in hepatosteatosis. By RT-PCR and qPCR, the level of Aqp9 mRNA in the liver of starved obese mice was comparable with the corresponding control lean littermates. By immunoblotting, the AQP9 protein at the hepatocyte sinusoidal plasma membrane of obese mice was markedly lower (33%) than lean mice, a finding fully confirmed by immunohistochemistry. By stopped-flow light scattering, the liver glycerol permeability of ob/ob mice was significantly lower (53%) than lean mice, a finding consistent with both the observed down-regulation of AQP9 protein and increased level 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 counteract further fat infiltration in liver parenchyma.

Altered distribution of caveolin-1 in early liver steatosis

Eur J Clin Invest 2011; 41 (6): 642–651

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.

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 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. Microsc. Res. Tech., 2013.

Histochemical characterization of the mucins of the alimentary tract of the grass snake, Natrix natrix (Colubridae).

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.

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. Microsc. Res. Tech. 75:1026–1032, 2012.

Comparative glycopattern analysis of mucins in the Brunner's glands of the guinea-pig and the house mouse (Rodentia).

The mucins secreted by the Brunners 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 Brunners 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 Brunners glandular ones, with sialic acid and N-acetylgalactosamine subterminal to sulfated groups. In the house mouse the Brunners 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 Brunners 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.

Histochemical and structural characterization of egg extra-cellular matrix in bufonid toads, Bufo bufo and Bufotes balearicus: Molecular diversity versus morphological uniformity.

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 eggs 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. Microsc. Res. Tech. 77:910–917, 2014.

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

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.

Seasonal changes in the liver of a non-hibernating population of water frogs, Pelophylax kl. esculentus (Anura: Ranidae)

Abstract 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.