Tiziano Verri

D-glucose transport in decapod crustacean hepatopancreas.

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.

Molecular and functional characterisation of the zebrafish (Danio rerio) PEPT1-type peptide transporter1

We report the molecular and functional characterisation of a novel peptide transporter from zebrafish, orthologue to mammalian and avian PEPT1. Zebrafish PEPT1 is a low‐affinity/high‐capacity system. However, in contrast to higher vertebrate counterparts in which maximal transport activity is independent of extracellular pH, zebrafish PEPT1 maximal transport rates unexpectedly increase at alkaline extracellular pH. Zebrafish pept1 is highly expressed in the proximal intestine since day 4 post‐fertilisation, thus preceding functional maturation of the gut, first feeding and complete yolk resorption. Zebrafish PEPT1 might help to understand the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, zebrafish pept1 can be a useful marker for screening mutations that affect gut regionalisation, differentiation and morphogenesis.

Cell shape and plasma membrane alterations after static magnetic fields exposure

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.

Regulation of opossum kidney (OK) cell Na/Pi cotransport by Pi deprivation involves mRNA stability

Renal proximal tubular Na-dependent phosphate transport (Na/Pi cotransport) has been studied extensively in the opossum kidney (OK) cell line. Recently, we cloned a complementary deoxyribonucleic acid (cDNA) (NaPi-4) from OK cells encoding an apical NaPi cotransport system. OK cells exposed to a low-Pi medium, as compared to high-Pi media, responded with an increase in Na/Pi cotransport, which was followed by an increase in NaPi-4 messenger ribonucleic acid (mRNA) abundance; maximal stimulation of Na/Pi cotransport was reached in 2 h, with no further increase for up to 16 h. NAPi-4 mRNA abundance was unaltered for 2 h, then increased to a maximum after 6–16 h in cells treated with low Pi medium. NaPi-4 mRNA decay rate was lowered by low-Pi media when compared to high-Pi media, with no increase in the NaPi-4 mRNA transcription rate. These data suggest that the upregulation of Na/Pi cotransport in OK cells by low-Pi media involves two regulatory mechanisms: an immediate (early) increase (after 2 h) in the expression of Na/Pi cotransport, independent of mRNA synthesis or stability, and a delayed (late) effect (after 4–6 h), resulting in an increase in NaPi-4 mRNA abundance, due to an increased stability.

The effect of plant protein-based diet supplemented with dipeptide or free amino acids on digestive tract morphology and PepT1 and PepT2 expressions in common carp (Cyprinus carpio L.).

Common carp (Cyprinus carpio) of average body mass 0.07+/-0.02 g were fed three formulated diets: wheat gluten protein-based diet supplemented with Lys-Gly dipeptide (PP), wheat gluten protein-based diet supplemented with free lysine and glycine (AA), and a wheat gluten protein-based control diet without lysine supplementation (CON), frozen zooplankton (Z) (restricted diet), and a commercial starter food Aglo Norse (AN). After 4 weeks of experimental feeding, fish fed AN diet showed the highest body mass and length. Significantly lower mass occurred in groups fed PP, AA, CON, and Z. Fish fed CON diet showed the lowest intestinal folds and the highest number of mucous cells. Fish fed PP diet showed a significantly higher number of gastrin/cholecystokinin (CCK) positive cells. The diameter of lipid vacuoles in hepatocyte cytoplasm of fish fed formulated diets (PP, AA and CON) was significantly higher than in fish fed zooplankton (Z) and the commercial diet (AN). Hepatocytes of fish fed AA and CON showed a higher nucleus proliferation rate than in the other experimental groups. The quantitative analysis of the number of proliferating cell nuclear antigen (PCNA) and caspase-3(rabbit polyclonal antibody CPP-32)-positive cells showed that the highest proliferation rate was accompanied by the high apoptosis in the intestine of fish fed AA and CON. After 4 weeks of experimental feeding the highest relative expression of PepT1 gene was observed in fish fed PP diet, while the lowest expression occurred in fish fed CON. Feeding carp plant protein-based diet supplemented with Lys-Gly dipeptide (PP) had a beneficial influence on fish growth and metabolism in the digestive tract as compared to fish fed control diet without lysine supplementation (CON).

Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspects.

SUMMARY A novel full-length cDNA that encodes for the Atlantic cod (Gadus morhua L.) PepT1-type oligopeptide transporter has been cloned. This cDNA (named codPepT1) was 2838 bp long, with an open reading frame of 2190 bp encoding a putative protein of 729 amino acids. Comparison of the predicted Atlantic cod PepT1 protein with zebrafish, bird and mammalian orthologs allowed detection of many structural features that are highly conserved among all the vertebrate proteins analysed, including (1) a larger than expected area of hydrophobic amino acids in close proximity to the N terminus; (2) a single highly conserved cAMP/cGMP-dependent protein kinase phosphorylation motif; (3) a large N-glycosylation-rich region within the large extracellular loop; and (4) a conserved and previously undescribed stretch of 8–12 amino acid residues within the large extracellular loop. Expression analysis at the mRNA level indicated that Atlantic cod PepT1 is mainly expressed at intestinal level, but that it is also present in kidney and spleen. Analysis of its regional distribution along the intestinal tract of the fish revealed that PepT1 is ubiquitously expressed in all segments beyond the stomach, including the pyloric caeca, and through the whole midgut. Only in the last segment, which included the hindgut, was there a lower expression. Atlantic cod PepT1, the second teleost fish PepT1-type transporter documented to date, will contribute to the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful tool for the study of gut functional regionalization, as well as a marker for the analysis of temporal and spatial expression during ontogeny.

Dietary protein hydrolysates and free amino acids affect the spatial expression of peptide transporter PepT1 in the digestive tract of Atlantic cod (Gadus morhua)

The effects of dietary inclusions of size-fractionated peptides and free amino acids (FAAs) on Peptide Transporter 1 (PepT1) mRNA levels were assessed along the length of the intestine of juvenile Atlantic cod (Gadus morhua). Five groups of fish (10-15g) were fed for 46days on diets containing approximately 42% protein, provided either as fish meal (FM, control diet) or as a combination of FM with whole fish hydrolysate (FH), retenate after ultrafiltration of FH (UFR), nanofiltered retenate of FH (NFR), or a mix of FAAs, at a 30% level of FM substitution. PepT1 mRNA expression was assessed in pyloric caeca (S1) and the remainder of the intestine divided into four equally long segments (S2-S5). PepT1 transcripts were found in all segments, indicating that the whole intestine is involved in peptide absorption. Differences in the regional expression profile of PepT1 were found. Under control diet (FM diet) conditions, fish exhibited a reduced expression in S5 compared to S2. In fish fed FAA and UFR diets, PepT1 mRNA levels were higher in S2 and S3 compared to other regions. These data suggest that PepT1 may be variably recruited along the whole intestine, including the most distal part, in response to changes in the luminal protein source content. This adaptive response might be functional to keep a maximal efficiency of protein absorption at the intestinal level.

The effect of peptide absorption on PepT1 gene expression and digestive system hormones in rainbow trout (Oncorhynchus mykiss).

The present study evaluates the effect of protein source (dipeptides, free amino acids, and intact protein) on development and growth of Salmonid fish alevin. Specifically, we follow the expression of oligopeptide transporter protein PepT1 in the intestine of rainbow trout (Oncorhynchus mykiss). Fish were fed exogenously one of four diets: three formulated (lysyl-glycine dipeptide supplemented diet - PP, free lysine and glycine supplemented diet - AA, control diet with no lysine - CON) or commercial starter (Aller Futura - AF). Fish increased mean body weight 8 fold with PP- and AA-supplemented diets resulting in significantly higher weight gain than fish fed CON. Statistical analysis revealed a significant increase in relative PepT1 expression of fish fed experimental diets. Immunohistochemical staining with PepT1 antibody showed the presence of the transporter protein in the brush border membrane of the proximal intestinal enterocytes of fish from all experimental groups. Leptin immunoreactivity occurred not only in the gastric glands but also in proximal intestine and pyloric caeca of fish fed PP, AA and AF diets. Leptin immunoreactivity was also observed in hepatocyte cytoplasm and pancreatic acinar cells. Gastrin/CCK immunoreactive cells were present in the proximal intestine and pyloric caeca.

Thyroid hormone stimulation of Na/Pi-cotransport in opossum kidney cells.

Thyroid hormone (T3), a known stimulator of renal proximal tubular brush border membrane Na-dependent phosphate (Pi) uptake (Na/Pi-cotransport), stimulated Na-dependent Pi transport in opossum kidney (OK) cells. Na/Pi-cotransport was stimulated in a time- and dose-dependent manner with maximal effects (57%) at 24 h and 10−10 M T3. This stimulation was related to an increase in the apparent capacity (Vmax) of Na/Pi-cotransport. Treatment with T3 had no effect on Na-independent transport of Pi or of l-arginine. The stimulation of Na/Pi-cotransport was paralleled by an increase in the messenger ribonucleic acid (mRNA) encoding the OK cell apical Na/Pi-cotransporter (termed NaPi 4); the mRNA levels related to the activity of Na-independent l-arginine transport (rBAT) were unaffected by T3. Actinomycin D (10−7M) completely prevented the stimulatory effect of T3 on OK cell Na/Pi-cotrransport and on NaPi-4 mRNA content. In conclusion, T3 stimulates apical Na/Pi-cotransport in OK cells most likely by enhancing its transcription.

Peptide transport and animal growth: the fish paradigm.

Protein digestion products are transported from the intestinal lumen into the enterocyte both in the form of free amino acids (AAs), by a large variety of brush border membrane AA transporters, and in the form of di/tripeptides, by a single brush border membrane transporter known as PEPtide Transporter 1 (PEPT1). Recent data indicate that, at least in teleost fish, PEPT1 plays a significant role in animal growth by operating, at the gastrointestinal level, as part of an integrated response network to food availability that directly supports body weight. Notably, PEPT1 responds to both fasting and refeeding and is involved in a phenomenon known as compensatory growth (a phase of accelerated growth when food levels are restored after a period of growth depression). In particular, PEPT1 expression decreases during fasting and increases during refeeding, which is the opposite of what observed so far in mammals and birds. These findings in teleost fish document, to our knowledge, for the first time in a vertebrate model, a direct correlation between the expression of an intestinal transporter, such as PEPT1, primarily involved in the uptake of dietary protein degradation products and animal growth.