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Biomedical subjects

M Bendayan

Publications and source records attributed to M Bendayan.

At least 19 recordsLinked to original sources
PubMed2007

Leptin secretion by white adipose tissue and gastric mucosa.

Leptin is a hormone that plays a central role in the regulation of food intake and energy expenditure. Originally discovered in mature white adipocytes, it was subsequently isolated from the gastric mucosa. This tissue contains a large number of epithelial endocrine and exocrine cells secreting leptin in the blood stream and in the gastric lumen, respectively. Light and electron microscopy have shown that adipocytes and gastric epithelial cells contain leptin along their rough endoplasmic reticulum-Golgi-granules secretory pathway. Both tissues synthesize a soluble form of the leptin receptor that is secreted bound to leptin in the blood and into the gastric juice. This soluble receptor protect leptin and enhances its half-life. Despite the similarities in the mechanisms of leptin secretion by adipocytes and gastric epithelial cells, they are in fact radically different. In gastric cells leptin follows a rapid regulated secretion pathway whereas adipocytes secrete leptin in a constitutive slow fashion. These differences can be explained by the specific roles play by leptin originating from these two different tissues. Gastric leptin is involved in the short-term regulation of digestion, including delay of gastric emptying, absorption of nutrients by the intestinal wall and secretion of gastric, intestinal and pancreatic hormones. On the other hand, leptin secreted by white adipocytes acts primarily on the hypothalamus for the long-term regulation of food intake. Therefore, the coordination of adipose and gastric leptins ensures the proper management of food processing and energy storage.

Adipocytes, White↗
PubMed2006

Clusterin induces differentiation of pancreatic duct cells into insulin-secreting cells.

AIMS/HYPOTHESIS: We recently reported that expression of the gene encoding clusterin (Clu) is upregulated in the regenerating pancreas, particularly in tissues undergoing differentiation. This led us to propose that clusterin participates in the cytodifferentiation of pancreatic tissue, particularly the endocrine islet cells. The aim of this study was to investigate whether clusterin induces the differentiation of duct-lining cells into insulin-secreting cells. METHODS: We isolated ductal tissue from rat pancreas and cultured it to develop epithelial cell explants for transfection of the Clu cDNA as well as for treatment of clusterin protein. RESULTS: The number of newly differentiated insulin cells increased 6.9-fold upon Clu overexpression compared with controls. Ins1 mRNA and peptide levels were also increased. Furthermore, glucose-stimulated insulin secretion was observed in the differentiated insulin cells. These cells were immunoreactive for insulin and C-peptide, but negative for other islet hormones and for cytokeratin-20, which indicates a fully differentiated state. Insulin cell differentiation was also increased in a dose-dependent manner by treating duct cells in culture with clusterin, indicating a growth-factor-like action of clusterin in insulin cell differentiation. CONCLUSIONS/INTERPRETATION: These results suggest that clusterin can be considered as a potential morphogenic factor that promotes differentiation of pancreatic beta cells.

Animals↗
PubMed2006

Expression and localization of MT1-MMP and furin in the glomerular wall of short- and long-term diabetic rats.

Diabetic glomerulopathy has been linked to shifts in balance between the synthetic and degradative pathways of the glomerular basement membrane (GBM), a key player in the permselectivity properties of the glomerular wall. The goal of this study was to trace the expression and localization of membrane type-1 metalloprotease (MT1-MMP) and its activating enzyme furin, key proteins involved in basement membrane turnover, in short- and long-term diabetic rat renal tissues. Quantitative immunogold was carried out for MT1-MMP and furin and their expression was evaluated in renal tissues of young and old, control and diabetic rats. To corroborate immunocytochemical findings, Western blots were performed on glomerular lysates. Electron microscopy revealed that the overall expression of MT1-MMP and furin is reduced in plasma membranes of all glomerular cell types of old normoglycemic animals, a phenomenon that is exacerbated in long-term diabetic animals. This observation supports the prevailing theory that diabetes fosters acceleration in the aging process. Interestingly, while biochemical results confirmed a decrease in MT1-MMP expression, an increase in furin was observed. Immunocytochemical studies resolved this discrepancy by tracing the increased furin expression in endoplasmic reticulum and Golgi membranes of podocytes, indicating that furin is retained in the secretory pathway in a diabetic environment. Disturbances at the molecular level of the otherwise tightly regulated MT1-MMP/furin interactions found at the cell surface must account for a lack in extracellular matrix remodeling, increased deposition of GBM material, and loss of glomerular filtration integrity.

Animals↗
PubMed2005

Glomerular handling of native albumin in the presence of circulating modified albumins by the normal rat kidney.

Persistent hyperglycemia, as occurring in diabetes, induces changes in circulating as well as in structural proteins. These changes involve substitution of lysine residues by glucose adducts resulting in early Amadori products that evolve into toxic and active substances, the advanced glycation end adducts. In previous studies, we demonstrated that early glycated (Amadori) albumin infused into the circulation of normal animals induces transitory alterations of glomerular filtration. Attempting to elucidate the mechanisms underlying these changes, various molecular modifications were introduced in vitro to serum albumin. Glycation, acetylation, carboxymethylation, methylation, and succinylation, involving either a few or a significant number of amino acid residues, produced heavier and more anionic albumin molecules compared with the native one. Native and each of the modified albumin molecules were injected intravenously into normal rats, followed, 30 min later, by hapten-tagged native BSA. Changes in glomerular filtration were evaluated by morphometrical analysis of gold immunolabelings. Compared with native albumin, all the modified forms of albumin induced a deeper penetration of the tracer through the glomerular basement membrane revealing alterations in glomerular permselectivity. This was more evident for severely modified albumin molecules which displayed high labelings in the urinary space and endocytic compartments of proximal tubule epithelial cells. These results indicate that modifications of serum albumin, even minimal, as those occurring in early diabetes, could immediately affect the permselectivity properties of the glomerular wall leading, with time, to severe glomerulopathies.

Animals↗
PubMed2004

Activation of nestin-positive duct stem (NPDS) cells in pancreas upon neogenic motivation and possible cytodifferentiation into insulin-secreting cells from NPDS cells.

Stem cells in adult pancreas and their specific marker are poorly characterized. We hypothesized that pancreatic stem cells could evolve from the duct system in response to neogenic stimulation and may transiently express nestin during tissue regeneration. After partial pancreatectomy (Px), we found extensive formation of ductules consisting of nestin-positive epithelial cells with higher replicating ability in the neogenic foci, particularly at day 3 after Px. Nestin was highly expressed in the earlier stages of ductule morphogenesis and then regressed as the cells evolved toward differentiated pancreatic cell types. The neogenic ductules were isolated for the culture of nestin-positive duct stem cells. These nestin-positive duct cells were numerous and displayed extensive self-replication in the duct cell explants after 2-3 days of culture, thus depicted as nestin-positive duct stem (NPDS) cells. As seen in the tissue of neogenic foci, NPDS cells were negative for cytokeratin-20 and vimentin, the marker for duct epithelial and mesenchymal cells, respectively. Endocrine cells, mostly insulin cells, were present in the explants at day 2 as single cells or as small clusters adjacent to the NPDS cells, and formed islet-like masses at day 3 of culture, suggesting islet cell differentiation from NPDS cells. In addition, insulin secretion from these beta cells responded to glucose stimulation. We found transient up-regulation of PDX-1 expression by reverse transcriptase-polymerase chain reaction at day 3 after Px in pancreatic tissue. Higher expression of PDX-1 was seen in the culture of neogenic ductules than that of ducts isolated from the sham-operated pancreas. In particular, a subpopulation of nestin-positive cells in the duct cell explants formed from the neogenic ductules expressed PDX-1 in their nuclei. Taken together, this information suggests that NPDS cells could be generated from adult pancreas by neogenic motivations and they may differentiate into insulin-secreting cells.

Animals↗
PubMed2003

Expression differences in mitochondrial and secretory chaperonin 60 (Cpn60) in pancreatic acinar cells.

In pancreatic acinar cells, chaperonin Cpn60 is present in all the cellular compartments involved in protein secretion as well as in mitochondria. To better understand the role Cpn60 plays in pancreatic secretion, we have evaluated its changes under experimental conditions known to alter pancreatic secretion. Quantitative protein A-gold immunocytochemistry was used to reveal Cpn60 in pancreatic acinar cells. Cpn60 immunolabelings in cellular compartments involved in secretion were found to decrease in acute pancreatitis as well as upon stimulation of secretion and in starvation conditions. A major increase in Cpn60 was recorded in diabetic condition. This was normalized by insulin treatment. Although in certain situations changes in secretory enzymes and in Cpn60 correlate well, in others, nonparallel secretion seemed to take place. In contrast, expression of mitochondrial Cpn60 in acinar cells appeared to remain stable in all conditions except starvation, where its levels decreased. Expression of Cpn60 in the secretory pathway and in mitochondria thus appears to behave differently, and Cpn60 in the secretory pathway must be important for quality control and integrity of secretion.

Animals↗
PubMed2003

Angiotensinogen localization and secretion in the rat pancreas.

Renin and angiotensinogen have been previously found in the rat pancreas, and angiotensin receptors have been located in the apical domain of duct cells. To evaluate the possibility that angiotensin II could be generated within the duct system, we decided to determine whether angiotensinogen is present in rat pancreatic juice and the angiotensinogen-immunoreactive pancreatic cell types that could be responsible for its production. Angiotensinogen was detected in significant amounts by Western blotting in pancreatic juice collected from several individual rats. Different isoforms between plasma and pancreatic juice angiotensinogens were demonstrated by isoelectric focusing. Immunocytochemical experiments revealed angiotensinogen-immunoreactive cells at the periphery of the islets of Langerhans, and confocal microscopy demonstrated that most angiotensinogen-immunoreactive cells were glucagon-secreting cells. Secretion of angiotensinogen did not follow the regulated secretory pathway since it was absent from the glucagon-containing granules. This was confirmed by electron microscopy immunocytochemistry. Duct and acinar cells did not express angiotensinogen at an immunocytochemical detectable level. The present findings indicated an exocrine secretion of angiotensinogen by glucagon-secreting cells and suggest that one of the final targets of the local pancreatic renin-angiotensin system may be the duct epithelium.

Angiotensinogen↗
PubMed2002

Morpho-functional studies of the blood-brain barrier in streptozotocin-induced diabetic rats.

AIMS/HYPOTHESIS: We undertook the characterization of the capillary bed of the rat frontal cortex and their permeability properties in short-term and long-term diabetic rats. METHODS: Diabetes was induced by strepozotocin injection. Rats were maintained hyperglycaemic without insulin treatment during 4 to 5 months (short-term) and 8 to 13 months (long-term). Rats from an additional short-term hyperglycaemic group received an injection of exogenous dinitrophenylated albumin 15 min before being killed. Tissues were processed for electron microscopy and quantitative immunocytochemistry. Endogenous and dinitrophenylated exogenous albumin were revealed with high resolution over the capillary wall using specific antibodies and the protein A-gold complex. Morphometrical analyses were carried out. RESULTS: Albumin is transported across endothelial cells by plasmalemmal vesicles or caveolae and larger vacuolar structures. This transport increased in diabetic rats by an increment in the number of vesicles. Albumin distribution across the capillary basement membrane showed that the restrictive properties of the basement membrane present in normoglycaemic rats are altered in the diabetic condition, as was its thickness. Similar alterations of the basement membrane structure and function were encountered in old normoglycaemic rats but to a lesser extent. CONCLUSION/INTERPRETATION: The results indicate that diabetes seems to accelerate the ageing process of the vascular wall and that the central nervous system capillary bed is also a target for diabetic microangiopathy.

Animals↗
PubMed2002

Sequential morphological and permeability changes in the rete capillaries during hyperglycaemia.

With the rete model of the eel swimbladder, we have studied the appearance and development of a microangiopathy during a 2-year period of hyperglycaemia. Hyperglycaemia was induced in the eel by chronic exposure to cold water. At 3-5 months, basement membrane thickness was twice the normal value and increased only slightly thereafter. Diffusion coefficients of permeability were measured in counter-current perfusion experiments for a variety of tracers that are believed to use different pathways of transcapillary transport. The permeability to sucrose was the first to significantly increase, at 6-8 months, followed by that of albumin, insulin, and inulin, at 9-11 months and that of sodium, at 18-24 months. The permeability to water and antipyrine remained stable throughout the study. The results indicate that in the rete model, chronic hyperglycaemia induces a rapid thickening of the capillary basement membrane and selective permeability increments in the various paths of transcapillary transport.

Air Sacs↗
PubMed2001

Transcytosis of pancreatic bile salt-dependent lipase through human Int407 intestinal cells.

In previous studies, we have shown that the bile-salt-dependent-lipase (BSDL), secreted by pancreatic acinar cells and secreted into the duodenal lumen, can be transcytosed through intestinal cells up to the lamina propria. In this study, we used an in vitro system to provide insights into the apical to basolateral transport of BSDL, across the intestinal barrier. The Int407 human epithelial cell line, grown under conditions that optimize polarity, was used as a tight epithelium model. We attempted to delineate uptake mechanisms and the transcytotic pathway followed by this pancreatic enzyme within the intestinal Int407 cells, which do not produce BSDL. When added to the apical reservoir of Transwell-grown Int407 cells, BSDL was shown to first interact with the apical membrane. Further, BSDL forms clusters that are internalized via clathrin-coated pits. Following endocytosis, BSDL is directed to a nocodazole- and colchicin-sensitive multivesicular compartment. Interestingly, this protein transits through the Golgi apparatus, where it was found to colocalize with the KDEL retrieval-receptor. Finally, enzymatically active intact BSDL was released at the basolateral membrane level. This is the first demonstration for an apical-to-basolateral transcytotic pathway of a secreted pancreatic digestive enzyme through polarized intestinal cells.

Animals↗
PubMed2001

Effect of human recombinant leptin on lipid handling by fully differentiated Caco-2 cells.

It has been established that leptin displays a number of effects on peripheral tissues. We have investigated the effect of the hormone on lipid synthesis, apolipoprotein biogenesis and lipoprotein secretion in Caco-2 cells. Immunocytochemistry revealed the presence of leptin receptors (Ob-Rb) on the basolateral membrane. Incubation of cells with 200 nM leptin resulted in a decreased export of triglycerides in the basolateral medium without affecting monoglyceride, diglyceride and cholesterol ester lipid classes. It also significantly reduced the output of de novo-synthesized apolipoprotein (Apo)B-100 and ApoB-48 as well as that of newly formed chylomicrons and of low-density lipoproteins. It also enhanced that of ApoA-I, ApoA-IV and ApoE. Our results support the hypothesis that leptin can affect energy balance at the gut level by reducing lipid release into the circulation.

Apolipoproteins↗
PubMed2001

Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines.

Intestinal and liver fatty acid binding proteins (I- and L-FABP) are thought to play a role in enterocyte fatty acid (FA) trafficking. Their modulation by cell differentiation and various potential effectors was investigated in the human Caco-2 cell line. With the acquisition of enterocytic features, Caco-2 cells seeded on plastic progressively increased L-FABP quantities, whereas I-FABP was not detectable even very late in the maturation process. On permeable filters that improved differentiation markers (sucrase, alkaline phosphatase, transepithelial resistance), Caco-2 cells furthered their L-FABP content and expressed I-FABP. Western blot analysis showed a significant increase in I- and L-FABP expression following an 8-hour incubation period with butyric acid, oleic acid, and phosphatidylcholine. However, in all cases, I-FABP levels were higher than L-FABP concentrations regardless of the lipid substrates added. Similarly, hydrocortisone and insulin enhanced the cellular content of I- and L-FABP whereas leptin triggered I-FABP expression only after an 8-hour incubation. Finally, tumor necrosis factor-alpha was more effective in increasing the cytosolic amount of I-FABP levels. In conclusion, our data demonstrate that I-FABP expression is limited to fully differentiated Caco-2 cells and can be more easily regulated than L-FABP by lipids, hormones, and cytokines.

Caco-2 Cells↗
PubMed2001

Association between islets of Langerhans and pancreatic ductal system in adult rat. Where endocrine and exocrine meet together?

AIMS/HYPOTHESIS: Studies on the functional and morphological relations between exocrine and endocrine pancreas have been conducted mainly to disclose the influence of islets of Langerhans on acinar parenchyma. Less attention has been paid to the relations occurring between islets and pancreatic ducts. METHODS: A series of consecutive sections of normal adult rat pancreas were double stained with islet (hormones) and duct (cytokeratin 20) markers. Electron microscopy was conducted to investigate the ultra-structural features of duct-islet relations and anti-insulin immunogold labelling was carried out to reveal the presence of insulin in the pancreatic duct system. RESULTS: Consecutive double-stained sections demonstrated that 73.60 +/- 2.97% of the islets were attached to the ducts. For each series, 93.48 +/- 5.43 % of the islets contacting the duct tree were associated with small-sized ducts or centroacinar cells. Electron microscopy revealed that some insulin and somatostatin cells do face the duct lumen. Insulin was detected within the duct lumen and in the endosomal compartment of the duct cells. CONCLUSIONS/INTERPRETATION: The finding that most islets are connected with the duct system in the adult pancreas is discussed in terms of hormone secretion into the ducts, islet histogenesis and the relation among the three tissue components of the pancreas, the endocrine, the exocrine and the duct system.

Animals↗
PubMed2001

Amplification methods for the immunolocalization of rare molecules in cells and tissues.

The needs to precisely assign macromolecules to specific locations and domains within tissues and cells and to reveal antigens which are present in low or even in trace amounts, led to the elaboration of a wide spectrum of immunocytochemical amplification procedures. These arise from the successive improvements of tissue preparation techniques, of antigen retrieval procedures and of immunological or non-immunological detection systems. Improvement of detection systems may be the most active in the development of amplification techniques. Since the early work of Coons, in which by the introduction of the indirect technique has started amplifying the signal, different systems have succeeded in increasing the sensitivity of antigens detection. Indeed, amplification techniques such as the multiple antibody layers, the multiple bridges, the enzyme complexes, the avidin-biotin, the silver intensification, and the numerous variations and combinations among these have increased the sensitivity for the detection of scarce tissue antigens. However, as shown by the recent progress carried out with new approaches such as the catalyzed reporter deposition (CARD) and the enhanced polymer one-step staining (EPOS), more efficient methods are still needed. In electron microscopy, few techniques have reached the resolution afforded by the post-embedding immunogold approach. In spite of this and in order to further increase its sensitivity, new probes and novel approaches are allowing combination of the gold marker with the amplification capacity of enzymes afforded by the CARD technique. Immunogold amplification strategies, such as the multiple incubations with the primary antibody and the use of an anti-protein A antibody have also led to enhanced signals displaying the advantages in terms of resolution and possibilities of quantification inherent to the colloidal gold marker.

Animals↗
PubMed2001

Alterations of vitronectin and its receptor alpha(v) integrin in the rat renal glomerular wall during diabetes.

Vitronectin, a multifunctional glycoprotein present in blood and extracellular matrix, is not only a member of the cell adhesion molecules, but also a regulator of proteolytic enzyme cascades, thereby providing a unique regulatory factor for proteolytic degradation of extracellular matrix and tissue remodeling. Vitronectin interacts with the cell surface through integrins of the alpha(v)-related system. Because vitronectin and its receptor may have a role in various renal physiological and pathological processes, we evaluated their expression in renal tissues of streptozotocin-induced short- and long-term hyperglycemic rats by applying quantitative immunoelectron microscopy and Western blot analysis. Vitronectin was shown over the glomerular basement membrane (GBM) and mesangial matrix (MM), whereas alpha(v) was located along the plasma membrane of endothelial, epithelial, and mesangial cells. Although distribution patterns of vitronectin and alpha(v) integrin labeling in renal tissues from short- and long-term hyperglycemic rats, as well age-matched normoglycemic rats, were similar, increases in their immunoreactive sites were detected in hyperglycemic conditions. Changes also were present in old compared with young normoglycemic animals. The diabetes-related increase in vitronectin was more significant in the GBM than MM, whereas the increase in alpha(v) integrin was as significant in podocytes as mesangial cells. Western blot analysis, performed on isolated glomerular material from normoglycemic and hyperglycemic animals, confirmed those changes. Our results suggest that vitronectin and its receptor, alpha(v) integrin, must have defined roles in molecular mechanisms involved in the pathogenesis of both diabetic and aging nephropathy.

Aging↗
PubMed2001

Temporary effects of circulating Amadori products on glomerular filtration properties in the normal mouse.

Previous studies have established a preferential glomerular filtration of glycated BSA (gBSA), as well as a facilitated filtration of BSA in the presence of gBSA. We intend to determine whether these modifications are permanent or transitory. gBSA was intravenously injected into anesthetized normal mice and maintained in circulation for 30 min, 1, 2, 24, and 48 h. Five minutes before death, FITC-BSA was injected. On immunocytochemical evaluations, increased glomerular filtration of FITC-BSA was found at all circulating time points. Changes at 24 and 48 h were less pronounced. Glomerular basement membrane (GBM)-to-lumen gBSA labeling ratios were similar at all time points suggesting no accumulation of gBSA in the GBM. Seventy percent of the gBSA was cleared from the circulation and the GBM after 24 h, and 95% after 48 h. This was confirmed in experiments with radiolabeled tracers. These results suggest that the alteration in GBM permeability to BSA in the normal mouse are due to the presence of gBSA and are gradually overcome along with its clearance from circulation. In early diabetes, increasing concentrations of circulating glycated proteins could be responsible for changes in glomerular permselectivity and probably for the alteration in glomerular filtration properties leading to diabetic nephropathy.

Animals↗
PubMed2001

Demonstration of acetylcholinesterase molecular forms in a continuous tubular lysosomal system of rat pancreatic acinar cells.

By applying the highly sensitive cytochemical Gautron's technique, we were able to reveal AChE activity in rat pancreatic acinar cells, particularly at the level of a complex membrane-bound network formed by tubules with varicosities located around the nuclei and close to the basolateral membrane. The Golgi apparatus was devoid of cytochemical reaction beside the trans-Golgi network cisternae, which showed a positive reaction. The RER of some acinar cells also presented a signal, demonstrating their capability of synthesizing AChE. Immunogold using a specific anti-AChE antibody yielded similar results. Double-labeling experiments corroborated the presence of enzyme cytochemical and immunocytochemical signals in the same lysosomal tubular network. Biochemical sedimentation assays confirmed the presence of AChE in acinar cells, which exists as two globular molecular forms, G(1) and G(4). These results were obtained with pancreatic tissue in situ as well as with isolated acinar cells maintained in culture and devoid of neural elements. The existence of a continuous tubular lysosomal network containing AChE is in agreement with previous reports on acinar and other cell types, and supports a more general hypothesis on dynamic continuities among cell structures. Whether AChE is being secreted by the acinar cells or internalized through this endo-lysosomal system was not defined. However, the capability of the acinar cells to synthesize AChE and to channel it through a tubular system is a good indication that the cells can modulate their cholinergic stimulation for optimal secretion of digestive enzymes.

Acetylcholinesterase↗