Liver Membranes Research Articles

Enhanced anti-oxidant protection of liver membranes in long-lived rats fed on a coenzyme Q 10-supplemented diet

Coenzyme Q 10 supplementation increases life-span of rats fed on a diet enriched with polyunsaturated fatty acids ( Quiles, J.L., Ochoa, J.J., Huertas, J.R., Mataix, J., 2004b. Coenzyme Q supplementation protects from age-related DNA double-strand breaks and increased lifespan in rats fed on a PUFA-rich diet. Exp. Gerontol. 39, 189–194). Our study was set as a first attempt to establish a mechanistic link between life span extension and CoQ 10 supplementation. When rats were fed on a PUFA n−6 plus CoQ 10 diet, levels of CoQ 10 were increased in plasma membrane at every time point compared to control rats fed on a PUFA n-6-alone diet. Ratios of CoQ 9 to CoQ 10 were significantly lower at every time point in both liver plasma membranes and homogenates of CoQ 10-supplemented animals. CoQ 10 supplementation did not affect cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1), which increased significantly with aging, but plasma membrane-bound NQO1 decreased significantly in the CoQ 10-supplemented group at 12 months, when maximal incorporation of exogenous CoQ 10 was observed. Neither aging nor the diet affected NADH-cytochrome b 5 reductase levels. Glutathione-dependent anti-oxidant activities such as cytosolic glutathione- S-transferase (GST) and microsomal Se-independent glutathione peroxidase decreased with aging and supplementation with CoQ 10 attenuated this decay. 2,2′ Azobis amidinopropane (AAPH)-induced oxidation of membranes was significantly higher in aged rats, and supplementation with CoQ 10 also inhibited this increase. Consistent with higher CoQ 10 levels and enhanced anti-oxidant protection, plasma membrane Mg 2+-dependent neutral sphingomyelinase was inhibited by dietary CoQ 10 in aged rats. Our results support the involvement of thiol-dependent mechanisms in the potentiation of the anti-oxidant capacity of membranes in CoQ 10-supplemented rats, further supporting the potentially beneficial anti-oxidative role of dietary CoQ 10 during aging. The possibility that a decreased CoQ 9/CoQ 10 ratio in animals fed on the PUFA n−6-rich plus CoQ 10 diet could also influence longevity is also discussed.

Interaction with PDZK1 Is Required for Expression of Organic Anion Transporting Protein 1A1 on the Hepatocyte Surface

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.

3H]linopirdine binding to rat brain membranes is not relevant for M-channel interaction

Linopirdine was developed as a cognitive enhancing molecule and demonstrated to specifically block the potassium current generated by the brain specific KCNQ2–KCNQ3 proteins (M-channel). In this study we investigated the relevance of [ 3H]linopirdine binding in rat brain extracts to the interaction with the M-channel proteins. Our results confirm the presence of a high affinity site for [ 3H]linopirdine in rat brain tissues ( K D = 10 nM) but we also identified a high affinity binding site for [ 3H]linopirdine in rat liver tissues ( K D = 9 nM). Competition experiments showed that [ 3H]linopirdine is displaced by unlabelled linopirdine with comparable affinities from its binding sites on rat brain and rat liver membranes. [ 3H]linopirdine was completely displaced by a set of cytochrome P450 (CYP450) ligands suggesting that [ 3H]linopirdine binding to rat brain and liver membranes is linked to CYP450 interaction. The testing of CYP450 ligands on the M-channel activity, using a Rb + efflux assay on cells expressing the KCNQ2–KCNQ3 proteins, demonstrated that [ 3H]linopirdine binding results cannot be correlated to M-channel inhibition. The results obtained in this study demonstrate that [ 3H]linopirdine binding to rat brain and rat liver membranes is representative for CYP450 interaction and not relevant for the binding to the M-channel proteins.

Alcoholic Liver Disease: From Pathophysiology to Therapy

This article presents the proceedings of a symposium held at the meeting of the International Society for Biomedical Research on Alcoholism in Mannheim, Germany, in October 2004. A salient feature of this symposium was to demonstrate how the striking advances made in our understanding of the pathophysiology of alcoholic liver disease are now raising prospects for better treatment as well. Genetic factors are now being elucidated, and F. Stickel (Germany) has summarized his own studies as well as those of others. M. Salaspuro (Finland) updated the possible role of gut bacteria in the pathogenesis of alcoholic liver disease, bringing us closer to antibacterial therapy as part of the treatment of alcoholic liver disease. Specifically, the gut bacterial flora may be important via the in situ production of acetaldehyde and the associated intestinal injury, which may favor the translocation of toxins from the gut lumen to the systemic circulation and the liver. The analytical progress in the assessment of alterations of phospholipid composition in liver membranes described by H. P. Schlemmer (Germany) may eventually give us an objective method to recognize patients in whom phospholipid therapy might be indicated. Other novel treatment modalities for severe alcoholic hepatitis were described by Y. Horie and H. Ishii (Japan), including plasma exchange. Finally, the pros and cons of nutraceutical therapy were analyzed by C. S. Lieber, with a demonstration that although some nutraceuticals may have toxicity exacerbated by alcohol and must be administered very carefully within a narrow therapeutic window, others are actually beneficial as demonstrated under controlled conditions.

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.

Cholesterol-lowering effects of dietary blue lupin (Lupinus angustifolius L.) in intact and ileorectal anastomosed pigs

The present study was undertaken to investigate the effect of cholesterol-enriched casein (CAS) and blue lupin seed (BL) diets on the cholesterol metabolism of intact (INT) and ileorectal anastomosed (IRA) pigs. For 3 weeks, four groups of six pigs were allocated to the treatments (CAS-INT, CAS-IRA, BL-INT, and BL-IRA). Diet-induced hypercholesterolemia was inhibited by the BL through a substantial decrease in plasma LDL-cholesterol. The BL also reduced liver esterified and total cholesterol, increased hepatic LDL receptor synthesis and HMG-CoA reductase activity, and stimulated intestinal bile acid reabsorption. The neutral sterol output was higher in BL- than in CAS-fed pigs. The bile acid output was lower in IRA than in INT pigs. Surgery also prevented steroid microbial transformation, but it did not influence plasma cholesterol levels. These results suggest that the hypocholesterolemic effect of the BL, compared with the CAS, is attributable to impaired intestinal cholesterol absorption, probably involving increased bile acid reabsorption and higher contents of dietary phytosterols, both factors that reduce the micellar solubilization of cholesterol. Furthermore, according to our data, the contribution of the large intestine to cholesterol metabolism is very weak.

Immunodetection of the expression of microsomal proteins encoded by the glucose 6-phosphate transporter gene

Glucose 6-phosphate transport has been well characterized in liver microsomes. The transport is required for the functioning of the glucose-6-phosphatase enzyme that is situated in the lumen of the hepatic endoplasmic reticulum. The genetic deficiency of the glucose 6-phosphate transport activity causes a severe metabolic disease termed type 1b glycogen storage disease. The cDNA encoding a liver transporter for glucose 6-phosphate was cloned and was found to be mutated in patients suffering from glycogen storage disease 1b. While related mRNAs have been described in liver and other tissues, the encoded protein(s) has not been immunologically characterized yet. In the present study, we report (using antibodies against three different peptides of the predicted amino acid sequence) that a major protein encoded by the glucose 6-phosphate transporter gene is expressed in the endoplasmic reticulum membranes of rat and human liver. The protein has an apparent molecular mass of approx. 33 kDa using SDS/PAGE, but several lines of evidence indicate that its real molecular mass is 46 kDa, as expected. The glucose 6-phosphate transporter protein was also immunodetected in kidney microsomes, but not in microsomes derived from human fibrocytes, rat spleen and lung, and a variety of cell lines. Moreover, little or no expression of the glucose 6-phosphate transporter protein was found in liver microsomes obtained from three glycogen storage disease 1b patients, even bearing mutations that do not directly interfere with protein translation, which can be explained by a (proteasome-mediated) degradation of the mutated transporter.

Changes in lipid composition of hepatocyte plasma membrane induced by overfeeding in duck

This experiment was carried out to examine the influence of overfeeding ducks with corn on the lipid composition of hepatocyte plasma membrane. Seventy-day-old male Mule ducks ( Cairina moschata × Anas platyrhynchos) were overfed with corn for 12.5 days in order to induce fatty livers. The cholesterol and phospholipid contents were approximately 50% higher in hepatocyte plasma membranes from fatty livers compared to those of lean livers obtained from non-overfed ducks. However, the cholesterol/phospholipids molar ratio did not differ between both groups. Overfeeding induced a significant change in phospholipid composition of hepatocyte plasma membrane with a decrease in phosphatidylcholine proportion and conversely an increase in phosphatidylethanolamine. The fatty acid profile of phospholipids was also altered. In fatty hepatocyte plasma membrane, the overall proportion of polyunsaturated fatty acids (PUFA) was decreased and this was due to the decrease of some of, but not all, the PUFA. In addition, the proportions of oleic acid and n-9 series unsaturated fatty acids were higher in fatty than in lean liver membranes. This study provides evidence that overfeeding with a carbohydrate-rich corn-based diet induces a de novo hepatic lipogenesis in Mule duck which predominates over dietary lipid intake to change the lipid composition of the hepatocyte plasma membrane.

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

BackgroundThe analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids.ResultsAfter initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed.ConclusionThis study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

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.

Triglyceride-rich lipoprotein metabolism in unique VLDL receptor, LDL receptor, and LRP triple-deficient mice

The very low density lipoprotein receptor (VLDLR), low density lipoprotein receptor (LDLR), and low density lipoprotein receptor-related protein (LRP) are the three main apolipoprotein E-recognizing endocytic receptors involved in the clearance of triglyceride (TG)-rich lipoproteins from plasma. Whereas LDLR deficiency in mice results in the accumulation of plasma LDL-sized lipoproteins, VLDLR or LRP deficiency alone only minimally affects plasma lipoproteins. To investigate the combined effect of the absence of these receptors on TG-rich lipoprotein levels, we have generated unique VLDLR, LDLR, and LRP triple-deficient mice. Compared with wild-type mice, these mice markedly accumulated plasma lipids and lipases. These mice did not show aggravated hyperlipidemia compared with LDLR and LRP double-deficient mice, but plasma TG was increased after high-fat diet feeding. In addition, these mice showed a severely decreased postprandial TG clearance typical of VLDLR-deficient (VLDLR-/-) mice. Collectively, although VLDLR deficiency in LRP- and LDLR-/- mice does not aggravate hyperlipidemia, these triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.

Knockdown of Hepatic ABCA1 by RNA Interference Decreases Plasma HDL Cholesterol Levels and Influences Postprandial Lipemia in Mice

To investigate the impact of hepatic ABCA1 on systemic lipoprotein metabolism in vivo by an adenovirus-mediated RNA interference approach. Efficiency of plasmid-based small interference RNA (siRNA)-induced knockdown of cotransfected murine ATP binding cassette transporter A1 (mABCA1) in HEK-293 cells was judged by RT-polymerase chain reaction, immunofluorescence, and Western blot analysis. The most effective plasmid was used to generate a recombinant adenovirus as a tool to selectively downregulate ABCA1 expression in mouse liver (C57BL/6). In comparison to controls, Western blot analysis from liver membrane proteins of Ad-anti-ABCA1 infected mice resulted in an approximately 50% reduction of endogenous ABCA1 and a clear upregulation of apolipoprotein E. Fast protein liquid chromatography analysis of plasma revealed that hepatic ABCA1 protein reduction was associated with an approximately 40% decrease of HDL cholesterol and a reduction of HDL-associated apolipoprotein A-I and E. In the fasted state, other lipoprotein classes were not affected. To analyze the influence of ABCA1 downregulation on postprandial lipemia, infected mice were given a gastric load of radiolabeled trioleate in olive oil. In Ad-anti-ABCA1 infected mice, the postprandial increase of chylomicrons and chylomicron-associated apolipoproteins B and E was significantly reduced as compared with controls. Hepatic ABCA1 contributes to HDL plasma levels and influences postprandial lipemia.

Modulation of regulatory factors involved in cholesterol metabolism in response to feeding of pravastatin- or cholesterol-supplemented diet in chickens

mRNA transcripts encoding multiple proteins from the cholesterol biosynthetic and uptake pathways in livers are controlled by sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound mammalian transcription factor. The aims of the present study were to investigate whether SREBP-2 responds to plasma cholesterol levels and modulates expression of factors involved in the cholesterol metabolism of chickens. Supplementing the diets of chickens with 0.1% pravastatin, a drug used to control hypercholesterolemia, decreased plasma LDL-cholesterol concentrations. It also increased levels of the nuclear forms of SREBP-2 and increased gene expression of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and low-density lipoprotein receptor (LDLr) in livers, relative to a control group. In contrast, feeding of 3% cholesterol-supplemented diet increased plasma total- and LDL-cholesterol concentrations but decreased levels of nuclear forms of SREBP-2 and reduced gene expression of HMGR and LDLr. However, the LDL-binding activities of chicken liver membranes were not affected by plasma cholesterol concentrations or by hepatic levels of the nuclear form of SREBP-2. LDL-binding proteins were detected as bands of 90 and 515 kDa on a ligand blot and the intensity of these bands was unaffected by pravastatin and cholesterol supplementation. These findings suggest that the cholesterol biosynthetic pathway is regulated by the nuclear form of SREBP-2 in chickens as well as in mammals, but that there may be species-specific differences in the regulatory mechanisms of hepatic cholesterol uptake.

Effect of different immunosuppressive therapies on the lipid pattern in kidney-transplanted rats

We analyzed the effect of oral administration of cyclosporine-methylprednisone (CsA-MP) and sirolimus (SRL) on the lipid pattern of kidney-transplanted rats after a 7-day survival. A significant increase in plasma cholesterol in CsA-MP group (control: 26 +/- 3 mg/dl vs. 59 +/- 8 mg/dl, P < 0.05) and in triglyceride levels in SRL group (control: 53 +/- 4 mg/dl vs. 114 +/- 3 mg/dl, P < 0.05), was shown. Kidney microsomal membranes from both treated groups showed that cholesterol and triglyceride values and the relative percentage of arachidonic acid in the total amount of n-6 fatty acids decreased. A diminution of linoleic acid occurred in testis (control: 9.4 +/- 0.1 mg/dl vs. CsA-MP: 6.0 +/- 0.3 mg/dl and vs. SRL: 6.8 +/- 0.2 mg/dl, P < 0.05), liver (control: 17.7 +/- 0.6 mg/dl vs. CsA-MP: 15.1 +/- 0.6 mg/dl and SRL: 13.5 +/- 0.8 mg/dl, P < 0.05) and erythrocyte membranes (control:11.7 +/- 0.1% vs. CsA-MP: 10.6 +/- 0.2% and SRL: 10.0 +/- 0.4%, P < 0.01). The immunosuppressive therapies improved the rejection rate of the graft, fact that was remarkable in the SRL-treated group. However, lipid abnormalities still remain in spite of immunosuppressive therapies (150).

The Inner Mitochondrial Membrane Has Aquaporin-8 Water Channels and Is Highly Permeable to Water

Mitochondria are remarkably plastic organelles constantly changing their shape to fulfil their various functional activities. Although the osmotic movement of water into and out of the mitochondrion is central for its morphology and activity, the molecular mechanisms and the pathways for water transport across the inner mitochondrial membrane (IMM), the main barrier for molecules moving into and out of the organelle, are completely unknown. Here, we show the presence of a member of the aquaporin family of water channels, AQP8, and demonstrate the strikingly high water permeability (Pf) characterizing the rat liver IMM. Immunoblotting, electron microscopy, and biophysical studies show that the largest mitochondria feature the highest AQP8 expression and IMM Pf. AQP8 was also found in the mitochondria of other organs, whereas no other known aquaporins were seen. The osmotic water transport of liver IMM was partially inhibited by the aquaporin blocker Hg2+, while the related activation energy remained low, suggesting the presence of a Hg2+-insensitive facilitated pathway in addition to AQP8. It is suggested that AQP8-mediated water transport may be particularly important for rapid expansions of mitochondrial volume such as those occurring during active oxidative phosphorylation and those following apoptotic signals.

Size of vasopressin receptors from rat liver and kidney.

Specific vasopressin receptors in rat liver membranes were recently characterized [Cantau et al., Journal of Receptors Research, in the press]. They differ from the receptors characterized earlier in kidney membranes as regards coupling with adenylate cyclase and specificity towards vasopressin structural analogues [Rajerison et al. (1974) J. Biol. Chem. 249, 6390-6400; Butlen et al. (1978) Mol. Pharmacol. 14, 1006-1017]. The object of the present work was to see whether these differences reflect a difference in the molecular size of liver and kidney vasopresin receptors. For this purpose, rat liver and kidney membranes were incubated with [3H]vasopressin and solubilized with Triton X-100 (0.3%). The properties of the macromolecular components of soluble extracts to which labelled vasopressin remained bound were observed to resemble those of the intact membrane receptors as regards binding reversibility at 30-37 degrees C and sensitivity to guanyl nucleotides. The hydrodynamic parameters of the soluble hormone-receptor complexes were estimated from Utrogel column filtration experiments and from sucrose density gradient ultracentrifugation experiments. The following values were obtained for liver and kidney receptors respectively: Stokes' radii: 5.4 and 5.6 nm; standard sedimentation coefficients: 3.7 and 3.7 S; partial specific volumes: 0.75 and 0.78 ml x g-1; molecular weight: 83000 and 80000. These results indicate that the marked functional differences between liver and kidney receptors are not accompanied by appreciable differences in molecular size.

Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.

Differential regulation of basolateral and canalicular transporter expression in rat liver in chronic renal failure

Background Renal failure (RF) decreases the hepatic elimination of substrates of basolateral Organic Anion Transporting Polypeptides (Oatp) and canalicular Multidrug Resistance Protein 2 (Mrp2), e.g. bromosulfophthalein, cerivastatin, and pravastatin. Therefore we investigated, the expression of these hepatocellular transporters in RF. Methods Male SD rats (120–140g) underwent 2-step 5/6-nephrectomy (6, N) or sham operation (8, C), were pair fed and killed after 6 weeks. Hepatic transporter expression was evaluated in a total liver membrane fraction (TLMF) by semiquantitative Western Blot analysis. Results N and C exhibited similar body weights, liver weights and transaminases. N had uremia, i. e. 314% increased serum creatinine, 698% increased serum urea and 73% decreased creatinine clearance as compared to C (100%). TLMF showed similar recovery and enrichment of canalicular marker enzyme activities in N and C. In N liver, transporter expression showed the following changes as compared to C (100%): Ntcp +20±13%, Oatp1a1 −61±6%, Oatp1a4 −43±13%, Oatp1b2 −72±10%, Mrp2 +25±41%, Mdr1 +7±18%, Mdr2 +96±34%. Thus chronic RF decreases the expression of basolateral Oatps in rat liver, whereas expression of canalicular transporters remains stable or increases. Conclusion Downregulation of hepatic basolateral Oatps might explain the decreased hepatic drug elimination, while upregulation of some canalicular transporters may compensate for reduced renal drug elimination in chronic RF. Clinical Pharmacology & Therapeutics (2005) 77, P34–P34; doi: 10.1016/j.clpt.2004.12.023

Approaches towards endogenous pancreatic regeneration.

The phenomenon of pancreatic regeneration in mammals has been well documented. It has been shown that pancreatic tissue is able to regenerate in several species of mammal after surgical insult. This tissue is also known to have the potential to maintain or increase its beta-cell mass in response to metabolic demands during pregnancy and obesity. Since deficiency in beta-cell mass is the hallmark of most forms of diabetes, it is worthwhile understanding pancreatic regeneration in the context of this disease. With this view in mind, this article aims to discuss the potential use in clinical strategies of knowledge that we obtained from studies carried out in animal models of diabetes. Approaches to achieve this goal involve the use of biomolecules, adult stem cells and gene therapy. Various molecules, such as glucagon-like peptide-1, beta-cellulin, nicotinamide, gastrin, epidermal growth factor-1 and thyroid hormone, play major roles in the initiation of endogenous islet regeneration in diabetes. The most accepted hypothesis is that these molecules stimulate islet precursor cells to undergo neogenesis or to induce replication of existing beta-cells, emphasizing the importance of pancreas-resident stem/progenitor cells in islet regeneration. Moreover, the potential of adult stem cell population from bone marrow, umbilical cord blood, liver, spleen, or amniotic membrane, is also discussed with regard to their potential to induce pancreatic regeneration.

The effect of glyphosate on the frequency of micronuclei in bovine lymphocytes in vitro

Glyphosate is a widely used broad-spectrum herbicide that has expanded its applications on plant varieties that are genetically modified to tolerate glyphosate treatment. The aim of this study was to determine the frequency of micronuclei (MNi) in bovine peripheral lymphocytes after exposure to glyphosate in vitro. The cytokinesis block micronucleus assay (CBMN) for estimation of genotoxic activity was used. The obtained results indicate that glyphosate weakly induced micronuclei in bovine peripheral lymphocytes. Significant elevations of MNi (p <0.05) were observed at concentrations of glyphosate of 280 μW and 560 μW, respectively. Treatment of bovine lymphocytes did not result in the induction of micronuclei in a dose-dependent manner. From cytotoxicity data it is evident that CBPI does not reflect the reduction of cell proliferation. The influence of metabolic activation on the genotoxic activity of glyphosate was investigated, too. When lymphocyte cultures were treated with glyphosate together with a liver membrane fraction (S9) from Aroclor 1245-induced rat liver, the number of micronuclei in binucleated cells did not increase significantly.