Oral peroxidases: From antimicrobial agents to ecological actors (Review).

Sialoperoxidase and myeloperoxidase are the two main peroxidase enzymes found in the oral cavity. Sialoperoxidase is present in salivary secretions and in the biofilms that line the oral surfaces, while myeloperoxidase is abundant in the dento­gingival sulcus area. In the presence of hydrogen peroxide (H2O2), oral peroxidases catalyze the oxidation of the pseudohalide anion thiocyanate (SCN­) to hypothiocyanite (OSCN­), a strong oxidant that serves an antimicrobial role. Furthermore, oral peroxidases consume bacteria­produced H2O2 and could help inactivate toxic carcinogenic and genotoxic substances. Numerous in vitro studies have reported the antibacterial, antimycotic and antiviral role of peroxidases, suggesting possible applications in oral therapy. However, the use of oral hygiene products incorporating peroxidase systems has not yet been shown to be beneficial for the treatment or prevention of oral infections. This paradox reflects our incomplete knowledge of the physiological role of peroxidases in a complex environment, such as the oral region. While hygiene is crucial for restoring oral microbiota to a symbiotic state, there are no data to suggest that the addition of a peroxidase per se can create a dysbiotic state. Recent investigations have associated the presence of peroxidase activity with gram­positive cocci microbial flora, and its insufficiency with dysbiosis has been linked to pathologies, such as caries, periodontitis or infections of the oral mucosa. Therefore, oxidants generated by oral peroxidases appear to be an essential ecological determinant for oral health through the selection of a symbiotic microbiota capable of resisting oxidative stress. The objective of the present review was to update the current knowledge of the physiological aspects and applications of oral peroxidases in clinical practice.

Effects of tyrosol and farnesol on Candida albicans biofilm.

The present in vitro study examined the effects of the quorum­sensing molecules farnesol and tyrosol on the development of Candida albicans biofilm in order to elucidate their role as novel adjuvants in oral hygiene. The investigation was conducted in C. albicans ATCC 10231 and C. albicans isolates from dentures and was performed in flat­bottomed 96­well polystyrene plates. Yeast growth and their capacity to form biofilms were evaluated following 24 and 48 h incubations at 37˚C in Sabouraud broth supplemented with 0.001­3 mM farnesol and/or 1­20 mM tyrosol. Yeast growth was assessed by turbidimetry and biofilms were quantitated by crystal violet staining, under aerobic and anaerobic conditions. The viability of the fungal cells was controlled by the culture of planktonic cells and by examination of the biofilms using fluorescence microscopy following staining with fluorescein diacetate and ethidium bromide. Farnesol at 3 mM exerted a stronger action when added at the beginning of biofilm formation (>50% inhibition) than when added to preformed biofilms (<10% inhibition). Similarly, tyrosol at 20 mM had a greater effect on biofilm formation (>80% inhibition) than on preformed biofilms (<40% inhibition). Despite significant reductions in attached biomass, yeast growth varied little in the presence of the investigated molecules, as corroborated by the turbidimetry, culture of supernatants on solid culture medium followed by counting of colony­forming units and viability tests using fluorescence microscopy. At the highest tested concentration, the molecules had a greater effect during the initial phases of biofilm formation. The effect of farnesol during anaerobiosis was not significantly different from that observed during aerobiosis, unlike that of tyrosol during anaerobiosis, which exhibited slightly reduced yeast biofilm inhibition. In conclusion, the present study demonstrated the specific anti­biofilm effect, independent of fungicidal or fungistatic action, of farnesol and tyrosol, as tested in C. albicans ATCC 10231 and 6 strains isolated from dentures. Prior to suggesting the use of these molecules for preventive purposes in oral hygiene, further studies are required in order to clarify the metabolic pathways and cellular mechanisms involved in their antibiofilm effect, as well as the repercussions on the oral microbiome.

Ex vivo decontamination of yeast-colonized dentures by iodine-thiocyanate complexes.

INTRODUCTION: Under well-defined experimental conditions, and in the presence of hydrogen peroxide, lactoperoxidase produces stable iodine-thiocyanate complexes that have antimicrobial properties. A novel process was developed to short circuit the consumption of hydrogen peroxide by microbial catalases by producing iodine-thiocyanate complexes prior to contact with microorganisms, with the aim of being able to decontaminate the ex vivo dentures colonized by yeasts. MATERIALS AND METHODS: Teabags containing lactoperoxidase adsorbed on inert clay beads were immersed for 1 minute in phosphate buffer solution (0.1 M pH 7.4) containing 5.2 mM potassium iodide, 1.2 mM potassium thiocyanate, and 5.5 mM hydrogen peroxide. After removing the adsorbed lactoperoxidase, the stability and efficacy of iodine-thiocyanate complexes for Candida-colonized denture decontamination were verified. Investigations were performed in vitro on Candida albicans ATCC 10231 and on clinical isolates from 46 dentures. A Candida plate count was performed after a 24-hour incubation at 37°C on Sabouraud-chloramphenicol or CHROMagar solid media; then, the yeast growth was evaluated in Sabouraud broth by turbidimetry and biofilm biomass by crystal violet staining. RESULTS: In vitro tests demonstrated the effectiveness of the oxidant solution in sterilizing a suspension of 106Candida cells per milliliter after a 5-minute incubation. A single ex vivo immersion of contaminated dentures in a solution of iodine-thiocyanate complexes led to a decrease of at least 1 log unit in the number of colony-forming units in 58.3% of the tested dentures, while immersing in water alone had no effect on denture colonization (significant c2: p = 0.0006). CONCLUSION: These data suggest a promising new strategy for decontamination of dentures.

Dose­dependent effect of lysozyme upon Candida albicans biofilm.

The present study investigated the in vitro effect of lysozyme (0-1,000 µg/ml) on Candida albicans (C. albicans) biofilm development. Investigations were conducted on C. albicans ATCC 10231 and on 10 clinical isolates from dentures. Strains were cultured aerobically at 37˚C in Sabouraud broth. Yeast growth was evaluated by turbidimetry. Biofilm biomass was quantified on a polystyrene support by crystal violet staining and on acrylic surfaces by counts of colony forming units. Lysozyme affected biofilm formation to a greater extent than it affected growth. For the ATCC 10231 reference strain, lysozyme acted as a biofilm promotor on polystyrene at the highest concentration tested (1,000 µg/ml, non­physiological). When the reference strain was investigated on acrylic resin support, lysozyme acted as a significant biofilm promotor on rough resin, but less on smooth resin. The attached biomass in the presence of physiological concentrations of lysozyme (10­30 µg/ml) was significantly decreased compared with the hypothetical value of 100% using a one­sample t­test, but a comparison between the different lysozyme conditions using analysis of variance and post hoc tests did not reveal significant differences. In 10 wild strains, different patterns of biofilm formation on polystyrene were observed in the presence of lysozyme. Some strains, characterized by large amounts of biofilm formation in the presence of 1,000 µg/ml lysozyme, were poor biofilm producers at low concentrations of lysozyme. In contrast, some strains that were poor biofilm producers with a high lysozyme concentration were more inhibited by low concentrations of lysozyme. The present study emphasizes the need to develop strategies for biofilm control based on in vitro experiments, and to implement these in clinical trials prior to approval of hygiene products enriched with exocrine proteins, such as lysozyme. Further studies will extend these investigations to other Candida species, and to fungi and bacteria present in oral biofilms.

Candida albicans susceptibility to lactoperoxidase-generated hypoiodite.

In vivo, lactoperoxidase produces hypothiocyanite (OSCN(-)) from thiocyanate (SCN(-)) in the presence of hydrogen peroxide (H2O2); in vitro, iodide (I(-)) can be oxidized into hypoiodite (OI(-)) by this enzyme. The aim of this study was to compare in vitro the anti-Candida effect of iodide versus thiocyanate used as lactoperoxidase substrate to prevent Candida biofilms development. Candida albicans ATCC 10231 susceptibility upon both peroxidase systems was tested in three different experimental designs: (i) in a liquid culture medium, (ii) in an interface model between solid culture medium and gel containing the enzymic systems, (iii) in a biofilm model onto titanium and acrylic resin. Yeast growth in liquid medium was monitored by turbidimetry at 600 nm. Material-adherent yeast biomass was evaluated by the tetrazolium salt MTT method. The iodide-peroxidase system has been shown to inhibit Candida biofilm formation at lower substrate concentrations (~200 fold less H2O2 donor) and for longer incubation periods than the thiocyanate-peroxidase system. In conclusion, efficiency of lactoperoxidase-generated OI(-) to prevent C. albicans biofilm development allows refining iodine antifungal use in ex vivo conditions.

Candida albicans biofilm on titanium: effect of peroxidase precoating.

The present study aimed to document Candida albicans biofilm development on titanium and its modulation by a peroxidase-precoated material which can generate antimicrobials, such as hypoiodite or hypothiocyanite, from hydrogen peroxide, iodide, or thiocyanate. For this purpose, titanium (powder or foil) was suspended in Sabouraud liquid medium inoculated with C. albicans ATCC10231. After continuous stirring for 2-21 days at room temperature, the supernatant was monitored by turbidimetry at 600 nm and titanium washed three times in sterile Sabouraud broth. Using the tetrazolium salt MTT-formazan assay, the titanium-adherent fungal biomass was measured as 7.50 ± 0.60 × 10(6) blastoconidia per gram of titanium powder (n = 30) and 0.50 ± 0.04 × 10(6) blastoconidia per cm(2) of titanium foil (n = 12). The presence of yeast on the surface of titanium was confirmed by microscopy both on fresh preparations and after calcofluor white staining. However, in the presence of peroxidase systems (lactoperoxidase with substrates such as hydrogen peroxide donor, iodide, or thiocyanate), Candida growth in both planktonic and attached phases appeared to be inhibited. Moreover, this study demonstrates the possible partition of peroxidase systems between titanium material (peroxidase-precoated) and liquid environment (containing peroxidase substrates) to limit C. albicans biofilm formation.

Denture contamination by yeasts in the elderly.

OBJECTIVES: The aim of this study was to investigate yeast carriage in healthy denture wearers by swabbing and to evaluate the effect of denture hygiene habits. MATERIALS AND METHODS: Denture wearers (n = 87) without evidence of denture stomatitis or any other oral disease were investigated by separately swabbing the fitting surface of the upper denture and the corresponding palatal mucosa in contact with the appliance. In a group of volunteers, a gel without any active compound was spread on the palatal side of the denture once in every morning for 2 weeks. RESULTS: Screening showed Candida colonisation of upper prosthesis in 75.9% of individuals. The most frequent species isolated were Candida albicans (77.9% of the positive cultures), Candida glabrata (44.1%) and Candida tropicalis (19.1%). Carriage of more than one yeast species was found in 48.5% of the contaminated dentures. There was a statistically significant association between denture contamination and palatal mucosa colonisation (chi-squared test: p < 0.0001). Repeated swabbings after 1 week as well as during a weekly follow-up for 1 month confirmed the denture contamination and its degree of severity. A daily gel application produced a yeast-count decrease to 10% of the initial value after 2 weeks (chi-squared test: p = 0.0134 and p = 0.2841 for prosthesis and palatal mucosa, respectively). CONCLUSION: This study documented the reliability of oral swabbing when investigating yeast carriage in healthy denture wearers. Moreover, just a diagnostic tool, sampling upper dentures for Candida could be the opportunity to verify the patient's compliance to hygiene advice.

Effects of plasma membrane Ca(2+)-ATPase overexpression upon D-glucose metabolism in insulin-producing BRIN-BD11 cells.

In order to investigate the possible link between PMCA (plasma-membrane Ca(2+)-ATPase) activity and D-glucose catabolism in insulin-producing cells, BRIN-BD11 cells were transfected with two isoforms of PMCA2. Transfection of insulin-producing BRIN-BD11 cells with PMCA2yb and PMCA2wb was documented by RT-PCR (reverse transcription-PCR), Western blot analysis, indirect immunofluorescence microscopy and (45)Ca(2+) uptake by microsomes. In the transfected cells, the overexpression of PMCA coincided with three major anomalies of D-glucose metabolism, namely a lower rate of D-[5-(3)H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-(14)C]oxidation and D-[5-(3)H]glucose utilization prevailing at a high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of (14)C-labelled acidic metabolites and amino acids and that of (3)H(2)O from D-[5-(3)H]glucose. These anomalies resulted in a decreased estimated rate of ATP generation (linked to the catabolism of the hexose) and a lowered ATP cell content, whether at low or high extracellular D-glucose concentrations. The net uptake of (45)Ca(2+) by intact cells was also decreased in the transfected cells, but to a greater extent than can apparently be attributed to the change in the ATP-generation rate. These findings document the relevance of PMCA activity to both D-glucose metabolism and Ca(2+) handling in insulin-producing cells, with emphasis on the key role of both cytosolic and mitochondrial Ca(2+) concentrations in the regulation of D-glucose catabolism. They also reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and consumption.

Possible role of carbonic anhydrase in rat pancreatic islets: enzymatic, secretory, metabolic, ionic, and electrical aspects.

The presence of carbonic anhydrase (type V) was recently documented in rat and mouse pancreatic islet beta-cells by immunostaining and Western blotting. In the present study, the activity of carbonic anhydrase was measured in rat islet homogenates and shown to be about four times lower than in rat parotid cells. The pattern for the inhibitory action of acetazolamide on carbonic anhydrase activity also differed in islet and parotid cell homogenates, suggesting the presence of different isoenzymes. NaN3 inhibited carbonic anhydrase activity in islet homogenates and both D-[U-14C]glucose oxidation and glucose-stimulated insulin secretion. Acetazolamide (0.3-10.0 mM) also decreased glucose-induced insulin output but failed to affect adversely D-[U-14C]glucose oxidation, although it inhibited the conversion of D-[5-3H]glucose to [3H]OH and that of D-[U-14C]glucose to acidic metabolites. Hydrochlorothiazide (3.0-10.0 mM), which also caused a concentration-related inhibition of the secretory response, like acetazolamide (5.0-10.0 mM), decreased H(14)CO3- production from D-[U-14C]glucose (16.7 mM). Acetazolamide (5.0 mM) did not affect the activity of volume-sensitive anion channels in beta-cells but lowered intracellular pH and adversely affected both the bioelectrical response to d-glucose and its effect on the cytosolic concentration of Ca2+ in these cells. The lowering of cellular pH by acetazolamide, which could well be due to inhibition of carbonic anhydrase, might in turn account for inhibition of glycolysis. The perturbation of stimulus-secretion coupling in the beta-cells exposed to acetazolamide may thus involve impaired circulation in the pyruvate-malate shuttle, altered mitochondrial Ca2+ accumulation, and perturbation of Cl- fluxes, resulting in both decreased bioelectrical activity and insulin release.

Fatty acid content and pattern of epididymal and parametrial adipose tissue lipids in streptozotocin (type 1) and Goto-Kakizaki (type 2) diabetic rats.

Attention was recently drawn to differences in the fatty acid pattern of liver phospholipids and triglycerides in animal models of type 1 and type 2 diabetes. The present study extends this knowledge to epididymal or parametrial adipose tissue lipids. The fatty acid pattern of such lipids was established in four fed female normal rats, four overnight fasted female normal rats, six fed female rats rendered diabetic by an injection of streptozotocin 3 days before sacrifice (STZ rats), and four female and four male Goto-Kakizaki rats (GK rats) also examined in the fed or fasted state. In addition to the fasting-induced and diabetes-related changes in plasma D-glucose and insulin concentrations, differences in either the weight percentage of fatty acids or the paired ratio between distinct fatty acids were often encountered. For instance, in the GK rats, gender differences were observed in the weight percentage of C18:2omega6, as well as C18:2omega6/C18:3omega6, C18:3omega6/C20:4omega6, C20:5omega3/C22:5omega3 and C22:5omega3/C22:6omega3 ratios. When compared to normal rats, the activity of Delta9-desaturase was markedly increased in GK rats and, to a lesser extent, in STZ rats. Starvation also increased to some extent the activity of Delta9-desaturase. The relative content of C22:6omega3 was also higher in diabetic than in normal rats. Further differences between GK and STZ rats concerned the generation of C18:3omega6 from C18:2omega6, C20:4omega6 from C18:3omega6, and C20:5omega3 from C18:3omega3. Several differences found in the adipose tissue of GK versus STZ rats were reminiscent of those recently identified in the liver triglycerides of these two types of diabetic animals, suggesting a common regulatory mechanism, possibly linked to the higher insulinemia of GK rats versus STZ rats.

Pancreatic islet function in omega3 fatty acid-depleted rats: Glucose metabolism and nutrient-stimulated insulin release.

In order to gain information on the determinism of the perturbation of fuel homeostasis in situations characterized by a depletion in long-chain polyunsaturated omega3 fatty acids (omega3), the metabolic and hormonal status of omega3-depleted rats (second generation) was examined. When required, these rats were injected intravenously 120 min before sacrifice with a novel medium-chain triglyceride-fish oil emulsion able to provoke a rapid and sustained increase of the omega3 content in cell phospholipids. The measurement of plasma glucose, insulin, phospholipid, triglyceride, and unesterified fatty acid concentration indicated modest insulin resistance in the omega3-depleted rats. The plasma triglyceride and phospholipid concentrations were decreased in the omega3-depleted rats with abnormally low contribution of omega3 in both circulating and pancreatic islet lipids. The protein, insulin, and lipid content of the islets, as well as their intracellular and extracellular spaces, were little affected in the omega3-depleted rats. The metabolism of D-glucose in the islets of omega3-depleted rats was characterized by a lesser increase in D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation in response to a given rise in hexose concentration and an abnormally low ratio between D-glucose oxidation and utilization. These abnormalities could be linked to an increased metabolism of endogenous fatty acids with resulting alteration of glucokinase kinetics. The release of insulin evoked by D-glucose, at a close-to-physiological concentration (8.3 mM), was increased in the omega3-depleted rats, this being considered as consistent with their insulin resistance. Relative to such a release, that evoked by a further rise in D-glucose concentration or by non-glucidic nutrients was abnormally high in omega3-depleted rats, and restored to a normal level after of the intravenous injection of the omega3-rich medium-chain triglyceride-fish oil emulsion. Because the latter procedure failed to correct the perturbation of D-glucose metabolism in the islets of omega3-depleted rats, it is proposed that the anomalies in the secretory behaviour of islets in terms of their response to an increase in hexose concentration or non-nutrient secretagogues is mainly attributable to alteration in K+ and Ca2+ handling, as indeed recently documented in separate experiments.

Opposite effects of D-fructose on total versus cytosolic ATP/ADP ratio in pancreatic islet cells.

D-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM D-glucose. Even in the absence of D-glucose, D-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of D-fructose could be attributed to an increase in the ATP content of islet cells. After 30-60 min incubation in the presence of D-glucose and/or D-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). D-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM D-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, D-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of D-glucose and/or D-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either (86)Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.

Fructokinase activity in rat liver, ileum, parotid gland, pancreas, pancreatic islet, B and non-B islet cell homogenates.

The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously documented. However, no information was so far available on the activity of this enzyme in islets relative to that in other tissues and on the respective contribution of insulin-producing B cells and non-B islet cells. The present study provides such an information. The activity of fructokinase, as assessed by the phosphorylation of 1.0 mM D-fructose, was compared to that of hexokinase isoenzyme(s), as measured in the presence of 1.0 mM D-glucose, and further characterized by its heat-resistance, K+ dependency and resistance to the inhibitory action of D-mannoheptulose. As judged from the results obtained in heated homogenates, the activity of fructokinase, expressed relative to protein content (nmol/min per mg protein) was highest in liver (21.5 +/- 2.5; n = 11) and lowest in parotid gland (0.16 +/- 0.09; n = 3), with in-between values in ileum (2.45 +/- 0.53; n = 3), pancreas (0.82 +/- 0.11; n = 11) and pancreatic islets (0.46 +/- 0.07; n = 6). The paired ratio between fructokinase and hexokinase isoenzyme activity was also highest in liver (548 +/- 45%; n = 8) and lowest in parotid gland (0.93 +/- 0.52%; n = 3). Such a ratio was not significantly different in pancreas, islets and purified B or non-B islet cells, with an overall mean value of 2.57 +/- 0.46% (n = 12). The present findings thus unambiguously document the presence of fructokinase activity in all cell types under consideration, except possibly parotid cells, with the following hierarchy: liver > ileum > pancreas. Relative to paired hexokinase activity, no obvious difference was found for fructokinase activity in B versus non-B islet cells.

Altered K+ fluxes and insulin release in pancreatic islets from omega3 fatty acid-depleted rats.

A low intake of long-chain polyunsaturated omega3 fatty acid often prevails in Western populations. Its consequences in terms of the control of fuel homeostasis led us to explore functional events in pancreatic islets isolated from either normal or omega3-depleted rats (second generation). In the latter rats, the inflow of K+ by both ouabain-sensitive and ouabain-resistant modalities was decreased, this coinciding with an impaired insulin secretory response to ouabain. The intravenous injection of a medium-chain triglyceride:fish oil emulsion to omega3-depleted rats 2 h before sacrifice restored a normal value for the inflow of K+ by the ouabainsensitive modality, i.e., that linked to the activity of the Na,K-ATPase, but failed to correct the entry of K+ by the ouabain-resistant modality and the defect of the insulin secretory response to ouabain. In conclusion, an impaired activity of the Na,K-ATPase in insulin-producing cells apparently represents a key determinant of altered islet function in omega3-depleted rats.

Effects of a medium-chain triglyceride:fish oil emulsion administered intravenously to omega3 fatty acid-depleted rats on cationic fluxes in aortic rings.

The handling of 45Ca and 86Rb by aortic rings obtained from rats depleted in long-chain polyunsaturated omega3 fatty acids (second generation) was examined in vitro over 10 to 60 min incubation at either increasing concentrations of extracellular K+ (5, 3 and 60 mM) in the case of 45Ca net uptake or in the absence and presence of ouabain (50 microM) in the case of 86Rb uptake. The omega3-depleted rats were injected intravenously 120 min before sacrifice with 1.0 ml of either an omega3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (MCT:FO) or a control medium-chain triglyceride:olive oil emulsion (MCT:OO). In the MCT:OO-injected rats, the rise in extracellular K+ concentration failed to stimulate 45Ca net uptake, whilst the prior injection of the MCT:FO emulsion restored the expected increase in 45Ca net uptake by aortic rings exposed to 60 mM K+. The absolute value for 86Rb net uptake after 10 or 60 min incubation and whether in the absence or presence of ouabain, which significantly decreased the uptake of 86Rb+ after 60 min incubation, only represented in the MCT:FO-injected rats 63.1+/-3.8% of the mean corresponding values found in MCT:OO-injected animals. These findings are consistent with the view that activity of cationic channels, such as the voltage-sensitive Ca2+ channel, the outflow of Ca2+ as mediated by either Na+-Ca2+ countertransport or a Ca2+-ATPase, the activity of Na+,K+-ATPase and the modality of K+ inflow by an oubain-resistant modality are all affected in aortic cells by the content of long-chain polyunsaturated omega3 fatty acids in membrane phospholipids.

Gut permeability and intestinal mucins, invertase, and peroxidase in control and diabetes-prone BB rats fed either a protective or a diabetogenic diet.

This study deals with the enteropathy recently identified in diabetes-prone BB rats (BBdp). Diabetes-resistant BB rats (BBc) and BBdp rats were fed from days 32-39 onward either a protective diabetes-retardant hydrolyzed casein diet (HC) or a plant-based diabetogenic (NTP) diet. The NTP diet decreased body weight and plasma insulin in BBc and BBdp rats. The BBdp rats displayed low intestinal invertase and increased intestinal peroxidase activity. In the BBdp rats fed the HC diet, the mucin content 30-35 cm below the pylorus was higher and the gut permeability lower than in the other three rat groups. There was a significant inverse correlation between gut permeability and the insulinogenic index in the BBdp rats fed the HC or NTP diet. Thus, in BBdp rats, the HC diet somehow prevents the increase in gut permeability and the decrease in the insulinogenic index otherwise found in some of these diabetes-prone animals.

Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D-glucose.

It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.

Quantitative and qualitative alterations of intestinal mucins in BioBreeding rats.

In diabetes-prone BioBreeding rats, an enteropathy often precedes the onset of auto-immune insulitis. The present study draws attention to quantitative and qualitative alterations of intestinal mucins in this animal model of type 1 diabetes mellitus. Male and female diabetes-resistant (BBc) and diabetes-prone (BBdp) BioBreeding rats fed, from one to two weeks after weaning onwards, either a plant-based diabetes-promoting diet (NTP) or a hydrolysed casein diabetes-protective diet (HC), were sacrificed at 11-14 weeks of age. Proteins and total mucins, as well as acid and neutral mucins, were measured in a segment of the intestinal tract, located 25-30 cm below the pylorus. No significant difference between BBc and BBdp rats was found when fed the HC diet. However, the NTP diet lowered both total and neutral mucins, whilst failing to affect significantly acidic mucins. The effects of the NTP diet were more pronounced in BBdp rats than in BBc rats. It is speculated that the quantitative and qualitative changes evoked by the NTP diet in BBdp rats may play a role in the alteration of gut permeability found under the same experimental conditions.

Peroxidase activity in the intestinal tract of Wistar-Furth, BBc and BBdp rats.

BACKGROUND: The development of immune-mediated diabetes in BB rats may involve an inflammatory lesion of the intestinal tract. METHODS: In order to further explore this issue, the activity of peroxidase was measured, in the absence and presence of either bromide or dapsone, at day 10, 30, 45, 70, 95 and 120 in three segments (top, mid and bottom) of the intestinal tract from Wistar-Furth rats and both diabetes-resistant and diabetes-prone BB rats fed after weaning either diabetes-promoting diets or a protective diet, which decreases the incidence of diabetes in the BB rats. RESULTS: In the present study, from day 30 onwards, an age-related increase in peroxidase activity was found in the intestine of diabetes-prone BB rats (BBdp rats) fed diabetogenic diets, when compared with either Wistar-Furth or diabetes-resistant BB rats (BBc rats). This increase was most pronounced in the distal segments of the intestinal tract. Even when fed a protective diet, higher peroxidase activity was found in BBdp than BBc rats. Yet, in BBc rats, and to a lesser extent in BBdp rats, the diabetogenic diets lowered peroxidase activity below the value found in rats of the same strain fed the protective diet. There was a tight correlation between the activity of peroxidase and its susceptibility to be increased by bromide. CONCLUSION: It is proposed that diabetogenic diets may decrease peroxidase activity in intestinal cells, this effect becoming masked in BBdp rats by an age-related increase in the contribution of inflammatory cells. The latter phenomenon affects preferentially the distal segments of the intestinal tract.

Dissimilar effects of D-mannoheptulose on the phosphorylation of alpha- versus beta-D-glucose by either hexokinase or glucokinase.

D-mannoheptulose inhibits D-glucose phosphorylation by hexokinase isoenzymes. The present study aims at investigating whether the pattern of such an inhibition differs in the case of alpha- versus beta-D-glucose. The phosphorylation of alpha- and beta-D-[U-14C]glucose was measured over 60-min incubation at 4 degrees C in the presence of bovine heart hexokinase and over 10 min at 24 degrees C in the presence of human liver glucokinase. The relative extent of the inhibitory action of D-mannoheptulose (0.02-10.0 mM) was always less marked with alpha- than beta-D-glucose. In the case of hexokinase, the experiments conducted at the high concentration of the D-glucose anomers (1.0 mM) revealed that D-mannoheptulose, at low concentrations (0.2-0.5 mM), may unexpectedly increase the phosphorylation of alpha-D-glucose. These findings thus document anomeric specificity in terms of the inhibitory action of D-mannoheptulose upon alpha- versus beta-D-glucose phosphorylation by either hexokinase or glucokinase.