Diabetic rat testes: morphological and functional alterations.

Reproductive dysfunction is a consequence of diabetes, but the underlying mechanisms are poorly understood. This study investigated the histological and molecular alterations in the testes of rats injected with streptozotocin at prepuperal (SPI rats) and adult age (SAI rats) to understand whether diabetes affects testicular tissue with different severity depending on the age in which this pathological condition starts. The testes of diabetic animals showed frequent abnormal histology, and seminiferous epithelium cytoarchitecture appeared altered as well as the occludin distribution pattern. The early occurrence of diabetes increased the percentage of animals with high number of damaged tubules. The interstitial compartment of the testes was clearly hypertrophic in several portions of the organs both in SPI and SAI rats. Interestingly, fully developed Leydig cells were present in all the treated animals although abnormally distributed. Besides the above-described damages, we found a similar decrease in plasma testosterone levels both in SPI and SAI rats. Oxidative stress (OS) is involved in the pathogenesis of various diabetic complications, and in our experimental models we found that manganese superoxide dismutase was reduced in diabetic animals. We conclude that in STZ-induced diabetes, the altered spermatogenesis, more severe in SPI animals, is possibly due to the effect of OS on Leydig cell function which could cause the testosterone decrease responsible for the alterations found in the seminiferous epithelium of diabetic animals.

Prevention of spontaneous autoimmune diabetes in NOD mice by transferring in vitro antigen-pulsed syngeneic dendritic cells.

To evaluate the effect of antigen-pulsed dendritic cell (DC) transfer on the development of diabetes, 5-week-old female NOD mice received a single iv injection of splenic syngeneic DC from euglycemic NOD mice pulsed in vitro with human y globulin (HGG). Eleven of 12 mice were protected from the development of diabetes up to the age of 25 weeks, and the insulitis score was significantly reduced. In contrast, NOD mice receiving unpulsed splenic DCs showed histological signs of insulitis and course of type 1 diabetes similar to untreated NOD mice. Treatment with HGG-pulsed DC was associated with profound modifications of cytokine secretory capacities within the islets. Thus, supernatants of islets from these mice contained increased levels of interleukin (IL)-4, IL-10, and, to a lesser extent, interferon-gamma and diminished levels of tumor necrosis factor-a compared with controls. Because exogenous IL-4 and IL-10 exert antidiabetogenic effect in NOD mice and early blockade of endogenous tumor necrosis factor-alpha prevents NOD mouse diabetes, these phenomena may be causally related to the antidiabetogenic effect of HGG-pulsed DC treatment.

Multiple low-dose and single high-dose treatments with streptozotocin do not generate nitric oxide.

Streptozotocin (STZ) is a widely used diabetogenic agent that damages pancreatic islet beta cells by activating immune mechanisms, when given in multiple low doses, and by alkylating DNA, when given at a single high dose. Actually, STZ contains a nitroso moiety. Incubation of rat islets with this compound has been found to generate nitrite; moreover, photoinduced NO production from STZ has been demonstrated. These reports have suggested that direct NO generation may be a mechanism for STZ toxicity in diabetogenesis. Several other studies have denied such a mechanism of action. This study has shown that (1) the multiple low-dose (MLDS) treatment does not stimulate NO production at the islet level; in fact, nitrite + nitrate levels and aconitase activity (also in the presence of an NO-synthase inhibitor, namely NAME) remain unmodified; RT-PCR analysis demonstrates that this treatment does not stimulate iNOS activity; (2) the high-dose (HDS) treatment does not stimulate NO production; in fact nitrite + nitrate levels remain unmodified and iNOS mRNA levels are not altered, although aconitase activity is significantly decreased. Moreover, we have confirmed that the MLDS treatment is able to decrease SOD activity by day 11 and that STZ, given in a single high dose, transiently increases superoxide dismutase (SOD) values (24 h from the administration), then dramatically lowers SOD levels. On the basis of our results, we conclude that STZ, "in vivo" is unable to generate NO, both as a MLDS or HDS treatment, thus excluding that NO exerts a role in streptozotocin-dependent diabetes mellitus.

Detection of dendritic cells in the non-obese diabetic (NOD) mouse islet pancreas infiltrate is correlated with Th2-cytokine production.

We investigate the role played by dendritic cells (DCs) in the non-obese diabetic (NOD) mouse pancreas. The early peri-islet, nondestructive infiltration phase, and intra-islet, destructive infiltration phase, which immediately precedes overt diabetes, are studied. Results show that infiltrating cells are Ia-b, ICAM-1, and, mainly, MIDC-8 immunoreactive (ir). These data from silica-treated animals and ultrastructural observations strongly support the hypothesis that DCs are both Ia-b-ir and ICAM-1-ir and that they exert a pivotal role during the period of early infiltration. This is a novel finding for NOD mice and increases the interest for this protective cell type during the rather complex islet infiltration process. Moreover, the cytokine profile demonstrates that Th2 protective cytokines are specific for peri-islet infiltrate. Disappearance of DCs from the infiltrate is concomitant with both the formation of intra-islet infiltration and the increase in proinflammatory Th1 cytokine levels. This further supports the hypothesis that DCs may exert a protective role against diabetes development.

Nicotinamide decreases MHC class II but not MHC class I expression and increases intercellular adhesion molecule-1 structures in non-obese diabetic mouse pancreas.

Pancreases of untreated and nicotinamide (NIC)-treated pre-diabetic (10-week-old) and overtly diabetic (25-week-old) female NOD (non-obese diabetic) mice and of NON (non-obese non-diabetic) control mice were studied, with the following results. (1) Islets and ducts of overtly diabetic untreated NOD mice (25-week-old) were found to express low levels of MHC class I and II molecules, like NON controls, and high levels of adhesive molecules. (2) NIC was able to slightly affect glycaemia and insulitis, slowing down diabetes progression. Moreover it significantly decreased MHC class II expression (but not class I) in vivo by week 10, and significantly enhanced intercellular adhesion molecule-1 (ICAM-1) expression, mainly by week 25, within the pancreas, where 5-bromo-2'-deoxyuridine positive nuclei and insulin positive cells were present, demonstrating that a stimulation of endocrine cell proliferation occurs. (3) In addition, NIC partly counteracted the fall of superoxide dismutase levels, observed in untreated diabetic NOD animals. (4) In vitro studies demonstrated that NIC: (i) was able to significantly reduce nitrite accumulation and to increase NAD+NADH content significantly, and (ii) was able to increase the levels of interleukin-4, a T helper 2 lymphocyte (Th2) protective cytokine, and of interferon-alpha (IFN-alpha), which is known to be able to induce MHC class I and ICAM-1 but not MHC class II expression, as well as IFN-gamma, which is also known to be able to induce MHC class I and ICAM-1 expression. The latter, although known to be a proinflammatory Th1 cytokine, has also recently been found to exert an anti-diabetogenic role. This study therefore clearly shows that adhesive mechanisms are ongoing during the later periods of diabetes in pancreatic ducts of NOD mice, and suggests they may be involved in a persistence of the immune mechanisms of recognition, adhesion and cytolysis and/or endocrine regeneration or differentiation processes, as both NIC-increased ICAM-1 expression and 5-bromo-2'-deoxyuridine positivity imply. The effects of NIC on MHC class II (i.e. a reduction) but not class I, and, mainly, on ICAM-1 expression (i.e. an increase), together with the increase in Th2 protective cytokine levels are very interesting, and could help to explain its mechanism of action and the reasons for alternate success or failure in protecting against type 1 diabetes development.

Macrophages and antioxidant status in the NOD mouse pancreas.

This study showed that citiolone (CIT), a free radical scavenger, significantly increased superoxide dismutase (P < 0.001 vs. untreated NOD, NMMA-treated, and silica-treated animals), catalase (P < 0.01 vs. untreated NOD), and glutathione peroxidase (P < 0.001 vs. untreated NOD and C57BL6/J) values. Silica treatment was capable of counteracting the plasma antioxidant capacity (TRAP) decrease observed in untreated NOD mice, although it did not block the blood glucose rise and insulitis progression in type 1 diabetes significantly. Conversely, early silica administration was able to deplete macrophages (as demonstrated by immunocytochemistry) and to block the rise in blood glucose levels and insulitis progression significantly. Silica-treated animals in this study showed the highest TRAP levels, demonstrating that depletion of macrophages also was able to improve the antioxidant status. This study suggested that macrophages are essential for type 1 diabetes development and showed that they also are involved when the antioxidant status is affected. The reported findings are significant in view of previous studies indicating that oxygen and/or nitrogen free radicals contribute to the islet beta-cell destruction in type 1 diabetes animal models.

Adhesion molecules and microvascular changes in the nonobese diabetic (NOD) mouse pancreas. An NO-inhibitor (L-NAME) is unable to block adhesion inflammation-induced activation.

The aim of the present study was to investigate the immunoreactivity of pancreatic microvasculature with emphasis on the adhesion molecule expression in NOD mice at a very early stage and after the start of infiltration, before the onset of the diabetic disease. Immunoreactivity for Ia-b, BM8 (mouse macrophages) and inter-cellular-adhesion-molecule-1 (ICAM-1) molecules in untreated control mice and in animals treated using an inhibitor of nitric oxide (NO) formation (L-arginine analogue), as well as islet culture, nitrite assay and ultrastructural studies were performed. Results showed that Ia-b and ICAM-1 immunoreactivities on endothelia are a very early phenomenon and that pancreatic blood vessels and, in particular, some peri-islet venules, as well as several venules of the exocrine parenchyma, undergo significant morphological changes. Several endothelial cells of both peri-islet and extra-islet compartments, often showed Ia-b and ICAM-1 immunoreactivities, demonstrating that these cells are important for the adhesion processes taking place during early autoimmune inflammation. Inhibition of NO formation does not significantly affect ICAM-1 and Ia-b immunoreactivity both in vivo and in vitro, BM8 immunoreactive cells were considerably less in number although these were detected either around islets or along pancreatic septa, but rarely within the epithelial layer.

Effects of butylated hydroxytoluene (BHT) enriched diet on serum antioxidant activity in pre-and overtly diabetic nod mice.

Preventive (antioxidant activity) and chain-breaking (total peroxyl radical-trapping parameter) antioxidants in the serum of controls and butylated hydroxytoluene (BHT)-diet enriched nonobese diabetic (NOD) and C57B16/J mice from 5 to 25 weeks of age are measured in this study. A significant decrease in the overall potency of both antioxidant types is demonstrated in NOD untreated controls but not in animals whose diet was BHT-enriched. Therefore, we show that alterations of the antioxidant status in NOD mice is efficaciously counteracted by BHT.

The vitamin-E derivative U-83836-E in the low-dose streptozocin- treated mouse: effects on diabetes development.

Low-dose streptozocin-treated (LDS) mice were administered an inhibitor of lipid peroxidation, U-83836-E (a derivative of vitamin E), in order to observe its ability to alter the onset of diabetes. Ten or 20 mg/kg body wt. per day of U-83836-E were given to mice for 7 days and they were killed after 21 days. Results revealed that there was a significant increase in glycaemia in treated groups up to day 14 after which no further increase was noticed. Superoxide dismutase (SOD) assay showed that: (1) the LDS treatment significantly reduces SOD activity when compared with untreated controls (P < 0.005); (2) U-83836-E increases SOD levels (when compared with untreated controls); and (3) U-83836-E counteracts LDS treatment, since SOD activity is significantly higher with respect to that found in LDS-controls (P < 0.05), and SOD levels were significantly higher with respect to that found in Group 2 animals (P < 0.05), but significantly lower with respect to those found in groups 3 and 4 (P < 0.005). Moreover, malondialdehyde (MDA), the end-product of lipoperoxidation, was found at much higher levels in LDS controls than in the other groups and the lowest values were found in U-83836-E controls and in normoglycaemic animals treated with both streptozocin and U-83836-E. Morphological observations demonstrated that islet beta cells were of normal appearance in normoglycaemic animals of the treated groups. In conclusion, the in vivo inhibition of lipid peroxidation by this compound produces a limited but significant prevention of the islet beta cell destruction.

Administration of a nitric oxide synthase inhibitor does not suppress low-dose streptozotocin-induced diabetes in mice.

Nitric oxide (NO) has been reported as being a key mediator of the autoimmune destruction of B-cells in type I diabetes, and studies have described a suppression of low-dose streptozotocin-induced (LDS) diabetes in mice after the use of NO synthase inhibitors. However, these studies disagree with regard to the outcome of hyperglycemia and insulitis after treatment with these L-arginine analogs. The present study tries to clarify this topic by administering N-nitro-L-arginine-methylester (NAME) (15 mg/d/mouse/15 d) after an LDS treatment in 108 male C57BL6/J mice. Glycemia measured at the end of the NAME treatment did show a slight, but significant, reduction when compared to LDS control animals (p < 0.001), but values returned to diabetic levels 2 wk after withdrawal of NAME. Morphological observations demonstrated that the degree of infiltration and islet B-cell damage was absolutely not inhibited by NAME. In conclusion, treatment with L-arginine analogs is not capable of protecting mice from LDS-induced diabetes.

Superoxide dismutase in the nonobese diabetic (NOD) mouse: a dynamic time-course study.

Superoxide dismutase (SOD) levels, thought to be the first cellular defence against free radicals, were studied in the nonobese diabetesprone (NOD-p) mouse, an animal model of type 1 diabetes in which about 100% of females and 20% of males become diabetic. Nonobese diabetes nonprone (NON-p) mice were used as controls. Animals were followed from 5th to 22nd week of life. Results show that SOD levels in female NOD-p mice are extremely low. In males, values are considerably higher than in females but still lower than values found in control mice. Moreover, SOD levels did not significantly change with age, degree of insulitis or level of diabetes. Islet beta cells in this strain, therefore, seem to be poorly protected against the negative effects of free radicals and this may predispose to diabetes. Furthermore, alterations of SOD may not be directly related to the development of the disease as the enzyme's activity is not further modified with age or the progression of diabetes.

An increase in superoxide dismutase counteracts islet vascular alterations in low-dose streptozocin-treated mice.

A decrease in superoxide dismutase (SOD), the first cellular defence against free radicals, occurs at about the same time as the activation of macrophages within the islets of low-dose streptozocin (LDS)-treated mice. Furthermore, a decrease in the total islet capillary area also has been shown to occur by 10 days after the first streptozocin (STZ) injection and this decline in capillary area is concomitant with the activation of macrophages as is the fall in SOD. Intracellular levels of SOD have been shown to increase after administration of acetyl-homocysteine-thiolactone (citiolone); therefore, the aim of the present study was to observe any relationship between the citiolone-induced increase in SOD levels and islet microvasculature area during LDS-induced diabetes. C57BL6/J male mice were pretreated with daily intramuscular injections of 50 mg citiolone/kg body wt. for 30 days and were then rendered diabetic with 45 mg STZ/kg body wt. given for 5 days; citiolone was given until the animals were killed (days 6, 11 and 18 after the first STZ injection). Further animals were used as non-diabetic and diabetic (STZ-only) controls. The results show that LDS-treated animals when given citiolone: (1) were generally normoglycaemic; (2) had SOD levels that were higher than those of STZ-only control animals; (3) had an islet capillary area that was larger than that of LDS-treated mice. Therefore, the administration of a free radical scavenger, namely citiolone, is able partly to counteract and delay the reduction of islet vascular area and oedema formation in LDS-treated mice.

Superoxide dismutase in low-dose-streptozocin-treated mice. A dynamic time-course study.

Superoxide dismutase (SOD) is a free-radical scavenger present in B cells. It is thought to be responsible for protection against the autoimmune processes that characterize type I diabetes mellitus. Streptozocin (STZ) has been used as a low-dose treatment (LDS) to induce diabetes in animal models. The aim of this study was to follow the islet SOD levels in a day-to-day study after an LDS treatment with STZ, 40 mg/kg body wt/d in C57BL6/J mice. Results reveal a progressive SOD decrease in pancreatic islets with increasing periods from the LDS treatment. This SOD decrease starts from the end of the STZ administration (d 5). In addition, it was noticed that glycemia starts to rise when SOD values have already reached their lowest levels. This indicates that a reduction of free-radical defense is a prerequisite for further cellular injuries. Furthermore, a difference was noticed between males and females: only 40% of female mice underwent a SOD decrement and an increase in glycemia, indicating an androgen-dependent mechanism.

Early macrophage infiltration in mice treated with low-dose streptozocin decreases islet superoxide dismutase levels: prevention by silica pretreatment.

It has been hypothesized that streptozocin (STZ) given in low doses for 5 consecutive days produces diabetes by induction of peroxidation phenomena similar to those induced by free radicals. Moreover, it has been demonstrated that macrophages are among the first to invade the pancreatic parenchyma and destroy islet B cells supposedly by the release of interleukin-1 that induces free radical formation. Superoxide dismutase (SOD) is a free radical scavenger present in cells, and islet B cells are known to have extremely low levels of this enzyme. Therefore, our aim was to observe SOD activity concomitantly with the appearance of intra-islet macrophages, in early diabetes induced by low-dose streptozocin (LDS). Silica-pretreated mice showed SOD values which were comparable to those found in control animals. In LDS-only-treated mice we found that SOD levels were decreased even after only 4 days from the last STZ injection and that it is at this time that the first 'recruited' macrophages appear in the islets. Moreover, the SOD levels found at this early stage (animals were still normoglycaemic and therefore not as yet diabetic) were similar to levels found by us in a previous work, in prediabetic Bio Breeding rats, thereby ascribing a crucial factor to the lowering of SOD levels even in LDS-induced diabetes.

Protection factors against free radical-induced ceroidogenesis.

The most important products of the combustion process are SO2, NOx, CO2 and the heavy metals. When these substances come into contact with the biotic components of the ecosystems they produce an oxidative damage by means of a free radical mechanism. One of the significant natural sources of these oxides and metals are the volcanic emissions that contribute, either locally or more diffusely, to enrich the atmosphere with these substances. The area of Campi Flegrei (Naples, Italy) is an experimental model fit for studying the contemporary effect of the aforesaid oxidative agents, because it is characterized by a continuous fumarolic activity, particularly in the area of the widest crater (Solfatara). We have made so two experiments utilizing rats and earthworms (Octolasium complanatum) to evaluate the following aspects in phylogenetically very different organisms: 1. the combined effect of the atmospheric pollutants, 2. the effect of the only heavy metals (Cu, Ni, Mn), 3. the protection action played by reduced glutathione in rats. The reduced glutathione being either a substrate of the glutathione proxidase or an oxyradicals scavenger, is one of the main protection agents against the above stress. Because many papers suggest that the mentioned atmospheric pollutants damage both animal and vegetable organisms by their oxidative properties, the reduced glutathione seems to be able to counteract efficaciously the damaging activity studied in terms of age pigments production.

Ceroidogenic effect of ionizing radiation.

The effect of the exposure of Torpedo marmorata to a single dose of 400 cGy of ionizing radiation and doses of 200 cGy/week for 4 weeks has been studied. The parameters measured were the TBA reactivity and lipofuscin production, a fluorescent pigment that is thought to derive from the reaction of malonaldehyde, a peroxide-degradation product, with free amino-groups. Acute irradiation was found to generate an increase of peroxidative damage in some tissues. This effect was inhibited by acetyl homocysteine-thiolactone, a drug that increases the activity of tissue superoxide dismutase. Chronic irradiation produces a severe increase in the generation of lipofuscin.

Effect of acetylhomocysteine thiolactone on nucleolar cytology and lipofuscinogenesis in electric lobe neurons.

Treatment with acetylhomocysteine thiolactone significantly reduces the cellular level of lipofuscin in neurons of the electric lobe of Torpedo marmorata. At the same time, this drug produces a 45% decrease in nucleolar volume in these neurons, reflecting decreased cellular synthetic activity.

Superoxide dismutase activity in the BB rat: a dynamic time-course study.

Diabetes produced spontaneously in the BB rat is similar to that observed in multiple low dose streptozocin-induced diabetes, both being characterized histologically by a lympho-monocytic infiltrate in the pancreatic islets (insulitis). Recent studies indicated that streptozocin acts through peroxidative patterns sensitive to superoxide dismutase (SOD) activity. We therefore conducted a time-course study to evaluate if SOD activity in the islets of Langerhans is related to the onset of diabetes in BB rats with varying degree of diabetes. It was found that SOD activity does not change with age nor with the onset of diabetes. However SOD activity in the islets of BB rats was significantly lower than in the control Wistars. This lower SOD activity may be a proneness factor that favors the development of the diabetic syndrome.

Influence of acetyl homocysteine thiolactone on erythrocyte superoxide dismutase activity.

The administration of 8 mg of acetylhomocysteinethiolactone/kg body wt determines a significant (P less than 0.001) increase in rat erythrocyte superoxide dismutase activity.