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Increasing evidence indicates that decreased functional β-cell mass is the hallmark of Type 2 diabetes mellitus. Therefore, the debate focuses on the possible mechanisms responsible for abnormal islet microenvironment, decreased β-cell number, impaired β-cell function and their multifactorial etiologies. The information available on the Goto-Kakizaki/Par rat line, one of the best characterized animal models of spontaneous Type 2 diabetes mellitus, are reviewed in such a perspective. We propose that the defective β-cell mass and function in the Goto-Kakizaki/Par model reflect the complex interactions of multiple pathogenic players, including several independent loci containing genes responsible for some diabetic traits (but not decreased β-cell mass), gestational metabolic impairment inducing an epigenetic programming of the pancreas (decreased β-cell neogenesis), which is transmitted to the next generation, and loss of β-cell differentiation due to chronic exposure to hyperglycemia, inflammatory mediators, oxidative stress and perturbed islet microarchitecture.

International audience; The adult non-obese Goto–Kakizaki (GK) rat model of type 2 diabetes, particularly females, carries in addition to hyperglycaemia a genetic predisposition towards dyslipidaemia, including hypercholesterolaemia. As cholesterol-induced atherosclerosis may be programmed in utero, we looked for signs of perinatal lipid alterations and islet microangiopathy. We hypothesise that such alterations contribute towards defective pancreas/islet vascularisation that might, in turn, lead to decreased beta cell mass. Accordingly, we also evaluated islet inflammation and endothelial activation in both prediabetic and diabetic animals. Blood, liver and pancreas were collected from embryonic day (E)21 fetuses, 7-day-old prediabetic neonates and 2.5-month-old diabetic GK rats and Wistar controls for analysis/quantification of: (1) systemic variables, particularly lipids; (2) cholesterol-linked hepatic enzyme mRNA expression and/or activity; (3) pancreas (fetuses) or collagenase-isolated islet (neonates/adults) gene expression using Oligo GEArray microarrays targeted at rat endothelium, cardiovascular disease biomarkers and angiogenesis, and/or RT-PCR; and (4) pancreas endothelial immunochemistry: nestin (fetuses) or von Willebrand factor (neonates). Systemic and hepatic cholesterol anomalies already exist in GK fetuses and neonates. Hyperglycaemic GK fetuses exhibit a similar percentage decrease in total pancreas and islet vascularisation and beta cell mass. Normoglycaemic GK neonates show systemic inflammation, signs of islet pre-microangiopathy, disturbed angiogenesis, collapsed vascularisation and altered pancreas development. Concomitantly, GK neonates exhibit elevated defence mechanisms. These data suggest an autoinflammatory disease, triggered by in utero programming of cholesterol-induced islet microangiopathy interacting with chronic hyperglycaemia in GK rats. During the perinatal period, GK rats show also a marked deficient islet vascularisation in conjunction with decreased beta cell mass.

In human diabetes, degenerative and functional disorders of the central nervous system, including depression, are common findings. Defective dentate gyrus (DG) neurogenesis is associated with affective-related disorders and depression. We previously demonstrated reduced DG neurogenesis in a pharmacological type 1 diabetes model, the streptozotocin (STZ)-treated mouse. Here, we explored DG neurogenesis in a spontaneous T1D model, the nonobese diabetic (NOD) mouse, at prediabetic and diabetic stages. Cell proliferation was assessed in the DG of 5, 8 and 12-week-old control C57BL/6 and BALB/c strains and NOD mice, killed 2 h after bromodeoxyuridine (BrdU) administration. Survival of the newly generated cells was studied in 15-week-old animals that were killed 21 days after BrdU injection. The number of proliferative BrdU-positive cells in the DG was, regardless of age, constantly and significantly lower in NOD than in control strains, showing the presence of hippocampal alterations far before clinical diabetes onset in NOD mice. Diabetes also strongly decreased cell survival in NOD DG. However, cell phenotype proportion, as assessed by co-localization with neuronal or glial markers and confocal microscopy, was not modified. Hippocampal neurogenesis is strongly diminished in the spontaneous NOD model, like in the STZ model. Notably, NOD hippocampal DG cell proliferation defect takes place during the prediabetic stage. Whether this early alteration might result, in this autoimmune strain, from hypothalamo-pituitary adrenal axis alterations and/or ongoing brain inflammatory process sharing many characteristics of aging is discussed and deserves further investigation.

1. A recently recognized complication of uncontrolled diabetes mellitus is the encephalopathy involving, among other regions, the hippocampus. Since estrogens bring neuroprotection in cases of brain injury and degenerative diseases, we have studied if estradiol (E2) administration counteracts some hippocampal abnormalities of streptozotocin (STZ)-diabetic adult mice. 2. We first report the ability of E2 to modulate neurogenesis in the dentate gyrus (DG) and subventricular zone (SVZ) of diabetic mice. Using bromodeoxyuridine (BrdU) to label newly generated cells, a strong reduction in cell proliferation was obtained in DG and SVZ of mice sacrificed 20 days after STZ administration. The reduction was completely relieved by 10 days of E2 pellet implantation, which increased 30-fold the circulating E2 levels. 3. Diabetic mice also showed abnormal expression of astrocyte markers in hippocampus. Thus, increased number of GFAP(+) cells, indicative of astrogliosis, and increased number of apolipoprotein-E (Apo-E)(+) astrocytes, a marker of ongoing neuronal dysfunction, was found in stratum radiatum below the CA1 hippocampal subfield of diabetic mice. Both parameters were reverted to normal by the E2 regime that upregulated cell proliferation. 4. The studies demonstrated that hippocampal neuropathology of uncontrolled diabetes is a reversible condition and sensitive to estrogen treatment. Studies in animal models may open up new venues for understanding the beneficial role of steroid hormones in diabetic encephalopathy.

Cerebral dysfunctions, including a high incidence of depression, are common findings in human type 1 diabetes mellitus. An association between depression and defective hippocampal neurogenesis has been proposed and, in rodents, antidepressant therapy restores neuronal proliferation in the dentate gyrus. Hippocampal neurogenesis is also deficient in diabetic mice, which led us to study whether the selective serotonin reuptake inhibitor fluoxetine influences cell proliferation in streptozotocin-diabetic animals. Diabetic and control C57BL/6 mice received fluoxetine (10 mg/kg/day, i.p., 10 days) and dentate gyrus cell proliferation was measured after a single injection of 5-bromo-2'-deoxyuridine (BrdU). Diabetic mice showed reduced cell proliferation. Fluoxetine treatment, although having no effect in controls, corrected this parameter in diabetic mice. The phenotype of newly generated cells was analysed by confocal microscopy after seven daily BrdU injections, using Tuj-1/beta-III tubulin as a marker for immature neurones and glial fibrillary acidic protein for astrocytes. In controls, the proportion of Tuj-1-BrdU-positive cells over total BrdU cells was approximately 70%. In vehicle-treated diabetic mice, immature neurones decreased to 56% and fluoxetine brought this proportion back to control values without affecting astrocytes. Therefore, fluoxetine preferentially increased the proliferation of cells with a neuronal phenotype. In addition, neurones were counted in the hilus of the dentate gyrus; a 30% decrease was found in diabetic mice compared with controls, whereas this neuronal loss was prevented by fluoxetine. In conclusion, fluoxetine treatment restored neuroplasticity-related hippocampal alterations of diabetic mice. These findings may be potentially important to counteract diabetes-associated depression in humans.

Previously, we reported elevated numbers of macrophages in the pancreas of NOD mice, a spontaneous animal model for T1D, during the early postnatal period. Extracellular matrix plays an important role in the tissue trafficking and retention of macrophages as well as in postnatal pancreas development. Therefore, we have examined the expression and distribution of laminin and fibronectin, two major extracellular matrix proteins and their corresponding integrin receptors, in the pre-weaning pancreases of NOD mice and control mouse strains. In addition, we have characterized the pancreas morphology during this period, since the morphology of the pre-weaning pancreas before the onset of lymphocytic peri-insulitis, when the pancreas is still subject to developmental changes, has been poorly documented. We show that laminin labeling is mainly associated with exocrine tissue, whereas fibronectin labeling was mostly localized at the islet-ductal pole, islet periphery and in intralobular septa. Moreover, the protein expression level of fibronectin was increased in NOD pancreases at the early stage of postnatal development, as compared to pancreases of C57BL/6 and BALB/c mouse strains. Interestingly, pancreatic macrophages were essentially found at sites of intense fibronectin labeling. The increased fibronectin content in NOD neonatal pancreas coincided with altered islet morphology, histologically reflected by enlarged and irregular shaped islets and increased percentages of total endocrine area as compared to that of control strains. In conclusion, increased levels of the extracellular matrix protein fibronectin were found in the early postnatal NOD pancreas, and this is associated with an enhanced accumulation of macrophages and altered islet morphology.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which insulin-producing beta cells of the pancreatic islets of Langerhans are destroyed. The nonobese diabetic (NOD) mouse is one of the rare spontaneous models that enable the study of prediabetic pancreatic events. The etiology of the autoimmune attack in human and animal T1D is still unknown, but genetic and environmental factors are involved in both cases. Although several autoantigens have been identified and defective immune-system regulation is implicated, this information does not satisfactorily explain the generally accepted beta-cell specificity of the disease or how so many and diverse environmental factors intervene in its pathogenesis. Based on data obtained from evaluating glucose homeostasis in a variety of situations, particularly stress and cytokine administration, in young prediabetic NOD mice, the author hypothesizes that the islet of Langerhans is a major actor, and its altered regulation through environmentally induced insulin resistance might reveal latent T1D. It is also postulated that T1D pathogenesis might be linked to abnormal pancreas development, probably due to disturbances of glutamic acid decarboxylase (GAD) + innervation phagocytosis by defective macrophages during the early postnatal period. Also discussed is the role of defective presentation of pancreatic hormones and GAD in the thymus, and its potential repercussion on T-cell tolerance. Observations have demonstrated that the diabetogenic process in the NOD mouse is extremely complex, involving neuroendocrine immune interaction from fetal life onward.

The mouse pancreas, an immature organ at birth, reaches its adult size and morphology after weaning (3 weeks of age). Around this time, apoptotic phenomena and various types of macrophages are normally present. During development, Fas-Fas ligand (FasL) interactions are known to play a role in apoptotic events involved in tissue remodeling and elimination of damaged cells, and macrophages are routinely observed near apoptotic cells. Apoptosis and Fas-FasL interactions are also thought to be involved in the pathogenesis of autoimmune diseases, particularly type 1 diabetes (T1D). Therefore, we used early postnatal mouse pancreata from three control strains (C57BL/6, DBA/2, BALB/c) and from two strains with the nonobese diabetic (NOD)-related genetic background (the spontaneous T1D NOD model and the lymphocyte-deficient NODscid strain) to study apoptotic phenomena together with the molecular and immunohistochemical expression of proapoptosis (Fas, FasL) and antiapoptosis (Bcl-2) proteins. First, although no major difference in the numbers of total pancreatic apoptotic cells was noted among strains, significantly more FasL(+) expression was detected immunohistochemically in mice with the NOD genetic background than in control pancreata from birth to 1 month of age. Second, FasL(+), Fas(+), and Bcl-2(+) structures seemed to be associated with innervation, regardless of the strain and age. Third, in control and NOD strains, nerves (identified by immunohistochemical labeling of peripherin or neurofilament 200), were often observed in periductular and peri-insular areas. Finally, some peripherin-positive nerves expressed the interferon-inducible protein-10 chemokine, and various types of macrophages were found to be in close proximity. These data highlight an overlooked, innervation-related aspect of normal mouse postnatal pancreas development with possible implications in T1D pathogenesis.

Type 1 diabetes mellitus is a T-cell-mediated autoimmune disease that results in the destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans. In spite of extensive genetic and immunological studies, mainly performed in the non-obese diabetic (NOD) spontaneous mouse model, the etiology of the autoimmune attack remains unknown. Several autoantigens have been identified and numerous studies have suggested a role for defective regulation of immune function. However, this account does not explain why the autoimmune process specifically affects the insulin-producing beta cells. Thus, abnormal immune regulation might explain the predisposition to autoimmunity in general, but additional factors should then determine the target of the autoimmune attack. Here, we review the evidence that abnormalities in islet cell differentiation and function exist that might trigger the immune system towards beta-cell autoimmunity in humans and NOD mice.

In the Nonobese diabetic (NOD) mouse, a spontaneous model of type 1 diabetes, the pathogenic process is classically thought to start at 3-4 weeks of age with an accumulation of antigen-presentingcells (APC), especially CD11c+ dendritic cells (DC), around the pancreatic islets of Langerhans. Concomitantly, hyperinsulinemia and slight hyperglucagonemia are observed, which may be either the cause or consequence of the initial APC infiltration. To determine whether infiltrating DC can affect islet activity in control (C57BL/6) and NOD mice, we performed experiments in which islets and DC were isolated and co-cultured. We first showed that, immediately after isolation, islets from 8-week-old prediabetic NOD mice had significantly higher insulin and glucagon contents than those from C57BL/6 controls. Moreover, as is the case in vivo, prediabetic NOD mouse islets secrete more insulin in vitro at 11.1 mM glucose than C57BL/6 ones. In DC-islet co-cultures, insulin secretion was significantly increased for NOD mice only, while that of glucagon was not significantly affected. These findings indicate that NOD DC are good candidates for stimulating the NOD mouse beta-cell hyperactivity that is observed both in vivo and in vitro, and might, consequently, sensitize NOD islets to an autoimmune attack.

Type 2 diabetes mellitus (T2D) arises when the endocrine pancreas fails to secrete sufficient insulin to cope with the metabolic demand because of β-cell secretory dysfunction and/or decreased β-cell mass. Defining the nature of the pancreatic islet defects present in T2D has been difficult, in part because human islets are inaccessible for direct study. This review is aimed to illustrate to what extent the Goto–Kakizaki rat, one of the best characterized animal models of spontaneous T2D, has proved to be a valuable tool offering sufficient commonalities to study this aspect. A comprehensive compendium of the multiple functional GK islet abnormalities so far identified is proposed in this perspective. The pathogenesis of defective β-cell number and function in the GK model is also discussed. It is proposed that the development of T2D in the GK model results from the complex interaction of multiple events: (i) several susceptibility loci containing genes responsible for some diabetic traits (distinct loci encoding impairment of β-cell metabolism and insulin exocytosis, but no quantitative trait locus for decreased β-cell mass); (ii) gestational metabolic impairment inducing an epigenetic programming of the offspring pancreas (decreased β-cell neogenesis and proliferation) transmitted over generations; and (iii) loss of β-cell differentiation related to chronic exposure to hyperglycaemia/hyperlipidaemia, islet inflammation, islet oxidative stress, islet fibrosis and perturbed islet vasculature.

Glucagon-producing α cells play a crucial role during the perinatal period. Because of their peri-islet localization near the early dendritic and macrophage cell infiltration, we thought it pertinent to investigate α cells in greater depth in nonobese diabetic (NOD) mice, a well-recognized spontaneous model for human type I diabetes. We determined α-cell distribution (glucagon immunohistochemistry and image analysis) and activity (real-time reverse transcriptase polymerase chain reaction [RT-PCR] and glucagon radioimmunoassay [RIA]), in relationship to glycemia in NOD and lymphocyte-deficient NODscid mice as compared to control mice (C57BL/6) from birth onward. NOD and NODscid mice, particularly at 1 day of age, had twice as many very small islets (

The thymus plays a central role in the selection of T lymphocytes that are tolerant to ‘self’ antigens and responsive to foreign pathogens. We and others have reported the expression of the pancreatic endocrine hormones, preproinsulin, proglucagon, prosomatostatin and propancreatic polypeptide in the human and mouse thymus. While mRNA expression is very low there is evidence for the presence of the translated product. In addition, we have investigated the cell types responsible for expression. In the thymus, hormone expression is enriched in the antigen-presenting cell population. Interestingly, while proglucagon, prosomatostatin and propancreatic polypeptide appear to be expressed in a macrophage population, preproinsulin expression was restricted to dendritic cells which are more potent antigen-presenting cells. The functional significance of the endogenous expression of insulin in the thymus has been indirectly investigated using transgenic models in which the transgene is introduced by the rat insulin promoter. The data suggest that thymic expression of the transgene is critical in the induction of T-cell tolerance to the transgene in the periphery. Taken together, the evidence suggests that the low-level pancreatic hormone expression in the thymus may be involved in central tolerance to proteins of restricted expression.

The expression of preproinsulin (ppIns), proglucagon, prosomatostatin, and propancreatic polypeptide was investigated in thymic extracts, thymic cells, and thymic cell lines from C57BL/6 mice by RT-PCR. The expression of pancreatic hormones was similar in thymic extracts taken from neonatal and 2-, 4-, and 8-week-old animals, but was decreased in 20-week-old animals. Pancreatic hormone expression was not observed in mouse liver, salivary gland, or spleen. Analysis of thymic cell populations revealed a 10- to 20-fold enrichment in expression of all hormones in low buoyant density cells. No expression was detected in high buoyant density cells (predominantly thymocytes) or in thymic epithelial cell lines, primary cultures of epithelial cells, or peripheral macrophages. In addition, immunoreactive insulin, measured by specific RIA, was detectable in the low buoyant density population, but not in high buoyant density cells. The enriched cell population was depleted of contaminating lymphocytes and sorted based on reactivity to the cell surface markers F4/80 (macrophage) or N418 (dendritic cells). Cells gated for N418 demonstrated expression for ppIns, but not the other pancreatic hormones. Conversely, expression for proglucagon, prosomatostatin, and propancreatic polypeptide, but not ppIns, was detected in F4/80-gated cells. Our data indicate that pancreatic endocrine hormones are differentially expressed by dendritic cells and macrophages in a normal mice., Instituto de Investigaciones Bioquímicas de La Plata

Because few data were available on glucose homeostasis at the early prediabetic stage in the nonobese diabetic (NOD) mouse, we investigated glycemia, insulinemia, and pancreatic insulin content under basal conditions in both sexes of 4-, 6-, and 8-week-old fed NOD mice, compared with sex- and age-matched fed C57BL/6 mice. We also investigated glucose tolerance in both sexes of fasting 8-week-old NOD and C57BL/6 mice. The main results obtained under basal fed conditions, when comparing both strains, were lower glycemia and higher insulinemia in NOD females at all ages investigated and in NOD males (particularly at 6 weeks of age). Glucose tolerance tests showed that: 1) the blood glucose response to 1 g/kg i.p. glucose was less sustained in both sexes of 8-week-old NOD mice than in their control counterparts; 2) the blood insulin response to glucose (1 g/kg i.p.) appeared earlier in both sexes of NOD mice than in sex-matched C57BL/6 mice; 3) an unusual sexual dimorphism existed in NOD mice, compared with controls, with females secreting, in response to glucose, twice as much insulin as males; 4) dose-response studies (1-6 g/kg glucose) confirmed the lower increase in blood glucose levels in both sexes of NOD mice and their unusual sexual dimorphism in insulin secretion; and 5) glucose tolerance tests in 4- to 8-week-old NOD mice showed that although the sexual dimorphism in insulin secretion was not observed in 4-week-old mice, it was particularly striking at 6 weeks of age. Taken together, these results suggest that beta-cell hyperactivity exists in the NOD mouse at the early prediabetic stage, especially in NOD females.

The progression of type I diabetes in the NOD mouse is modulated by, among other things, stressful events and steroids. We measured in 2-month-old prediabetic NOD mice various circulating steroids (progesterone, corticosterone, dehydroepiandrosterone, delta4-androstenedione, testosterone, estrone and estradiol) under basal and stressful conditions (1.5h immobilization). Basal progesterone concentrations were low but measurable in randomized cycling NOD females and under the detection limit in NOD males. Immobilization increased progesterone concentrations in both sexes. Serum corticosterone concentrations also increased after immobilization but with the sexual dimorphism normally observed in rodents. Dehydroepiandrosterone concentrations were similar in both sexes and remained unaffected by stress. Testosterone and delta4-androstenedione were drastically reduced after immobilization in NOD males. Serum estrone and estradiol were not found to be statistically different in NOD females and males, but slightly higher to that described in the literature, and immobilization increased estrone concentrations in NOD males. In conclusion, while nonspecific to the NOD mouse, the modulation of circulating corticosteroids, estrogens and androgens induced by environmental factors may be part of the mechanism(s) by which these factors modulate the progression of type I diabetes. The hormonal changes may act in a complex manner at different levels: the immune system, the islet of Langerhans and the other structures involved in glucose homeostasis.

International audience; Regenerating (REG) proteins are associated with islet development, b-cell damage, diabetes and pancreatitis. Particularly, REG-1 and REG-3-beta are involved in cell growth/survival and/or inflammation and the Reg1 promoter contains interleukin-6 (IL-6)-responsive elements. We showed by transcriptome analysis that islets of Goto-Kakizaki (GK) rats, a model of spontaneous type 2 diabetes, overexpress Reg1, 3a, 3b and 3c, vs Wistar islets. Goto-Kakizaki rat islets also exhibit increased cytokine/chemokine expression/release, particularly IL-6. Here we analyzed Reg1 and Reg3b expression and REG-1 immuno-localization in the GK rat pancreas in relationship with inflammation. Isolated pancreatic islets and acinar tissue from male adult Wistar and diabetic GK rats were used for quantitative RT-PCR analysis. REG-1 immunohistochemistry was performed on paraffin sections with a monoclonal anti-rat REG-1 antibody. Islet cytokine/chemokine release was measured after 48 h-culture. Islet macrophage-positive area was quantified on cryostat sections using anti-CD68 and major histocompatibility complex (MHC) class II antibodies. Pancreatic exocrine-to-endocrine Reg1 and Reg3b mRNA ratios were markedly increased in Wistar vs GK rats. Conversely, both genes were upregulated in isolated GK rat islets. These findings were unexpected, because Reg genes are expressed in the pancreatic acinar tissue. However, we observed REG-1 protein labeling in acinar peri-ductal tissue close to islets and around large, often disorganized, GK rat islets, which may retain acinar cells due to their irregular shape. These large islets also showed peri-islet macrophage infiltration and increased release of various cytokines/ chemokines, particularly IL-6. Thus, IL-6 might potentially trigger acinar REG-1 expression and secretion in the vicinity of large diabetic GK rat islets. This increased acinar REG-1 expression might reflect an adaptive though unsuccessful response to deleterious microenvironment.

Prophylactic insulin treatment has been shown to have beneficial effects in type 1 diabetes, both in humans and in various animal models of the disease. In experimental models, the protective effect of prophylactic insulin treatment was observed in two parameters: (1) progression of insulitis and (2) diabetes incidence. The mechanism of protection still remains to be investigated. We therefore analysed by immunohistochemistry the effect of prophylactic insulin treatment vs placebo treatment (from 4 to 13 weeks of age) on ER-MP23+ macrophage infiltration in and around pancreatic islets in the non-obese diabetic (NOD) mouse, a spontaneous model for type 1 diabetes. BALB/c mice were used as diabetes-free controls. Using conventional haematoxylin-eosin staining, we detected a protective effect of prophylactic insulin treatment in NOD females on the lymphocytic insulitis, significant at 13 weeks, but not at 9 weeks of age. However, when assessed by immunostaining for early infiltration of ER-MP23+ macrophages around islets, the reduction in severity of insulitis could already be detected as early as 9 weeks of age. With regard to the early accumulation of ER-MP23+ cells, we observed that their numbers per mm2 surface area of the exocrine pancreas and per micron at the circumference of the islet were higher in placebo-treated NODs (197 +/- 13.8 and 14 +/- 0.9, respectively) as compared to age-matched BALB/c mice (123.1 +/- 7.1 and 3.5 +/- 0.9, respectively). Prophylactic insulin treatment of NODs lowered the attraction of ER-MP23+ macrophages to the exocrine pancreas and to the circumference of the islets (156.3 +/- 8.5 and 7.9 +/- 1, respectively). Interestingly also, the islet size was found to be larger in placebo-treated NODs (51% was larger than 10 microns2) than in age-matched BALB/c mice (9% larger than 10 microns2). Prophylactic insulin treatment of NODs reduced their islet size to sizes found in the control BALB/c strain. In conclusion, the decrease in islet size by early insulin administration, and the lower attraction of ER-MP23+ macrophages to the islets are morphological indications that prevention of diabetes development by prophylactic insulin treatment results from a down-regulation of islet metabolism and growth, with a concomitant decline in the release of islet factors attracting macrophages.

The nonobese diabetic (NOD) mouse spontaneously develops insulin-dependent diabetes (IDDM or type I diabetes), resulting from T-lymphocyte-mediated destruction of pancreatic beta cells. This autoimmune phenomenon includes mononuclear cell infiltration of the islets of Langerhans (insulitis) and the presence of circulating autoantibodies. The specificity of the autoantibodies and of the autoreactive T cells was investigated and several autoantigens were proposed, in particular glutamic acid decarboxylase (GAD). This enzyme exists in two forms (GAD 65 and GAD 67) encoded by two independent genes. To explain the role of GAD in type I diabetes, we prepared recombinant rat GAD 65 as fusion protein, produced in an Escherichia coli expression system, and we treated NOD female mice from 4 to 7 weeks of age by repeated intraperitoneal injections of 5 micrograms fusion protein (3 injections per week); control groups received the fusion partner, maltose binding protein (MBP) or dissolving agent (NaCl 0.9%). We investigated two parameters, the degree of insulitis 5 weeks after the last injection and the overall incidence of the disease. Histological examination of the pancreata from GAD-treated mice revealed a significant reduction in the severity of insulitis compared with the two control groups. Furthermore, we observed that the time of onset and the frequency of diabetes in NOD females injected with GAD fusion protein differed significantly from the control groups receiving MBP or NaCl (P < 0.0001). These results show that a 3-week treatment of NOD female mice starting at 4 weeks of age protects them from diabetes, again emphasizing the crucial role of GAD as autoantigen in type I diabetes.

The non-obese diabetic mouse (NOD) is one of the few available models of spontaneous autoimmune insulin-dependent diabetes mellitus (IDDM). The authors determined the free fatty acid (FFA) levels and the concentrations and relative percentages of the various classes of FFA before the onset of diabetes in both sexes at 2 and 4 months of age and in diabetic females. A circadian rhythm of FFA concentrations was found in prediabetic mice, with lower values in the evening. Moreover, there was a sex difference in FFA concentrations in the morning, with 2-month-old females having higher concentrations than males. Sex and age-related differences were also observed in the concentrations of the various classes of FFA, with higher polyunsaturated fatty acid concentrations in 2-month-old females and increases in di- and triunsaturated fatty acids concentrations in both sexes with age. Hormonal manipulation such as adrenalectomy and/or castration modulated total FFA and the concentrations of the various classes of FFA in 2-month-old mice. These FFA differences between males and females should be taken into account in the onset of type I diabetes.

Diabetes mellitus is associated with increased risk for cardiovascular disorders, which are major causes of mortality in this disease. Hyperhomocysteinemia, defined by high plasma homocysteine levels, is an independent risk factor for the development of cardiovascular diseases. Type 2 diabetic patients have higher circulating homocysteine levels than healthy subjects and these levels are even higher in plasma of obese than nonobese diabetic patients. Homocysteine metabolism that has been studied in 2 animal models of type 2 diabetes with obesity led to conflicting data. The aim of the present study was to analyze homocysteine metabolism in a spontaneous nonobese model of type 2 diabetes, the Goto–Kakizaki rats at various successive and well characterized stages of the disease: during early postnatal normoglycemia, at the onset of hyperglycemia (around weaning), and during chronic mild hyperglycemia with progressive insulin resistance. Compared to age-matched Wistar controls, Goto–Kakizaki rats showed lower plasma levels of homocysteine and a falling trend in its major byproduct antioxidant, glutathione, from the prediabetic stage onwards. Concomitantly, Goto–Kakizaki rats exhibited increased liver activity of cystathionine beta synthase, which catalyzes the condensation of homocysteine with serine in the first step of the transsulfuration pathway. These results emphasize a strong association between homocysteine metabolism and insulin via the first step of the hepatic transsulfuration pathway in Goto–Kakizaki rats.

The effects of long-term chronic stress (induced by repeated restraint, overcrowding or both), short-term chronic stress (induced by a triad of stressors over a short period of time early in life) and adrenalectomy were investigated on the prevalence, on the degree of insulitis and various physiological and immunological parameters in the NOD mouse, a spontaneous model of type I-insulin-dependent diabetes mellitus (IDDM). Long-term chronic stress, obtained by restraint once a week or overcrowding, significantly protected NOD females, while both applied concomitantly had only a tendency to protect against diabetes. In contrast, short-term chronic stress had no significant effect on diabetes expression, whereas adrenalectomy resulted in a trend toward accelerated diabetes onset. The various long-term chronic stress paradigms exerted different effects on the progression of insulitis: repeated restraint tended to protect against insulitis, overcrowding had no effect but, when associated with restraint, significantly counteracted the beneficial effect of restraint alone. Adrenalectomy and short-term chronic stress had no significant effect on the development of insulitis. Various parameters, such as body, thymus and spleen weights, thymus and spleen cellularities, mitogen-induced spleen cell proliferation and serum corticosterone levels were also studied under the various experimental conditions. Taken together, the observations suggest that stressors modulate the expression of spontaneous autoimmune diabetes by exerting pleiotropic effects on immune and/or inflammatory components at the pancreas level and on peripheral glucose metabolism.

A multiparametric study of the thymus was performed in normal aging mice (12-15 months old) submitted to a mild oral zinc supplementation during 3-6 months as compared to age-matched control mice. First, this study demonstrated that in rodents, zinc levels are significantly reduced with aging and can be restored to values close to those observed in young animals after 6 months of zinc supplementation. Second, our data showed that oral zinc administration stimulates thymus growth and partially restores the microenvironmental as well as lymphoid compartments of the organ. Regarding thymic endocrine function, a significant increase in thymulin levels and a concomitant decrease in plasma thymulin inhibitors were observed, suggesting that the age-related decline of thymic function might at least partially be due to extrinsic factors, such as zinc deficiency. The total number of thymic lymphocytes was consistently increased, without significant changes in CD4/CD8 defined thymocyte subsets. Finally, structural changes of the thymus epithelium were also detected, including the disappearance of epithelial cysts frequently observed in old animals, reappearance of a normal pattern of the thymic epithelial cell network, and a decrease in the extracellular matrix network. Taken together, these data suggest that aging-related physiological zinc deficiency induces some relevant changes in thymus structure and function which can be partially corrected by a mild oral zinc supplementation.

Type 2 diabetes mellitus (T2D) arises when the endocrine pancreas fails to secrete sufficient insulin to cope with the metabolic demand because of beta-cell secretory dysfunction and/or decreased beta-cell mass. Defining the nature of the pancreatic islet defects present in T2D has been difficult, in part because human islets are inaccessible for direct study. This review is aimed to illustrate to what extent the Goto-Kakizaki rat, one of the best characterized animal models of spontaneous T2D, has proved to be a valuable tool offering sufficient commonalities to study this aspect. A comprehensive compendium of the multiple functional GK islet abnormalities so far identified is proposed in this perspective. The pathogenesis of defective beta-cell number and function in the GK model is also discussed. It is proposed that the development of T2D in the GK model results from the complex interaction of multiple events: (i) several susceptibility loci containing genes responsible for some diabetic traits (distinct loci encoding impairment of beta-cell metabolism and insulin exocytosis, but no quantitative trait locus for decreased beta-cell mass); (ii) gestational metabolic impairment inducing an epigenetic programming of the offspring pancreas (decreased beta-cell neogenesis and proliferation) transmitted over generations; and (iii) loss of beta-cell differentiation related to chronic exposure to hyperglycaemia/hyperlipidaemia, islet inflammation, islet oxidative stress, islet fibrosis and perturbed islet vasculature.

BACKGROUND: Unlike other strains, spontaneously type 1 non-obese diabetic (NOD) mice experience transient hyperinsulinemia after weaning. The same applies for NOD/SCID mice, which lack functional lymphocytes, and unlike NOD mice, do not develop insulitis and diabetes like NOD mice. AIMS: Given that β-cell stimulation is a natural feature of gestation, we hypothesized that glucose homeostasis is disturbed in gestate pre-diabetic NOD and non-diabetic NOD/SCID mice, which may accelerate the onset of diabetes and increase diabetes prevalence. METHODS: During gestation and postpartum, mice were analyzed under basal feed conditions, and following glucose injection (1 g/kg, i.p.) after overnight fast, using glucose tolerance test (GTT). Glycemia, corticosteronemia, blood and pancreatic insulin, glucagon levels, islet size, and islet morphology were evaluated. Glycemia and mortality were assessed after successive gestations in NOD mice mated for the first time at 2 different ages. RESULTS: 1. Basal glucagonemia rose markedly in first-gestation fed NOD mice. 2. β-cell hyperactivity was present earlier in first-gestation non-diabetic fasted NOD and NOD/SCID mice than in age-matched C57BL/6 mice, assessed by increased insulin/glucose ratio after GTT. 3. Overnight fasting increased corticosteronemia rapidly and sharply in pre-diabetic gestate NOD and NOD/SCID mice. 4. Islet size increased in non-diabetic gestate NOD mice compared with C57BL/6 mice. 5. Successive gestations accelerated diabetes onset, and contributed to increased mortality in NOD mice. CONCLUSIONS: First-gestation pre-diabetic NOD and non-diabetic NOD/SCID mice exhibited β-cell hyperactivity and deregulation of glucagon and/or corticosterone secretion. This amplified normally occurring insulin resistance, further exhausted maternal β-cells, and accelerated diabetes in NOD mice.

The inbred nonobese diabetic (NOD) mouse spontaneously develops an autoimmune diabetes, which is now recognized as an experimental model for human type I insulin-dependent diabetes mellitus (IDDM). The autoimmune reaction, specifically directed against pancreatic beta cells (insulitis), involves both macrophages and T lymphocytes. The study of the production of cyclooxygenase and lipoxygenase derivatives of arachidonic acid metabolism shows that in some conditions, and in particular in the presence of zymosan A, macrophages from NOD mice produced significantly more 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and leukotriene C4 (LTC4) than macrophages from age- and sex-matched C57BL/6 mice. Moreover, zymosan A-stimulated macrophages from NOD females produced significantly more LTC4 than did macrophages from NOD males. These results may be of interest, given the bidirectional relationship between the various cytokines involved in the destruction of beta cells of the islets of Langerhans and different eicosanoids.

The NOD mouse is a recognized model for studying immunologically mediated insulin-dependent diabetes mellitus (IDDM). In most colonies, the disease appears with a greater preponderance in females than in males and castration alters the expression of the disease. The prevalence of diabetes may also vary depending upon environmental factors such as stress. Therefore, we measured in the NOD mouse serum glucocorticoid concentrations in basal and stress conditions. We observed in NOD as well as in C57BL/6 mice, taken as controls, a circadian rhythm of corticosterone, with females having higher values than males. After a single restraint stress, female and male NOD mice exhibit a comparable response, whereas after repeated stress, males respond significantly less than females, suggesting an adaptation phenomenon. In contrast, there is no difference in the pattern of corticosterone response of C57BL/6 females and males to both types of stress, but females always respond better than males. Moreover, whatever the stress considered, NOD mice generally exhibit a higher corticosterone response than C57BL/6 mice. The sexual dimorphism in diabetes expression in NOD mice may be related to the levels of corticosterone, a hyperglycemic hormone, in both basal and stress conditions. However, the understanding of corticosteroid effects in this model of type I IDDM is rather complex given their well known anti-inflammatory and immunosuppressive effects in other models of autoimmune diseases.

Metabolic and vascular consequences of diabetes mellitus induce several CNS complications. The dentate gyrus of the hippocampus, a well-recognized target for diabetic alterations, is a neurogenic area associated with memory and learning processes. Here, we explored the hippocampal neurogenesis and its microenvironment (astrocytes, vascularisation and glucocorticoid influence) in a spontaneous model of type 2 diabetes, the Goto-Kakizaki rat. The number of proliferative Ki67(+) cells and young doublecortin(+) neurons was 2-fold higher in the hippocampus from diabetic rats than in normoglycemic control Wistar at 4 months of age. However, there was no difference in cell survival, studied 3 weeks after bromodeoxyuridine administration. Labeling of endothelial cells against von Willebrand factor, demonstrated a 50% decrease in the granular cell layer fractional area covered by blood vessels and a diminished capillary branching in diabetic rats. Finally, Goto-Kakizaki rats exhibited decreased glucocorticoid receptor immunolabeling in CA1, associated with higher corticosteronemia. In conclusion, diabetic rats showed increased cell proliferation and neuronal differentiation without concomitant survival modification. A high proliferation rate, potentially reflecting a compensatory mechanism for neuronal suffering, also exists in various pathological situations. However, endothelial alteration induced by chronic hyperglycemia, hyperleptinemia and insulin resistance and associated with deleterious glucocorticoid effects might impair effective neurogenesis in diabetic Goto-Kakizaki rats. Fil: Beauquis, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina Fil: Homo Delarche, Françoise. Universite Paris Diderot - Paris 7; Francia Fil: Giroix, Marie Heléne. Universite Paris Diderot - Paris 7; Francia Fil: Ehses, Jan. University Hospital of Zürich; Suiza Fil: Coulaud, Josiane. Universite Paris Diderot - Paris 7; Francia Fil: Roig, Paulina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina Fil: Portha, Bernard. Universite Paris Diderot - Paris 7; Francia Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina Fil: Saravia, Flavia Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina

BACKGROUND:Inflammation followed by fibrosis is a component of islet dysfunction in both rodent and human type 2 diabetes. Because islet inflammation may originate from endothelial cells, we assessed the expression of selected genes involved in endothelial cell activation in islets from a spontaneous model of type 2 diabetes, the Goto-Kakizaki (GK) rat. We also examined islet endotheliuml/oxidative stress (OS)/inflammation-related gene expression, islet vascularization and fibrosis after treatment with the interleukin-1 (IL-1) receptor antagonist (IL-1Ra). METHODOLOGY/PRINCIPAL FINDINGS:Gene expression was analyzed by quantitative RT-PCR on islets isolated from 10-week-old diabetic GK and control Wistar rats. Furthermore, GK rats were treated s.c twice daily with IL-1Ra (Kineret, Amgen, 100 mg/kg/day) or saline, from 4 weeks of age onwards (onset of diabetes). Four weeks later, islet gene analysis and pancreas immunochemistry were performed. Thirty-two genes were selected encoding molecules involved in endothelial cell activation, particularly fibrinolysis, vascular tone, OS, angiogenesis and also inflammation. All genes except those encoding angiotensinogen and epoxide hydrolase (that were decreased), and 12-lipoxygenase and vascular endothelial growth factor (that showed no change), were significantly up-regulated in GK islets. After IL-1Ra treatment of GK rats in vivo, most selected genes implied in endothelium/OS/immune cells/fibrosis were significantly down-regulated. IL-1Ra also improved islet vascularization, reduced fibrosis and ameliorated glycemia. CONCLUSIONS/SIGNIFICANCE:GK rat islets have increased mRNA expression of markers of early islet endothelial cell activation, possibly triggered by several metabolic factors, and also some defense mechanisms. The beneficial effect of IL-1Ra on most islet endothelial/OS/immune cells/fibrosis parameters analyzed highlights a major endothelial-related role for IL-1 in GK islet alterations. Thus, metabolically-altered islet endothelium might affect the beta-cell microenvironment and contribute to progressive type 2 diabetic beta-cell dysfunction in GK rats. Counteracting islet endothelial cell inflammation might be one way to ameliorate/prevent beta-cell dysfunction in type 2 diabetes.

Recent studies suggest an inflammatory process, characterized by local cytokine/chemokine production and immune cell infiltration, regulates islet dysfunction and insulin resistance in type 2 diabetes. However, the factor initiating this inflammatory response is not known. Here, we characterized tissue inflammation in the type 2 diabetic GK rat with a focus on the pancreatic islet and investigated a role for IL-1. GK rat islets, previously characterized by increased macrophage infiltration, displayed increased expression of several inflammatory markers including IL-1β. In the periphery, increased expression of IL-1β was observed primarily in the liver. Specific blockade of IL-1 activity by the IL-1 receptor antagonist (IL-1Ra) reduced the release of inflammatory cytokines/chemokines from GK islets in vitro and from mouse islets exposed to metabolic stress. Islets from mice deficient in IL-1β or MyD88 challenged with glucose and palmitate in vitro also produced significantly less IL-6 and chemokines. In vivo, treatment of GK rats with IL-1Ra decreased hyperglycemia, reduced the proinsulin/insulin ratio, and improved insulin sensitivity. In addition, islet-derived proinflammatory cytokines/chemokines (IL-1β, IL-6, TNFα, KC, MCP-1, and MIP-1α) and islet CD68 + , MHC II + , and CD53 + immune cell infiltration were reduced by IL-1Ra treatment. Treated GK rats also exhibited fewer markers of inflammation in the liver. We conclude that elevated islet IL-1β activity in the GK rat promotes cytokine and chemokine expression, leading to the recruitment of innate immune cells. Rather than being directly cytotoxic, IL-1β may drive tissue inflammation that impacts on both β cell functional mass and insulin sensitivity in type 2 diabetes.

The nonobese diabetic (NOD) mouse is a recognized model for studying immunologically-mediated insulin-dependent diabetes mellitus. The disease appears with a greater preponderance in females than in males. Castration at weaning led to a significant increase in the prevalence of diabetes in NOD males, whereas a tendency to a decreased prevalence was observed in NOD females. Castration combined with thymectomy was found to further increase the prevalence of diabetes in NOD males, whereas in females castration reversed the effect of thymectomy. These results on changes in diabetes prevalence were corroborated by the degree of lymphocytic infiltration directed toward the pancreatic islets of Langerhans. Taken together these results indicate a direct relationship between the endocrine and immune systems, whereby orchidectomy has a deleterious effect on the immunopathogenesis of diabetes. In addition, we examined whether the distribution of lymphocyte subpopulations, mitogen reactivity, lymphokine production, and in vivo response to a thymus-dependent antigen, such as sheer red blood cell, were dependent or independent of the sex steroid environment.

One of the expressions of the activity of phagocytic cells such as monocytes or macrophages in a burst of increased oxidative activity on stimulation. The free oxygen radicals liberated, mainly O − 2 and H 2 O 2 , lead to chemoluminescence, which is thus a measure of activation. Chemoluminescence also depends on arachidonic acid metabolism, and this depends on phospholipase A 2 (PLA 2 ). We modified monocyte activity in monkeys by injecting them i.v. with this enzyme and observed that 30 min after injection, the phagocytic activity of peripheral blood monocytes and the chemoluminescence they emitted was greater than that of controls. We suggest that PLA 2 may act as an in vivo immunomodulator in mammals.

Interest in the field of neuroimmunoendocrinology is in full expansion. With regard to this, steroid influence on the immune system, in particular sex steroids and glucocorticoids, has been known for a long time. Sex steroids are part of the mechanism underlying the immune sexual dimorphism, as particularly emphasized in autoimmune diseases. Immunosuppressive and anti-inflammatory effects of glucocorticoids are now considered a physiological negative feedback loop to cytokines produced during an immune and/or inflammatory response. Psychosocial factors may play a role in the development of immunologically-mediated diseases, e.g. autoimmune diseases. The nonobese diabetic (NOD) mouse, that develops an immunologically-mediated insulin-dependent diabetes mellitus (IDDM) is an interesting model to study the role of endogenous steroids. Insulitis is present in both sexes, but diabetes has a strong preponderance in females. Hormonal alteration, such as castration, modulates the incidence of diabetes, whereas environmental factors, such as stress, accelerate the disease. In the present paper, we have reviewed the role of gender, sex steroid hormones, stress and glucocorticoids in autoimmunity as well as analyzed their different levels of actions and interrelationships, focusing particular attention on the immunologically-mediated IDDM of the NOD mouse.

Increasing evidence indicates that decreased functional beta-cell mass is the hallmark of type 2 diabetes (T2D) mellitus. Nowadays, the debate focuses on the possible mechanisms responsible for abnormal islet microenvironment, decreased beta-cell number, impaired beta-cell function, and their multifactorial aetiologies. This review is aimed to illustrate to what extend the Goto-Kakizaki rat, one of the best characterized animal models of spontaneous T2D, has proved be a valuable tool offering sufficient commonalities to study these aspects. We propose that the defective beta-cell mass and function in the GK model reflect the complex interactions of multiple pathogenic players: (i) several independent loci containing genes responsible for some diabetic traits (but not decreased beta-cell mass); (ii) gestational metabolic impairment inducing an epigenetic programming of the pancreas (decreased beta-cell neogenesis and/or proliferation) which is transmitted to the next generation; and (iii) loss of beta-cell differentiation due to chronic exposure to hyperglycemia/hyperlipidemia, inflammatory mediators, oxidative stress and to perturbed islet microarchitecture.

Type 1 diabetes (T1D) is linked to an 'encephalopathy' explained by some features common to the aging process, degenerative and functional disorders of the central nervous system. In the present study we describe a manifest hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis in two different experimental mouse models of T1D including the pharmacological one induced by streptozotocin and the spontaneous NOD (nonobese diabetic mice). The high expression of hypothalamic hormones like oxytocin and vasopressin were part to this alteration, together with elevated adrenal glucocorticoids and prominent susceptibility to stress. In the hippocampus of diabetic animals a marked astrogliosis, often associated with neural damage, was present. Dentate gyrus neurogenesis was also affected by the disease: proliferation and differentiation measured by bromodeoxyuridine immunodetection were significantly reduced in both experimental models used. Several facts, including changes associated with chronic hyperglycemia, hyperstimulation of the HPA axis, increased levels of circulating glucocorticoids in combination with brain inflammation and low production of new neurons, contribute to emphasize the impact of diabetes on the central nervous system. Fil: Beauquis, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Homo Delarche, Francoise. Université Paris Diderot - Paris 7; Francia. Inserm; Francia Fil: Revsin, Yanina. Leiden University; Países Bajos Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina Fil: Saravia, Flavia Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina

Steroids secreted from peripheral endocrine glands and acting on the brain are called neuroactive steroids. Under physiological conditions, neuroactive steroids modulate multiple brain functions, and in aging, trauma or neurodegeneration their role varies from neuroprotective to neurotoxic depending on the chemical properties of the steroid. In this regard, it is known that excess levels of adrenal steroids (gluco and mineralocorticoids) sensitize the hippocampus to the deleterious effects of a pathological environment, whereas the sex hormone estradiol is a powerful hippocampal neuroprotectant. We studied the protective role of estrogens in the ailing hippocampus in animal models of aging and age-associated diseases such as diabetes mellitus and hypertension of genetic (SHR) or mineralocorticoid origin. These models present in common a glucocorticoid or mineralocorticoid overdrive, a process that exacerbates hippocampal neuropathology, according to: (1) decreased proliferation of neuronal progenitors in the subgranular zone (SGZ) of the dentate gyrus (DG); (2) astroglial reactivity, with increased expression of the glial fibrillary acidic protein (GFAP); and (3) decreased neuronal density in the hilus of the DG. These pathological changes were reversible by treatment with estrogens, which ameliorated the hippocampal parameters in middle age mice and models of diabetes and hypertension. Thus, estradiol treatment stimulated progenitor proliferation in the SGZ, normalized the density of GFAP+ astrocytes, and avoided the loss of hilar neurons. Given the important role of the hippocampus in learning, memory and neuroendocrine events, estrogens may offer therapeutic advantages for the treatment of brain disturbances accompanying systemic diseases and aging. Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Pietranera, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Beauquis, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Homo Delarche, Francoise. Universite de Paris; Francia Fil: Saravia, Flavia Eugenia. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Arachidonic acid (AA) can be released from membrane phospholipids by the action of phospholipase A2 (PLA2). There is evidence that unsaturated fatty acids, particularly AA, released from membrane phospholipids are required to activate the respiratory burst of macrophages. The data reported here indicate that peritoneal macrophages harvested 30 min after i.p. injection of PLA2 can phagocytose Candida albicans more efficiently and emit more chemoluminescence (CL) than normal cells when stimulated by zymosan. PLA2 injection also enhances the CL of peritoneal cells from mice already stimulated by immunomodulators such as trehalose dimycolate (TDM), bestatin, or oncostatic drugs such as aclacinomycin (ACM). CL is not sensitive to potassium cyanide (KCN), but is inhibited by catalase, superoxide dismutase (SOD), nordihydroguaiaretic acid (NDGA) and high doses of indomethacin (10(-3) M). In vivo PLA2 treatment stimulates the synthesis of both cyclooxygenase and lipoxygenase derivatives of AA metabolism (PGE2, 6-keto, PGF1 alpha TXB2 and LTC4). Inhibitors of AA metabolism (NDGA, indomethacin) modulate the production of free oxidizing radicals in this experimental model, partly because of their effect on AA metabolism, as determined by the measuring immunoreactive products. However, this work indicates that the effects of these inhibitors, which have been extensively used in CL studies, should be interpreted with caution, since their specificity for AA metabolism is relative.

Prostaglandins (PGs) have been assumed to play a role in the biological activity of thymic hormones (TH). Indeed, it has been shown that type E-PGs are able to mimic the action of several TH. Moreover, indomethacin interferes in the rosette assay, which still represents the most commonly used bioassay for the evaluation of TH and, in particular thymulin levels, in biological fluids. Previously, our attempt to modulate PG production by different TH showed that none of the TH tested affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized (Tx) mice, while indomethacin was able to inhibit the spontaneous PG production. Here, we investigated a possible role for each endogenously produced PG in the experimental conditions of the rosette assay, in order to define: 1) whether or not there was a specificity of action of a given PG; and 2) to analyze the pattern of action between thymulin and the endogenously produced PGs. We demonstrated that PGE2 and 6-keto-PGF1 alpha are the PGs which are physiologically involved in the rosette assay, according to their levels of endogenous production, and that they are able to synergize with thymulin. This synergy was demonstrated in two ways: 1) by adding anti-PGE2 and anti-6-keto PGF1 alpha-antibodies, which prevent part of the thymulin effect, or 2) by simultaneous addition of PG and thymulin, at concentrations far lower than those which correspond to their thymulin-like effect. Moreover, PGE2 addition, at concentration close to that found to be endogenously produced, partially reversed the indomethacin-induced effect in the rosette assay. In conclusion, if PGs do not act as mediators of thymulin, they are able to synergize in one of its biological action.

Male and female mice from different control strains (C57BL/6, DBA/2, BALB/c) and the nonobese diabetic (NOD) strain, a spontaneous model of type 1 diabetes, were subjected to various stressors (restraint, lipopolysaccharide or interleukin-1 injection). Significant differences were measured among strains in blood glucose, insulin and corticosterone levels and, for restraint, IL-6. Addition of dexamethasone, a glucocorticoid receptor agonist, to inhibit the expression of several proteins by LPS-stimulated bone marrow-derived macrophages in vitro showed a gradient among control strains: C57BL/6>DBA/2>BALB/c corroborating the pattern of corticosensitivity suggested by their stress-induced glucose responses at the systemic level.

The effects of bromocriptine (10 mg/kg), known to inhibit prolactin secretion and lower autoimmune processes, were studied on glucose homeostasis in non-fasted non-obese diabetic mice, a spontaneous model of type 1 diabetes. Hyperglycemia was observed 120 and 240 min after i.p. but not s.c. injection. Bromocriptine administration i.p. led to rapid and marked hyperglycemia characterized by sexual dimorphism with males having higher glycemia than females. Bromocriptine induced a rapid but transient decrease in insulinemia in males only and biphasic increases in glucagon levels and a sustained stimulatory effect on circulating corticosterone in both sexes. Bromocriptine-induced hyperglycemia involved D2-dopaminergic receptors, as demonstrated by the inhibitory effect of the D2-dopamine antagonist, metoclopramide (10 mg/kg). Simultaneous injection of bromocriptine and metoclopramide also blocked the rise in blood corticosterone. In conclusion, by inducing hyperglycemia, i.p. bromocriptine administration to prediabetic autoimmune mice may counteract its beneficial anti-immunostimulatory effects.

Activation of the innate immune system in obesity is a risk factor for the development of type 2 diabetes. The aim of the current study was to investigate the notion that increased numbers of macrophages exist in the islets of type 2 diabetes patients and that this may be explained by a dysregulation of islet-derived inflammatory factors. Increased islet-associated immune cells were observed in human type 2 diabetic patients, high-fat–fed C57BL/6J mice, the GK rat, and the db/db mouse. When cultured islets were exposed to a type 2 diabetic milieu or when islets were isolated from high-fat–fed mice, increased islet-derived inflammatory factors were produced and released, including interleukin (IL)-6, IL-8, chemokine KC, granulocyte colony-stimulating factor, and macrophage inflammatory protein 1α. The specificity of this response was investigated by direct comparison to nonislet pancreatic tissue and β-cell lines and was not mimicked by the induction of islet cell death. Further, this inflammatory response was found to be biologically functional, as conditioned medium from human islets exposed to a type 2 diabetic milieu could induce increased migration of monocytes and neutrophils. This migration was blocked by IL-8 neutralization, and IL-8 was localized to the human pancreatic α-cell. Therefore, islet-derived inflammatory factors are regulated by a type 2 diabetic milieu and may contribute to the macrophage infiltration of pancreatic islets that we observe in type 2 diabetes.

Cerebral dysfunctions, including a high incidence of depression, are common findings in human type 1 diabetes mellitus. An association between depression and defective hippocampal neurogenesis has been proposed and, in rodents, antidepressant therapy restores neuronal proliferation in the dentate gyrus. Hippocampal neurogenesis is also deficient in diabetic mice, which led us to study whether the selective serotonin reuptake inhibitor fluoxetine influences cell proliferation in streptozotocin-diabetic animals. Diabetic and control C57BL/6 mice received fluoxetine (10 mg/kg/day, i.p., 10 days) and dentate gyrus cell proliferation was measured after a single injection of 5-bromo-2'-deoxyuridine (BrdU). Diabetic mice showed reduced cell proliferation. Fluoxetine treatment, although having no effect in controls, corrected this parameter in diabetic mice. The phenotype of newly generated cells was analysed by confocal microscopy after seven daily BrdU injections, using Tuj-1/beta-III tubulin as a marker for immature neurones and glial fibrillary acidic protein for astrocytes. In controls, the proportion of Tuj-1-BrdU-positive cells over total BrdU cells was approximately 70%. In vehicle-treated diabetic mice, immature neurones decreased to 56% and fluoxetine brought this proportion back to control values without affecting astrocytes. Therefore, fluoxetine preferentially increased the proliferation of cells with a neuronal phenotype. In addition, neurones were counted in the hilus of the dentate gyrus; a 30% decrease was found in diabetic mice compared with controls, whereas this neuronal loss was prevented by fluoxetine. In conclusion, fluoxetine treatment restored neuroplasticity-related hippocampal alterations of diabetic mice. These findings may be potentially important to counteract diabetes-associated depression in humans.

Article suite au 41st Annual Meeting of the European-Association-for-the-Study-of-Diabetes. 10-15 September 2005, Athens, Greece; International audience; The molecular pathways leading to islet fibrosis in diabetes are unknown. Therefore, we studied gene expression in islets of 4-month-old Goto-Kakizaki (GK) and Wistar control rats. Of 71 genes found to be overexpressed in GK islets, 24% belong to extracellular matrix (ECM)/cell adhesion and 34% to inflammatory/immune response families. Based on gene data, we selected several antibodies to study fibrosis development during progression of hyperglycemia by immunohistochemistry. One-month-old GK and Wistar islets appeared to be similar. Two-month-old GK islets were strongly heterogenous in terms of ECM accumulation compared with Wistar islets. GK islet vascularization, labeled by von Willebrand factor, was altered after 1 month of mild hyperglycemia. Numerous macrophages (major histocompatibility complex class II(+) and CD68(+)) and granulocytes were found in/around GK islets. These data demonstrate that marked inflammatory reaction accompanies GK islet fibrosis and suggest that islet alterations in this nonobese model of type 2 diabetes develop in a way reminiscent of microangiopathy.

Type 1 diabetes mellitus correlates with several brain disturbances, including hypersensitivity to stress, cognitive impairment, increased risk of stroke and dementia. Within the central nervous system, the hippocampus is considered a special target for alterations associated with diabetes. Neurogenesis is a plastic event restricted to few adult brain areas: the subgranular zone of the dentate gyrus and the subventricular zone (SVZ). First, we studied the ability for neurogenesis in the dentate gyrus and SVZ of chronic diabetic mice induced by streptozotocin (STZ). Using bromodeoxyuridine (BrdU) labelling of cells in the S-phase, we observed a strong reduction in cell proliferation rate in both brain regions of diabetic mice killed 20 days after STZ administration. Second, because oestrogens are active neuroprotective agents, we investigated whether 17beta-oestradiol (200 micro g pellet implant in cholesterol during 10 days) restored brain cell proliferation in the diabetic mouse brain. Our results demonstrated a complete reversibility of dentate gyrus cell proliferation in oestrogen-treated diabetic mice. This plasticity change was not exclusive to the hippocampus because oestrogen treatment restored BrdU incorporation into newborn cells of the SVZ region of diabetic animals. Oestrogen treatment did not alter the hyperglycemic status of STZ-diabetic mice. Moreover, oestrogen did not modify BrdU incorporation in control animals. These data show that oestrogen treatment strongly stimulates brain neurogenesis of diabetic mice and open up new venues for understanding the potential neuroprotective role of steroid hormones in diabetic encephalopathy. Fil: Saravia, Flavia Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina Fil: Revsin, Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lux, Victoria Adela R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Beauquis, Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Homo Delarche, Francoise. Inserm; Francia Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina

The influence of diabetes mellitus on brain pathology is increasingly recognized. Previous contributions of our laboratory demonstrated in models of type 1 diabetes (nonobese diabetic and streptozotocin (STZ)-treated mice), a marked astrogliosis and neurogenesis deficit in hippocampus and increased expression of hypothalamic neuropeptides. In the present investigation, we further analyzed alterations of astroglia and neurons in the hippocampus of mice 1 month after STZ-induced diabetes. Results showed that these STZ-diabetic mice presented: (a) increased number of astrocytes positive for apolipoprotein-E (Apo-E), a marker of ongoing neuronal dysfunction; (b) abnormal expression of early gene products associated with neuronal activation, including a high number of Jun + neurons in CA1 and CA3 layers and dentate gyrus, and of Fos-expressing neurons in CA3 layer; (c) augmented activity of NADPH-diaphorase, linked to oxidative stress, in CA3 region. These data support the concept that uncontrolled diabetes leads to hippocampal pathology, which adjoin to changes in other brain structures such as hypothalamus and cerebral cortex. Fil: Revsin, Yanina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Saravia, Flavia Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina Fil: Roig, Paulina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lima, Analia Ethel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: de Kloet, E. Ronald. Leiden University; Países Bajos Fil: Homo Delarche, Francoise. Inserm; Francia Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina