Taurine supplement in early life altered islet morphology, decreased insulitis and delayed the onset of diabetes in non-obese diabetic mice
- 739 Downloads
We hypothesised that nutritional taurine, which is important for the development of the endocrine pancreas and reduces cytokine-induced apoptosis in pancreatic beta cells, would prevent or delay the onset of autoimmune diabetes, if given early in life to the non-obese diabetic (NOD) mouse.
Pregnant NOD mice received a diet supplemented with taurine throughout gestation or until weaning, and the pancreas of the offspring was examined using immunohistochemistry. This was done at postnatal day 14 and after 8 weeks (assessment of insulitis). The animals were also monitored until they became diabetic.
At 14 days, pancreatic islet mass was significantly greater in animals treated with taurine than in controls. This finding was associated with a greater incidence of islet cell proliferation and a lower incidence of apoptosis. At age 8 weeks the number of islets manifesting insulitis was reduced by more than half, and the area of insulitis was reduced by 90%. Taurine treatment delayed the mean onset time of diabetes from 18 to 30 weeks in females, and from 30 to 38 weeks in males, while 20% of treated females remained free of diabetes after one year.
Taurine supplementation in early life altered islet development, reduced insulitis and delayed the onset of diabetes in NOD mice.
KeywordsBeta cell Diabetes IGF-II Insulitis Islet NOD mouse Taurine
proliferating cell nuclear antigen
In the development of autoimmune diabetes the NOD mouse shares several immunopathogenic features with human Type 1 diabetes [1, 2]. In both, infiltration of the pancreatic islets by mononuclear leucocytes or insulitis precedes the selective destruction of islet beta cells . In NOD mice, diabetes is preceded by the progressive invasion of the pancreatic islets by CD4 and CD8 T cells and macrophages over a prolonged pre-diabetic period , with a discrepancy between the onset of insulitis and diabetes. We previously noted an altered developmental morphology in the endocrine pancreas of the diabetes-prone, female NOD mouse in early life and prior to the process of insulitis , suggesting that this might be a pre-disposing feature for diabetes. We therefore sought to alter pancreatic islet development in early life to increase beta cell mass using the amino acid taurine, which has been shown to have mitogenic and anti-apoptotic actions on beta cells, and through this determine if this delayed the onset of diabetes.
Taurine is a non-essential, sulfated amino acid generated from the precursors methionine and cysteine, and is a normal constituent of the diet [6, 7]. It is present in almost all mammalian tissues but is concentrated in certain tissues including the brain, pro-inflammatory cells and pancreas [8, 9]. Taurine is necessary for normal development, and defects in growth, tissue differentiation and immune development occur when taurine is deficient [10, 11]. The cellular actions of taurine are numerous, including the regulation of cell volume, extracellular and intracellular calcium mobilisation, and an inhibition of apoptosis in hepatocytes, endothelial cells and macrophages [6, 12, 13, 14, 15]. Taurine has trophic effects in the developing endocrine pancreas. When deficient, as seen during maternal protein restriction , taurine supplementation prevents a reduction in beta cell mass via enhanced beta cell proliferation, IGF-II expression and islet vascularisation, and by reducing islet cell apoptosis [17, 18, 19]. In addition, taurine added to the culture medium or to maternal low-protein diet prevented IL-1 and nitric oxide (NO)-induced apoptosis in isolated fetal islets while restoring insulin secretion [20, 21]. Low-protein diet in early life resulted in a long-term susceptibility of beta cells to cytokine-induced apoptosis in adult life, and this was reversed by diet supplementation with taurine .
The combination of developmental actions in the endocrine pancreas and the ability to suppress cytotoxic cytokine release from mononuclear cells make taurine a potential candidate for delaying or preventing the onset of Type 1 diabetes. The aim of this study was to determine if nutritional taurine supplementation to the NOD mouse in early life could alter the morphology of the developing endocrine pancreas, change the degree of insulitis in later life, and delay the onset of diabetes.
Materials and methods
Animals and treatments
Virgin NOD females of 4 to 5 weeks and male mice were obtained from the breeding colony at the Robarts Research Institute, London, Ontario, Canada and were allowed free access to food and water. All procedures were performed with ethical approval of the Animal Care Committee of the University of Western Ontario in accordance with the guidelines published by the Canadian Council on Animal Care. At 8 weeks of age breeding pairs were caged in individual cages and females were checked daily for vaginal plugs. When pregnancy was confirmed, 2.5% (w/v) of taurine was added to their drinking water until parturition or continued until weaning. The animals received normal Purina rat chow, and were housed in the Animal Care Facility at the Lawson Health Research Institute and maintained at 25 °C with a 12-h light/dark cycle. Pups were separated by sex at weaning, and killed either 14 days after birth or, in a second group, at 8 weeks of age by CO2 asphyxiation after an overnight fast. Pancreata were collected, weighed and fixed with 10% formalin in PBS for histology. Before death, blood was also collected from the tail vein at these times points, and blood glucose measured using 2 µl of total blood with a fast-take glucometer (Lifescan, Burnaby, BC, Canada). A third group of mice from six to eight different litters each of taurine-treated and control animals was monitored until the onset of diabetes. The primary indicator of diabetes was polyurea on two consecutive days with a confirmation of glucosurea (6–14 mmol/l) using urine glucose strips (Diastix, Ames, Toronto, Canada). Diabetes was confirmed by a blood glucose value in excess of 11 mmol/l using blood drawn from the tail vein. Once diabetic, the animals were killed, blood glucose again recorded and the pancreas removed for histological examination.
Histological sections of pancreas (5 µm) were cut from paraffin blocks and mounted on glass microscope slides (Superfrost Plus, fisher Scientific, Nepean, ON, Canada). Immunohistochemistry was performed on pancreas sections to localise insulin, glucagon, proliferating cell nuclear antigen (PCNA), CD3 and IGF-II within the islets by a modified avidin–biotin peroxidase method  using the following primary antibodies: guinea pig anti-insulin (1:50 dilution) and rabbit anti-porcine glucagon (1:100 dilution; C-terminal specific 04A antiserum) (provided by Dr T.J. McDonald, Department of Medicine, University of Western Ontario, Canada); mouse anti-PCNA (1:750 dilution) (Sigma Chemical, St. Louis, Mo., USA); rat monoclonal CD3 antibody (1:100 dilution) (Santa Cruz Biotechnology, Santa Cruz, Calif., USA); and rabbit anti-IGF-II (1:200 dilution) (GroPep, Adelaide, Australia). All antisera were diluted in 0.1 mol/l PBS (pH 7.5) containing 0.25% (w/v) bovine serum albumin, 0.3% (v/v) Triton X100, and 0.01% (w/v) sodium azide (100 µl per slide). Biotinylated horse anti-mouse, goat anti-rabbit and goat anti-guinea pig (1:100 dilution: Vector Laboratories, Burlingame, Calif., USA) were used as secondary antibodies. Peptide immunoreactivity was localised by incubation in fresh diaminobenzidine tetrahydrochloride (Biogenex, San Ramon, Calif., USA). Tissue sections were counter-stained with Carazzi’s haematoxylin or methyl green 0.01% in acetate buffer. Controls included substitution of primary antisera with non-immune serum, the omission of the secondary antiserum, and for insulin, glucagon and IGF-II, an absence of staining following pre-incubation of the antiserum with excess antigen.
Visualisation of apoptosis
Apoptosis was visualised by the TUNEL method, with the In Situ Cell Death Detection kit (fluorescein) from Roche Diagnostics, Germany, and by using a confocal microscope (MRC-1024 UV, Bio-Rad, Hemel Hempstead, UK) with FITC (excited at 488 nm and emission peak at 552 nm). Total nuclei were labelled with ethidium bromide (excited at 510 nm and emission peak at 595 nm).
Morphometric and statistical analysis
Pancreata from up to 20 animals from each sex, and from at least 3 to 4 different litters were examined at each age. Morphometric analysis was performed using a Carl Zeiss transmitted light microscope at a magnification of ×250 or ×400. Automatic image analysis of the pancreatic sections for calculation of tissue areas was performed with Northern Eclipse, version 6.0, morphometric analysis software (Empix Imaging, Mississauga, Ontario, Canada). The number of small (<5000 µm2) or large (>10,000 µm2) islets, and the percent of islet cells immunoreactive for insulin, glucagon, PCNA or IGF-II, or demonstrating apoptotic nuclei, were calculated for each group from 5 sections per pancreas taken at 50 to 60 section intervals to represent the entire pancreas at 14 days of age. For islet cell apoptosis and IGF-II presence at 14 days, only female pancreata were analysed. The minimum size for an islet was taken to be a cluster of three or more insulin-positive cells. Calculation of the percent of islet cells immunopositive for PCNA was also determined at 8 weeks of age using 5 sections per pancreas representing the head region. For the estimation of insulitis 5×5-µm sections were cut at 10-section intervals, stained with haematoxylin–eosin, and the area of insulitis calculated.
Differences between mean values for variables within individual experiments were compared statistically by two-way analysis of variance, and were considered to be significantly different at a p value of 0.05 or less. Values are given as mean values ± SEM. Differences in the conversion of NOD mice to diabetes with and without taurine supplementation were calculated from Kaplan–Meier cumulative plots and evaluated using the Wilcoxon test for equality of survival.
The offspring of NOD mice supplemented with dietary taurine during pregnancy and lactation were of similar birth size, gestational length and litter size to control-fed mice, and suckled normally. Similarly, at 8 weeks of age there were no differences in body or pancreatic weight between control and taurine-supplemented animals, when male and female animals were separately compared. Mean fasting blood glucose levels did not differ statistically between taurine-treated or control females (5.0±0.5 mmol/l vs 4.2±0.3 mmol/l, n=12) or males (4.6±0.3 mmol/l vs 4.5±0.4 mmol/l) at 8 weeks age.
Islet characteristics in pancreata from NOD mice at 14 days of age
Volume-weighted mean islet volume, (µm3×10−5)a
Islet mass, (µg)a
Islet beta cell area (%)
Exposure to taurine supplementation in utero and prior to weaning had long-lasting beneficial effects on beta cell survival in the NOD mouse model of autoimmune diabetes. It seems likely that these effects were due to an altered development of the islet cells, the peripheral immune system, or both. While it is well documented that dietary factors can either precipitate or delay the onset of diabetes in the NOD mouse, this is usually achieved with changes in complex proteins rather than an individual amino acid , and with treatment commencing after weaning.
Taurine supplementation in gestation resulted in a greater incidence of islet cell DNA synthesis and a greater islet cell mass in both sexes, an increased number of islet cells containing immunoreactive IGF-II, and a reduction in islet cell apoptosis in mice at 14 days after birth, and prior to any evidence of insulitis. We reported previously that taurine supplementation of the pregnant rat was able to reverse the loss of beta cell mass in the offspring caused by exposure to a low-protein diet, with a corresponding increase in islet cell proliferation, a reduction in Fas presence and rate of cell apoptosis, and increased presence of IGF-II . Multiple direct cellular signalling pathways might be involved. In retinal cells taurine enhanced proliferation by invoking Ca2+ flux , in macrophages a prevention of apoptosis was mediated by a reduction in inducible nitric oxide synthase and NF-kappa B , and in hepatocytes an activation of phosphatidylinositol 3-kinase led to inhibition of Fas-mediated apoptosis .
An indirect trophic action of taurine via the expression of islet IGF-II is possible, as was already observed and discussed in a previous study in which the maternal low-protein diet was supplemented with taurine . Changes in islet size distribution in the NOD neonatal mouse, as well as the altered trophic indicators, were more prominent in females. We previously found that NOD female mice differ in the ontogeny of the endocrine pancreas during neonatal life compared to males or control strains. The neonatal wave of developmental apoptosis within beta cells which peaks at postnatal day 11 in mice was significantly more pronounced in female NOD animals than in BALB/c controls . This resulted in the female NOD having a lower mean islet size with a relative deficiency in beta cells subsequent to this developmental remodelling. Similarly, in the present study islet cell mass was significantly smaller in control females than in males at postnatal day 14. We have postulated that the increased beta cell apoptotic activity seen in females might act as an early initiator of the autoimmune cascade .
Peri-insulitis is first found in the NOD female at around 4 to 5 weeks and affects 70 to 90% of islets by 9 to 10 weeks . When examined at 8 weeks of age, taurine-supplemented female mice had a 90% reduction in the mean area of insulitis per islet. This suggests that the immune infiltration within the islets was impaired, which could imply that recognition of the beta cell auto-antigens by macrophages or dendritic cells was reduced, leading to diminished T cell recruitment or action. Taurine is abundant in neutrophils, and taurine or taurine chloramines, which are produced during inflammation by reaction of hypochlorous acid with taurine, reduce inflammation and the number of inflammatory cells [28, 29]. Despite discontinuation of taurine supplementation at three weeks of age, insulitis was reduced at 8 weeks, suggesting that the metabolic consequences of increased taurine availability were prolonged, and/or that the phenotypes of either the beta cells or sentinel macrophages/dendritic cells within islets had been changed. At 8 weeks of age taurine-supplemented female mice also demonstrated an increase in the number of islet cells undergoing DNA synthesis, which might indicate an attempt at regeneration to maintain beta cell mass. Since an increased incidence of DNA synthesis was also seen in the pancreatic ductal epithelium, such a regenerative response might also involve neogenesis of new endocrine cells from ductal precursors. No increase in the incidence of islet cell DNA synthesis was seen in male NOD mice previously supplemented with taurine at 8 weeks of age. Since the onset of diabetes is relatively delayed in males, this may reflect a lower beta cell loss and smaller requirement for a regenerative response.
The onset of diabetes in the female NOD mouse occurs at 11 to 15 weeks in most colonies (range 4–25 weeks), with a mean incidence of conversion of 19 weeks (range 7–37 weeks) . While male mice develop insulitis at the same time as females, the onset of diabetes is delayed (mean 14 weeks, range 6–25 weeks), and the peak incidence of conversion is 23 weeks, although all males ultimately become diabetic . In this study control females first became diabetic at 10 weeks, and males at 18 weeks, which is more rapid than in many colonies, but well within the range of experience. The retardation in peak onset of diabetes caused by taurine supplementation was over 12 weeks in females and 8 weeks in males. This, together with the lack of diabetes in 1 in 5 female NOD mice after 1 year, subsequent to taurine exposure in early life, is unlikely to be explained by transient metabolic effects of taurine, and suggests a long-term change either in beta cell responsiveness to autoimmune attack or in autoimmune inter-cellular communication.
In conclusion, the findings show that taurine supplementation in fetal and neonatal life delays the onset of diabetes in the NOD mouse and is associated with an altered beta cell development prior to the onset of insulitis.
We are grateful to the Canadian Diabetes Association, the Juvenile Diabetes Research Foundation, the Canadian Institutes of Health Research, The Stem Cell Network Centre of Excellence, the Ontario Research and Development Challenge Fund, Fond National de la Recherche Scientifique of Belgium, and the Parthenon Trust, London, UK for financial support. We thank Catherine Currie for technical support and Dr Sandra Thyssen for help in statistical analysis.
- 6.Huxtable RJ (1992) Physiological actions of taurine. Physiol Rev 72:101–163Google Scholar
- 7.Huxtable R, Fanconi F, Gironi A (1987) The biology of taurine. Methods and mechanisms. Plenum Press, New YorkGoogle Scholar
- 9.Bustamente J, Lobo MVT, Alonso NTA et al. (2001) An osmotic sensitive taurine pool is located in rat pancreatic islet cells containing glucagon and somatostatin. Am J Endocr Metab 281:E1275–E1285Google Scholar
- 11.Schuller-Levis G, Mehta PD, Rudelli R, Sturman J (1990) Immunological consequences of taurine deficiency in cats. J Leucocyte Biol 47:321–331Google Scholar
- 16.Reusens B, Dahri S, Snoeck A et al. (1995) Long term consequences of diabetes and its complications may have a fetal origin: experimental and epidemiological evidences. In: Cowett RM (ed.) Nestlé Nutrition Workshop Series, vol 25. Raven Press, New York, pp 187–198Google Scholar
- 17.Petrik J, Reusens B, Arany E, Remacle C, Hoet JJ, Hill DJ (1999) A low protein diet alters the balance of islet cell replication and apoptosis in the fetal and neonatal rat, and is associated with a reduced pancreatic expression of insulin-like growth factor-II. Endocrinology 140:4861–4873CrossRefPubMedGoogle Scholar
- 22.Merezak S, Reusens B, Renard A et al. (2004) Early low protein diet increases the vulnerability of rat adult rat islet to cytokines: preventive role of taurine. Diabetologia 47:669–675Google Scholar