Did the gradual loss of GLUT2 cause a shift to diabetic disorders in the New Zealand obese mouse (NZO/Hl)?

The New Zealand obese mouse (NZO/Hl) is characterised by hereditary obesity and type-2 diabetes, including insulin resistance, hyperinsulinaemia, and glucose intolerance. In other diabetic models, it has been revealed that the proper functioning of the glucose transporter isoform 2 (GLUT2) is essential for adequate secretion of insulin. The aim of this study was to compare the distribution of islet cells and GLUT2, as well as the expression of GLUT2-mRNA, in the pancreas of NZO mice and metabolically unimpaired NMRI (Naval Medical Research Institute) mice. Pancreas tissue was obtained from different stages of development. For molecular determination of the expression level of GLUT2-mRNA, total-RNA was extracted from the pancreas and analysed by quantitative real-time RT-PCR. All investigated NZO mice displayed increased weight, elevated hyperinsulinaemia, and slightly enhanced blood glucose levels compared with the NMRI control mice. By means of immunofluorescence microscopy drastically reduced insulin levels were detected, which might be compensated by the observed islet cell hyperplasia and hypertrophy. Furthermore, the normally peripheral localisation of the alpha-cells within islets was disturbed. By contrast, there were no changes in somatostatin cell distribution. However, considerable differences appeared with regard to GLUT2: whereas the beta-cells of NMRI mice showed dense immunostaining of the GLUT2 transporter on the cell surface, in all age groups of NZO mice, GLUT2 on the plasma membranes was reduced and dispersed in the cytoplasm. These findings agree with the molecular biological results, which displayed decreased mRNA-expression of GLUT2. In summary, the observed alteration of islet morphology and of GLUT2 expression in diabetic mice complements our previous results from a superfusion protocol and further clarifies the mechanisms of diabetogenesis in NZO mice.

Alpha2-adrenoceptor antagonism is neither sufficient nor necessary for the distinctive action of atypical neuroleptics on intracranial self-stimulation in the rat.

Response rates in variable-interval intracranial self-stimulation (ICSS) in rats can provide a continuous record of drug-induced changes in brain function. Use of this procedure has been found to distinguish between typical and atypical neuroleptics, with the latter producing a similarly intense but much briefer depression of responding. This difference has been ascribed to the alpha2-adrenoceptor antagonist properties of atypical neuroleptics, but the evidence is ambiguous. The role of alpha2-adrenoceptors was examined in the present study using ventral tegmental ICSS to track the depressant effects of the typical and atypical neuroleptics, haloperidol (0.075 mg/kg) and olanzapine (0.9 mg/kg), injected alone or in combination with an alpha2-adrenoceptor agonist or antagonist. Neither haloperidol nor olanzapine (despite its atypical features) shows appreciable affinity for the alpha2-adrenoceptor. In the present study, olanzapine was found to depress self-stimulation responding dose-dependently, but with considerable recovery after 4 h. Simultaneous administration of alpha2-adrenoceptor receptor agonists or antagonists (respectively clonidine 0.015 mg/kg or idazoxan 3.0 mg/kg) failed significantly to increase or decrease the action of either haloperidol or olanzapine. These results indicate that alpha2-adrenoceptor antagonism does not necessarily promote recovery from neuroleptic-induced depression, and that 'atypical' features do not necessarily depend on alpha2-adrenoceptor antagonism. Various other explanations remain possible, but the accelerated time course of the atypical agents appears to support more recent explanations, based on their rapid dissociation from the D2 receptor.

Genotypic and phenotypic characterization of six new recombinant congenic strains derived from NOD/Shi and CBA/J genomes.

Recombinant Congenic Strains (RCS) are useful for dissecting complex polygenic traits. Here, we describe genetic and phenotypic characterization of six new RCS generated from outcrosses between NOD/Shi and CBA/LsLt, followed by sib mating of first backcross progeny (to CBA) for 20 generations, whereupon genetic and phenotypic analysis commenced. Four of the RCS were selected on the basis of residual heterozygosity present at F20 in one of the three original RCS. Contrary to expectations for RCS developed at first backcross, all derived at least 50% of the polymorphic markers typed from the NOD parental strain. Development of autoimmune insulin-dependent diabetes mellitus (IDDM) in NOD is a strain-specific characteristic. The major genetic component predisposing NOD mice to IDDM, their H2(g7) haplotype, was present in all RCS. Nevertheless, the presence of variable amounts of CBA genome at non-MHC loci conferred complete resistance in all RCS to spontaneous IDDM development, and rendered them strongly resistant to cyclophosphamide-induced IDDM. Although the RCS more resemble NOD in regard to certain strain-specific characteristics, such as prolificacy, an immunologic phenotype that was significantly reduced when compared to both parental strains was the number of peripheral CD8(+) T cells. Given the genetic characterization presented, these new RCS should prove valuable to investigators interested in studying genes controlling differential susceptibilities distinguishing the NOD and CBA inbred strain backgrounds.

Rapid recovery of self-stimulation responding from depression by clozapine is prevented by the alpha 2-adrenoceptor agonist, clonidine.

Typical and atypical antipsychotic agents were tested on rats responding for variable-interval electrical stimulation of the ventral tegmental area (VTA). The typical neuroleptics, chlorpromazine and haloperidol, led to prolonged depression of responding lasting at least 4 h, whereas response rates after similarly effective doses of the atypical agents, clozapine and risperidone, recovered to control levels in the same period. The role of alpha 2-adrenoceptors in producing these differences was investigated by administering clozapine together with an alpha 2-adrenoceptor agonist, clonidine, and chlorpromazine together with an alpha 2-adrenoceptor antagonist, idazoxan. The addition of clonidine extended the response-depressant activity of clozapine, resulting in prolonged depression comparable to that produced by chlorpromazine or haloperidol. Conversely, the addition of idazoxan to chlorpromazine shortened the duration of chlorpromazine's suppressant action to a level comparable to that of clozapine or risperidone. These results suggest that the brevity of clozapine's effects on operant behaviour (a feature which may be related to its reduced liability to extrapyramidal side-effects) may be a consequence of its alpha 2-adrenoceptor antagonist properties.

Hyperleptinemia and leptin receptor variant Asp600Asn in the obese, hyperinsulinemic KK mouse strain.

KK obese mice exhibit a multigenic syndrome of moderate obesity, hyperinsulinemia and hyperglycemia. Here we show that the syndrome is accompanied by a marked elevation of leptin protein in adipose tissue, as well as leptin levels in serum, which corresponds with the degree of obesity. The cDNA sequence of leptin is normal in KK mice, whereas three nucleotide polymorphisms were found in the cDNA of the leptin receptor, one of them resulting in exchange of an aspartate residue for asparagine (Asp600Asn) in a highly conserved part of the second extracellular cytokine-receptor homology module. In female (but not male) F2 mice of a C57BL/6JxKK intercross, the weight of gonadal, retroperitoneal and mesenteric adipose tissue was positively correlated with the number of alleles inherited from the KK parental strain at a microsatellite marker (D4Mit175) which maps close (0.7 centimorgan proximal) to the leptin receptor gene. It is suggested that the Asp600Asn leptin receptor variant contributes to the obesity syndrome in KK female mice, but that its contribution is only a part of the multigenic syndrome.

NIDDM genes in mice: deleterious synergism by both parental genomes contributes to diabetogenic thresholds.

We used mouse genetics to model how polygenic thresholds for the transition from impaired glucose tolerance (IGT) to NIDDM are reached. NON/Lt and NZO/Hl are inbred mouse strains selected for IGT and polygenic obesity, respectively. Their F1 male progeny consistently developed NIDDM. Genetic analysis of F2 males from both cross directions identified an NON-derived diabetogenic locus, Nidd 1, on chromosome (Chr) 4 near the leptin receptor. This locus was associated with reduced plasma insulin, increased non-fasted blood glucose, and lower body weight. Another NON-derived diabetogenic locus on Chr 18 (Nidd2) that controls blood glucose was identified. An NZO-derived diabetogenic region on Chr 11 (Nidd3), possibly comprising two separate loci, reduced ability to sustain elevated plasma insulin and significantly reduced weight gain over time. Thus, the diabetogenic synergism between genetic loci from strains separately exhibiting subthreshold defects perturbing glucose homeostasis underscores the likely complexity of the inheritance of obesity-associated forms of NIDDM in humans.

Long-term effects of CCK-agonist and -antagonist on food intake and body weight in Zucker lean and obese rats.

The present study evaluates long-term effects of the CCK-agonist caerulein and the CCK-A antagonist loxiglumide in obese and lean Zucker rats. Caerulein and loxiglumide altered food intake neither in obese nor in lean rats. By as yet unknown mechanisms, however, weight increase was accelerated by loxiglumide and reduced by caerulein in obese and lean rats. Caerulein increased pancreatic weight only in lean but not in obese rats. Thus, obese rats show a resistance of pancreatic CCK-A receptors. The failure of CCK-agonist and -antagonist to alter food intake suggests that this CCK-resistance is not responsible for obesity in the genetically altered rats.

Apoptosis in the adrenal gland of non-obese diabetic (NOD) mice.

Apoptosis appears to play an important role in the development of diabetes in the non-obese diabetic (NOD) mouse. Since the autoimmune process leading to the manifestation of insulin dependent diabetes mellitus (IDDM) can also affect the sympathochromaffin system, we analyzed the role of apoptosis and infiltration of the adrenal medulla as features of this autoimmune process in parallel with the development of diabetes. Prediabetic and diabetic NOD mice aged 3 to 30 weeks were studied and compared with control mice. Apoptosis was assessed by in situ end-labeling method and ultrastructural analysis. Adrenals were screened for lymphocytic infiltration by conventional hematoxylin-eosin staining. Chromaffin cells were characterized by immunohistochemical staining against synaptophysin and tyrosine hydroxylase. Apoptotic nuclei were detected in all mice studied at a very low level, mainly occuring within the connective tissue between medulla and cortex. The maximum score was achieved at 3 weeks (1.91+/-0.48 apoptotic cells/1000 counted cells; n = 4). There was no significant difference between NOD mice and control mice. No correlation could be found between blood glucose levels and apoptosis. On the ultrastructural level, apoptotic cells presented typical features of apoptosis, i.e. condensed nuclei and cytoplasm. Neither in NOD mice nor in controls lymphocytic infiltration or fibrosis of the adrenal was detected. Even NOD mice with overt diabetes did not exhibit morphological signs of medullitis. In summary, no signs of immune destruction of the adrenal medulla in NOD mice aged 3 to 30 weeks could be detected.

Hyperleptinemia, leptin resistance, and polymorphic leptin receptor in the New Zealand obese mouse.

New Zealand Obese (NZO) mice exhibit a polygenic syndrome of hyperphagia, obesity, hyperinsulinemia, and hyperglycemia similar to that observed in young diabetes mutant mice on the C57BLKS/J background (C57BLKS/J-Lepr(db)/Lepr(db)). Here we show that in NZO this syndrome is accompanied by a marked elevation of the leptin protein in adipose tissue and serum. The promoter region and the complementary DNA of the ob gene of NZO mice, including its 5'-untranslated region, are identical with the wild-type sequence (C57BL, BALB/c), except that the transcription start is located 5 bp upstream of the reported site. In contrast to C57BLKS/J+/+ and C57BL/6J-Lep(ob)/Lep(ob) mice, NZO mice failed to respond to recombinant leptin (7.2 microg/g) with a reduction of food intake. Leptin receptor messenger RNA as detected by PCR appears as abundant in hypothalamic tissue of NZO mice as in tissue from lean mice. Ten nucleotide polymorphisms are found in the complementary DNA of the leptin receptor, resulting in two conservative substitutions (V541I and V651I) in the extracellular part of the receptor and one nonconservative substitution (T1044I) in the intracellular domain between the presumed Jak and STAT binding boxes. However, these mutations are also present in the related lean New Zealand Black strain (body fat at 9 weeks: New Zealand Black, 6.2 +/- 1.3%; NZO, 17.0 +/- 1.7%). Thus, the polymorphic leptin receptor seems to play only a minor, if any, role in the obesity and hyperleptinemia of the NZO mouse. It is suggested that the main defect in NZO is located distal from the leptin receptor or at the level of leptin transport into the central nervous system.

CCK-resistance in Zucker obese versus lean rats.

Obese Zucker rats are less sensitive to the satiety effect of CCK than lean litter mates. The present studies further characterised this CCK resistance. Subcutaneous injection of the CCK agonist caerulein dose-dependently decreased food intake in Zucker obese and lean rats whereas the CCK-B agonist gastrin-17 did not. Caerulein at 4 microg/kg, which resulted in CCK plasma bioactivity slightly above postprandial levels, decreased food intake in lean rats but not in obese rats. The decrease in food intake was also more marked at higher caerulein doses (20-100 microg/kg) in lean versus obese rats. In lean animals the satiety effects of the "near physiological" 4 microg/kg caerulein dose was abolished after blockade of vagal afferents with capsaicin, whereas the effects of higher caerulein doses were not. CCK-stimulated amylase secretion from pancreatic acini and binding capacity of 125I-labelled CCK-8 were decreased in obese versus lean rats. The CCK-A antagonist loxiglumide at 20 mg/kg, a dose which abolished the action of all caerulein doses on food intake, failed to alter the food intake either in obese or in lean rats when given without an agonist. The results suggest that the satiety effects of "near physiological" doses of caerulein in lean rats are mediated by vagal afferents whereas pharmacological doses act via non-vagal mechanisms. The differences in CCK's satiety effect between lean and obese rats may be due to differences in CCK-receptor binding and action at peripheral vagal sites. However, the failure of the CCK-A antagonist to increase food intake questions whether any of the effects of exogenous CCK are of physiological relevance.

Spontaneous epileptiform seizures but increased resistance to kindled seizures in a mutant Sprague-Dawley rat (mf/mf).

Approximately 30% of a breeding colony of Sprague-Dawley rats homozygous for an autosomal recessive mutation mf ("mutilated foot") associated with a peripheral sensory neuropathy have been found unexpectedly to suffer spontaneous epileptiform attacks. Seizures ranged from brief episodes of compulsive running to tonic-clonic convulsions lasting for up to 30 s, recurring at intervals of hours or days. EEG recordings during seizures showed high-voltage 8-10 Hz spike trains that abated over the ensuing 1-2 min. Interictal records were usually normal. Twice-daily kindling of the amygdala (200 microA sinewave for 1.0 s) was unexpectedly ineffective. Most of the rats that had suffered spontaneous seizures failed to develop kindled afterdischarges, even after 30 kindling stimulations. Other mf rats developed prolonged high-amplitude kindled afterdischarges that were arrested at stage 2 and failed to evolve into convulsive seizures. Hippocampal dentate granule cells of kindled mf rats, stained for zinc by Timm's method, showed significantly less mossy fibre sprouting than wild-type Sprague-Dawley rats after the same number of kindled afterdischarges. A minority of the mf rats tested (2 of 14) kindled normally. Auditory stimulation (n = 23) or stroboscopic flicker (n = 14) failed to elicit seizures or running fits in any mf rat. Peripheral neuropathy corresponding to that in the mf rat, with resistance to kindling and diminished mossy fibre sprouting, have also been reported in transgenic mice with defective p75NGFR neurotrophin receptors. A homologous genetic defect in the rat could account for most of the features of the mf phenotype.

Rapid recovery of self-stimulation from depression produced by the atypical neuroleptic risperidone is not prevented by 5-HT2 receptor stimulation.

Behavioral effects of the antipsychotic drug risperidone were tested in rats responding for variable-interval stimulation of the ventral tegmental area (VTA). Risperidone (0-0.9 mg/kg) produced a dose-dependent depression of responding in the 60 min after injection. Self-stimulation tests delayed for 30 or 120 min after injection showed that inhibition of responding by risperidone was limited in duration, with response rates recovering to pre-injection levels in a time-dependent manner. Recovery occurred regardless of opportunity to engage in self-stimulation, and was virtually complete at a time when receptor occupancy has been shown to be almost undiminished. The atypical properties of risperidone have been ascribed to its potent antagonist activity at 5-HT2 receptors; however, spontaneous recovery from the effects of risperidone was not prevented by simultaneous administration of a selective 5-HT2 agonist (DOI), even though DOI when given alone produced a 50-70% reduction in response rates. These results show that the inhibitory effect of risperidone on operant performance may be self-limiting in a manner that is not accounted for by its pharmacokinetic properties nor by its antagonist activity at central 5-HT2 receptors.

Evidence that reduced leptin levels, but not an aberrant sequence of leptin or its receptor, contribute to the obesity syndrome in NON mice.

NON mice exhibit a polygenic syndrome of mild obesity which is less pronounced than that of the ob and db strains. Here, we have shown that the syndrome is accompanied by a rise in leptin mRNA levels in adipose tissue, corresponding with the increase in adipose tissue mass. Surprisingly, levels of the leptin protein in adipose tissue and serum were comparable to those of lean control animals (BL57/Ksj-+/+), and markedly lower than those in db/db-mice. The coding regions of the cDNA sequences of both leptin and the leptin receptor from NON mice were identical with those of the wild-type sequences. We suggested that low levels of leptin in adipose tissue and serum contribute to the obesity of NON mice.

Kindled seizures do not affect adenosinergic inhibition of DA or ACh release in rat accumbens or PFC.

Epileptic seizures are thought to terminate largely as a result of the extracellular accumulation of the purinergic neuromodulator, adenosine, released by discharging neurons. However, the postictal surge in extracellular adenosine and its widespread inhibitory effects are limited in time to only a few minutes and cannot directly account for increased resistance to seizures and the complex behavioural and motivational effects that may persist for hours or days after a seizure. The present study examined whether kindled seizures might alter the sensitivity or efficacy of inhibitory presynaptic adenosine receptors, and thereby induce more enduring changes in downstream transmitter systems. Rats were kindled in the amygdala of the dominant cerebral hemisphere, contralateral to the preferred direction of rotation, and their brains were removed either 2 h or 28 days after completion of kindling. Inhibition of electrically stimulated release of dopamine (DA) and acetylcholine (ACh) by the A1 adenosine-receptor agonist, R-phenylisopropyladenosine (R-PIA) was then measured in the prefrontal cortex (PFC) and nucleus accumbens. R-PIA (1.0 microM) inhibited [1H]DA release from PFC and nucleus accumbens tissue, and [14C]ACh release from nucleus accumbens tissue, but release was unaffected by prior kindling, regardless of the intervening interval. These results do not support suggestions that DA or ACh might mediate the effects of seizure-induced changes in purinergic inhibitory tone so as to cause long-term shifts in seizure threshold and postictal behavior.

Long-term and rapid regulation of ob mRNA levels in adipose tissue from normal (Sprague Dawley rats) and obese (db/db mice, fa/fa rats) rodents.

Increased levels of mRNA transcribed from the ob gene in adipose tissue of obese/hyperinsulinaemic Zucker (fa/fa) rats were detectable as early as 3 weeks after birth and continued to rise there after in parallel with body weight and serum insulin. mRNA levels of two other fat-specific genes (ARL4, FST44) were unaltered. In C57BL/KsJ db/db mice, ob mRNA levels also increased in parallel with body weight and serum insulin, and remained elevated in older animals when insulin levels decreased. In heterozygous control animals (db/+; fa/Fa), mRNA levels were comparable with those in the homozygous controls. In normal Sprague Dawley rats, the ob mRNA increased continuously, but more slowly than in Zucker rats, in parallel with body weight and insulin levels, and reached 15 times higher levels in the heaviest rats (400 g) studied. In Sprague Dawley rats made diabetic by an injection of streptozotocin, ob mRNA levels were reduced by approximately 50% after 24 h. A 24-h fasting period reduced the ob mRNA by 50% in lean Sprague Dawley and Fa/Fa, but not in obese Zucker fa/fa rats, although insulin levels were reduced in both groups. These data indicate that ob mRNA levels increase in both normal and obese rodents in parallel with age, body weight and serum insulin, reflecting an early (Zucker rats, db-mice) or slowly developing (Sprague Dawley rats) resistance to leptin and insulin. This increase does not appear to be mediated by the recently described rapid regulation of ob mRNA by insulin, but seems to be due to a different, long-term control mechanism which signals the size of the fat depots.

Nitric oxide synthesis, epileptic seizures and kindling.

Nitric oxide (NO) has been implicated in synaptic changes underlying long-term potentiation and some forms of learning. It is unclear, however, whether NO contributes to long-term changes associated with the kindling of epileptic seizures. In the present study rats were treated, on the first 6 days of kindling, with L-arginine (L-Arg), the endogenous donor from which NO derives, or with L-nitro-arginine (L-No-Arg), a competitive inhibitor of NO synthesis, or with vehicle. Drugs were given in doses previously shown to affect learning or other behaviour. L-Arg (750 mg/kg IP) did not affect kindling or seizure severity. L-No-Arg (100 mg/kg) prolonged the duration of afterdischarges and convulsions on treatment days but did not advance kindling or affect seizures on subsequent days. A second experiment examined the possible role of NO in the development of resistance to seizures following prior seizures. Six or more stimuli were administered at 10-min intervals to fully-kindled rats after injection of L-No-Arg or vehicle. Vehicle-treated rats became progressively more resistant to afterdischarges and convulsions with successive stimulations but L-No-Arg-treated rats failed to do so. Rats injected with L-NO-Arg also showed an unexpected high mortality in the ensuing 24 h. L-No-Arg appeared to have no direct effect on the course of kindling but impaired the development of postictal resistance, and increased the duration and lethal after-effects of closely repeated seizures. The results do not support suggestions that antagonists of NO might prove clinically useful as anticonvulsants.

α 2-Adrenoreceptor antagonism may contribute to the atypical properties of risperidone: experimental support for the Nutt case.

The therapeutic efficacy and reduction in side effects claimed for new antischizophrenic drugs such as clozapine and risperidone have been ascribed to their heightened affinity for serotonin 5-HT(2) receptors rather than D-2 receptors. A case for α(2)-adrenoreceptor antagonism has recently been argued. We have confirmed that at least one atypical property of risperidone (a rapid decrement in its ability to depress self-stimulation) can be partly prevented by an α(2)-adrenoreceptor agonist (clonidine) but not by a 5-HT( 2) receptor agonist (DOI). This result supports the suggested role of α( 2)-adrenoreceptor antagonism in counteracting extrapyramidal effects during treatment with risperidone.

Impaired FAD-glycerophosphate dehydrogenase activity in islet and liver homogenates of fa/fa rats.

The mitochondrial FAD-linked enzyme glycerophosphate dehydrogenase plays a key role in the pancreatic B-cell glucose sensing device. In the present study, the activity of this enzyme was examined in islets of fa/fa rats in which inherited diabetes mellitus is associated with obesity, hyperinsulinism and severe insulin resistance. The specific activity of both FAD-linked glycerophosphate dehydrogenase and glutamate dehydrogenase were decreased in islet and liver homogenates prepared from fa/fa, as compared to Fa/Fa, rats, this coinciding with a low ratio between glutamateoxalacetate and glutamate-pyruvate transaminase activity in both islet and liver extracts, islet hyperplasia, hyperinsulinemia and hepatic steatosis in the hyperglycemic fa/fa rats. It is speculated that a low activity of FAD-linked glycerophosphate dehydrogenase in the pancreatic B-cell may participate to the perturbation of glucose homeostasis in fa/fa rats, like in other animal models of non-insulin-dependent diabetes mellitus.

Kindled epileptic seizures, postictal refractoriness, status epilepticus, and electrical self-stimulation.

A single stimulus applied once daily to the limbic system commonly leads to convulsive seizures yet seizures are relatively infrequent during intracranial self-stimulation (ICSS), a procedure that involves many hundreds of similar stimuli. The present study examined the possible role of electrode site, interstimulus interval, afterdischarge and reinforcement thresholds and postictal refractoriness in accounting for this paradox. Electrode location was an overriding factor: seizures were never seen with hypothalamic implants posterior to the level of the ventromedial nucleus but were elicited by the majority of more rostral reward sites. Frequent repeated stimulation by ICSS did not in itself prevent subsequent kindling or reverse the effects of earlier kindling; on the contrary, seizures induced by ICSS showed a progressive increase in severity similar to the progression produced by conventional kindling. Individual convulsive seizures, as in previous studies, conferred transient protection against further seizures whether from ICSS or from kindling. More prolonged protection occassionally developed after repeated convulsive seizures: protection was accompanied by continuous EEG slow-waves corresponding in presentation to clinical petit mal status. Prolonged resistance to seizures has also been reported after tonic-clonic status epilepticus causing temporal lobe damage. The relative infrequency of seizures during ICSS ordinarily appears to depend on the siting of the electrodes, on distinct short- and long-term postictal refractory states, and on the rat learning to restrict stimulus input to subseizural levels.

GAD65 is recognized by T-cells, but not by antibodies from NOD-mice.

Since the 64kDa-protein glutamic acid decarboxylase (GAD) is one of the major autoantigens in T-cell mediated Type 1 diabetes, its relevance as a T-cell antigen needs to be clarified. After isolation of splenic T-cells from non-obese diabetic (NOD) mice, a useful model for human Type 1 diabetes, we found that these T-cells proliferate spontaneously when incubated with human GAD65, but only marginally after incubation with GAD67, both recombinated in the baculovirus system. No effect was observed with non-diabetic NOD mice or with T-cells from H-2 identical NON-NOD-H-2g7 control mice. It has been published previously that NOD mice develop autoantibodies against a 64kDa protein detected with mouse beta cells. In immunoprecipitation experiments with sera from the same NOD mice and 35S-methionine-labelled GAD, no autoantibody binding could be detected. We conclude firstly that GAD65 is an important T-cell antigen which is relevant early in the development of Type 1 diabetes and secondly that there is an antigenic epitope in the human GAD65 molecule recognized by NOD T-cells, but not by NOD autoantibodies precipitating conformational epitopes. Our results therefore provide further evidence that GAD65 is a T-cell antigen in NOD mice, being possibly also involved in very early processes leading to the development of human Type 1 diabetes.