Maternal undernutrition programs the apelinergic system of adipose tissue in adult male rat offspring.

Based on the Developmental Origin of Health and Disease concept, maternal undernutrition has been shown to sensitize adult offspring to metabolic pathologies such as obesity. Using a model of maternal 70% food restriction in pregnant female rats throughout gestation (called FR30), we previously reported that obesity-prone adult male rat offspring displayed hyperleptinemia with modifications in leptin and leptin receptor messenger RNA (mRNA) levels in white adipose tissue (WAT). Apelin is a member of the adipokine family that regulates various aspects of energy metabolism and WAT functionality. We investigated whether apelin and its receptor APJ could be a target of maternal undernutrition. Adult male rat offspring from FR30 dams showed increased plasma apelin levels and apelin gene expression in WAT. Post-weaning high-fat diet led to marked increase in APJ mRNA and protein levels in offspring's WAT. We demonstrate that maternal undernutrition and post-weaning diet have long-term consequences on the apelinergic system of adult male rat offspring.

Maternal perinatal undernutrition modifies lactose and serotranferrin in milk: relevance to the programming of metabolic diseases?

A close link between intrauterine growth restriction and development of chronic adult diseases such as obesity, diabetes, and hypertension has been established both in humans and animals. Modification of growth velocity during the early postnatal period (i.e., lactation) may also sensitize to the development of metabolic syndrome in adulthood. This suggests that milk composition may have long-lasting programming/deprogramming metabolic effects in the offspring. We therefore assess the effects of maternal perinatal denutrition on breast milk composition in a food-restricted 50% (FR50) rat model. Monosaccharides and fatty acids were characterized by gas chromatography, and proteins were profiled by surface-enhanced laser desorption/ionization-time-of-flight analysis in milk samples from FR50 and control rat dams. Milk analysis of FR50 rats demonstrated that maternal undernutrition decreases lactose concentration and modulates lipid profile at postnatal day 10 by increasing the unsaturated fatty acids/saturated fatty acids and diminishes serotransferrin levels at postnatal day 21. Our data indicate that maternal perinatal undernutrition modifies milk composition both quantitatively and qualitatively. These modifications by maternal nutrition open new perspectives to identify molecules that could be used in artificial milk to protect from the subsequent development of metabolic diseases.

Placental expression of the obesity-associated gene FTO is reduced by fetal growth restriction but not by macrosomia in rats and humans.

Genetic variants in the FTO (fat mass- and obesity-associated) gene have the highest association of all obesity-associated genes. Its placental expression was shown to relate to birth weight, suggesting that it may participate in the control of fetal weight gain. To gain more insight into the implication of FTO in fetal growth, we measured its placental expression in samples including extremes of abnormal fetal growth, such as after intrauterine growth restriction (IUGR) or macrosomia in both rats and humans. In rats, fetal growth was modulated by maternal nutritional modifications. In humans, placental villi were collected from pathological pregnancies (i.e. with IUGR or fetal macrosomia). Placental FTO mRNA expression was reduced by IUGR but was not significantly affected by macrosomia in either rats or humans. Our data suggest that placental FTO may participate in interactions between the in utero environment and the control of fetal growth under IUGR conditions by modulating epigenetic processes.

Maternal perinatal undernutrition has long-term consequences on morphology, function and gene expression of the adrenal medulla in the adult male rat.

Epidemiological studies suggest that maternal undernutrition sensitises to the development of chronic adult diseases, such as type 2 diabetes, hypertension and obesity. Although the physiological mechanisms involved in this 'perinatal programming' remain largely unknown, alterations of stress neuroendocrine systems such as the hypothalamic-pituitary-adrenal (HPA) and sympathoadrenal axes might play a crucial role. Despite recent reports showing that maternal perinatal undernutrition disturbs chromaffin cells organisation and activity in male rats at weaning, its long-term effects on adrenal medulla in adult animals are unknown. Using a rat model of maternal perinatal 50% food restriction (FR50) from the second week of gestation until weaning, histochemistry approaches revealed alterations in noradrenergic chromaffin cells aggregation and in cholinergic innervation in the adrenal medulla of 8-month-old FR50 rats. Electron microscopy showed that chromaffin cell granules exhibited ultrastructural changes in FR50 rats. These morphological changes were associated with reduced circulating levels and excretion of catecholamines. By contrast, catecholamine plasma levels were significantly increased after a 16 or 72 h of fasting, indicating that the responsiveness of the sympathoadrenal system to food deprivation was accentuated in FR50 adult rats. Among 384 pituitary adenylate cyclase-activating polypeptide-sensitive genes, we identified 129 genes (33.6%) that were under expressed (ratio < 0.7) in FR50 animals. A large number of these genes are involved in cytoskeleton remodelling and vesicle trafficking. Taken together, our results show that maternal perinatal undernutrition programmes adrenomedullary function and gene expression in adult male rats. Because catecholamines contribute to metabolic homeostasis, as well as arterial blood pressure regulation, the alterations observed in the adrenal medulla of adult male FR50 rats may participate in the programming of chronic adult diseases.

Maternal perinatal undernutrition impairs chromaffin cells proliferation in the postnatal rat.

Numerous data show that malnutrition during early life programs chronic diseases in adulthood. Many of these disorders may result from alterations in the development of neuroendocrine systems, such as the hypothalamo-pituitary-adrenal axis and the sympathoadrenal system. We have previously reported that maternal 50% food restriction during late pregnancy and lactation reduces adrenal weight and impairs chromaffin cell differentiation in male rats at weaning. In addition, maternal undernutrition modifies the expression of several genes involved in proliferation and apoptosis. This study therefore investigated the impact of maternal food restriction on adrenal cell growth in the late postnatal rat. Histological analysis showed that the number of proliferating chromaffin cells assessed by nuclear labelling with BrdU was reduced by 45%, whereas the level of apoptosis visualised by caspase-3 immunoreactivity was increased by 340% in adrenal medulla of offspring from undernourished mothers. In contrast, maternal food restriction did not affect proliferation and apoptosis in cortical cells of rats. These developmental changes were associated with overexpression of TGFbeta2. These data show that perinatal undernutrition impairs the balance between chromaffin cell proliferation and apoptosis. These modifications may lead to "malprogramming" of adrenal medulla development, which could contribute to the pathogenesis of chronic diseases in adulthood.

Metyrapone-induced glucocorticoid depletion modulates tyrosine hydroxylase and phenylethanolamine N-methyltransferase gene expression in the rat adrenal gland by a noncholinergic transsynaptic activation.

The hypothalamic corticotropin-releasing hormone system and the sympathetic nervous system are anatomically and functionally interconnected and hormones of the hypothalamic-pituitary-adrenocortical axis contribute to the regulation of catecholaminergic systems. To investigate the role of glucocorticoids on activity of the adrenal gland, we analysed plasma and adrenal catecholamines, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNA expression in rats injected with metyrapone or dexamethasone. Metyrapone-treated rats had significantly lower epinephrine and higher norepinephrine production than control rats. Metyrapone increased TH protein synthesis and TH mRNA expression whereas its administration did not affect PNMT mRNA expression. Dexamethasone restored plasma and adrenal epinephrine concentrations and increased PNMT mRNA levels, which is consistent with an absolute requirement of glucocorticoids for PNMT expression. Adrenal denervation completely abolished the metyrapone-induced TH mRNA expression. Blockage of cholinergic neurotransmission by nicotinic or muscarinic receptor antagonists did not prevent the metyrapone-induced rise in TH mRNA. Finally, pituitary adenylate cyclase activating polypeptide (PACAP) adrenal content was not affected by metyrapone. These results provide evidence that metyrapone-induced corticosterone depletion elicits transsynaptic TH activation, implying noncholinergic neurotransmission. This may involve neuropeptides other than PACAP.

Perinatal malnutrition programs sympathoadrenal and hypothalamic-pituitary-adrenal axis responsiveness to restraint stress in adult male rats.

In humans, an altered control of cortisol secretion was reported in adult men born with a low birth weight making the hypothalamic-pituitary-adrenal (HPA) axis a possible primary target of early life programming. In rats, we have recently shown that maternal food restriction during late pregnancy induces both an intrauterine growth retardation and an overexposure of fetuses to maternal corticosterone, which disturb the development of the HPA axis in offspring. The first aim of this work was to investigate, in adult male rats, whether perinatal malnutrition has long-lasting effects on the HPA axis activity during both basal and stressful conditions. Moreover, as the HPA axis and sympathetic nervous system are both activated by stress, the second aim of this work was to investigate, in these rats, the adrenomedullary catecholaminergic system under basal and stressful conditions. This study was conducted on 4-month-old male rats malnourished during their perinatal life and on age-matched control animals. Under basal conditions, perinatal malnutrition reduced body weight and plasma corticosteroid-binding globulin (CBG) level but increased mineralocorticoid receptor (MR) gene expression in CA1 hippocampal area. After 30 min of restraint, perinatally malnourished (PM) rats showed increased plasma noradrenaline, adrenocorticotropin hormone (ACTH) and corticosterone concentrations similarly as controls, but calculated plasma-free corticosterone concentration was significantly higher and adrenaline level lower than controls. During the phase of recovery, PM rats showed a rapid return of plasma ACTH and corticosterone concentrations to baseline levels in comparison with controls. These data suggest that in PM rats, an elevation of basal concentrations of corticosterone, in face of reduced CBG and probably increased hippocampal MR lead to a much larger impact of corticosterone on target cells that mediate the negative-feedback mechanism on the activities of both the HPA axis and sympathoadrenal one.

Activities of the pituitary-adrenal and gonadal axes during the estrous cycle in adult female rats prenatally exposed to morphine.

The present investigation concerns 80-90-day-old female rats born from morphine-exposed mothers (2x10 mg/kg per day from day 11-18 of gestation) or saline-treated ones (controls). The former showed reduced size and activity of the adrenals at birth. At adult stage, they present: (1) higher increase of plasma adrenocorticotrophic hormone level on proestrus; (2) significant rise of plasma corticosterone level on diestrus morning and estrus evening; (3) adrenal atrophy which was significant only on diestrus and estrus morning; (4) more corticosterone binding sites of type I (mineralocorticoid receptors) on proestrus morning in the hippocampus; (5) more corticosterone binding sites of type II (glucocorticoid receptors) in the hippocampus on proestrus morning and in the hypothalamus on estrus morning. In both experimental groups, B(max) for hypothalamic mineralocorticoid receptors were drastically higher on estrus morning than on the other stages of the estrous cycle. The activity of the pituitary-gonadal axis is poorly affected by prenatal morphine-exposition. In both experimental groups drastic and comparable surges of both plasma progesterone and luteinizing hormone were observed during proestrus. Nevertheless morphine-exposed females showed higher levels of plasma estradiol on diestrus morning but lower levels on metestrus morning. In conclusion, prenatal exposition to morphine has long-term effects mainly on pituitary-adrenal axis as well as on binding sites for corticosterone in the hypothalamus and the hippocampus which are dependent on the estrous cycle stages in adult females.

Hypothalamic-pituitary-adrenocortical and gonadal axes and sympathoadrenal activity of adult male rats prenatally exposed to morphine.

The present investigation concerns 80-90 day-old male rats born from morphine-exposed mothers (2 x 10 mg/kg per day from days 11 to 18 of gestation which showed at birth reduced size and activity of the adrenals). This prenatal treatment did not significantly disturb under resting conditions: (1) the postnatal body growth up to week 10 after birth, (2) the activity of the pituitary gonadal axis (circulating luteinizing hormone (LH) and testosterone (T), weight of the testicles and seminal vesicles), (3) the activity of the hypothalamo-pituitary-adrenal axis (HPA) (hypothalamic corticoliberin (CRF) content, plasma adrenocorticotrophic hormone (ACTH) level, adrenal weight and corticosterone (B) content, plasma B level) as well as Bmax and Kd of mineralocorticoid (type I) and glucocorticoid (type II) receptors to B in both the hippocampus and the hypothalamus. In contrast these rats showed reduced content of adrenals in noradrenaline (NA) and adrenaline (A) but increased circulating levels of A.

Circulating neuropeptide Y (NPY) and catecholamines in rat under resting and stress conditions. Arguments for extra-adrenal origin of NPY, adrenal and extra-adrenal sources of catecholamines.

Neuropeptide Y (NPY) is found in cell bodies of neurons in the brain and co-localized with noradrenaline (NA) in sympathetic nerves as well as with NA and adrenaline (A) in the adrenal chromaffin cells. The purpose of the present work is to determine whether NPY and catecholamines found in the plasma of the rat under resting and stress conditions (ether inhalation, restraint) arise from the adrenals or from extra-adrenal sites. We used adrenalectomized (adx) rats and sham-adx ones. Adrenalectomy increased plasma adrenocorticotrophic hormone (ACTH) levels but decreased drastically circulating corticosterone (B) and A (-97%). However, resting NA was slightly but not significantly decreased and NPY not affected. Ether inhalation (3 min) increased plasma levels of ACTH, B, NA and A in sham-adx rats, ACTH, NA and, weakly, A in adx ones. Restraint (30 min) increased B, NA and A in sham-adx rats, NA and, poorly, A, in adx ones. In contrast, plasma levels of NPY were not significantly affected by these stress conditions. The present data suggest that NA found in rat plasma at rest and during ether or restraint stress could arise from both adrenal medulla and noradrenergic nerve endings while A arises mainly from the adrenergic chromaffin cells of the adrenals. In contrast, NPY found in the circulation, at rest and under stress conditions, is not derived from the adrenals but emanates mainly from an extra-adrenal source.

Effect of cholinergic blockade on glucocorticoid regulation of NPY and catecholamines in the rat adrenal gland.

Catecholamines and neuropeptide Y (NPY) levels were determined in the adrenals of rats treated for 2.5 days with chlorisondamine (6 mg/day), a nicotinic ganglionic blocking agent, metyrapone (66 mg/day), an inhibitor of the 11 beta-hydroxylase activity or both metyrapone and chlorisondamine. Chlorisondamine induced a significant increase in adrenal weight (31%) without significant rise in hypothalamic CRH content, plasma ACTH level and plasma corticosterone concentration. This drug was unable to affect significantly dopamine (DA), norepinephrine (NE) and epinephrine (E) content of the adrenals; in contrast, it induced a significant decrease (90%) of plasma NE and E levels. Chlorisondamine induced no change in adrenal NPY content as well as NPY mRNA level determined by Northern blot but significantly increased NPY plasma level. Metyrapone-treatment induced a significant drop of plasma corticosterone level and elicited a significant reduction of hypothalamic CRH content, a rise (460%) of the plasma ACTH concentration associated with a significant increase (18%) of the adrenal weight. A marked increase of DA (240%) and significant decrease of E (22%) in the adrenal gland were observed in response to metyrapone treatment. In addition, metyrapone induced a drop (23%) in plasma E level. In both the adrenals and the plasma, the ratio E/NE was significantly reduced by metyrapone treatment. Metyrapone elicited a significant increase of adrenal NPY content (88%) as well as of NPY mRNA revealed by Northern blot analysis but was unable to significantly affect NPY plasma level. The effects of chlorisondamine, given in combination with metyrapone on both hypothalamic CRH content and plasma ACTH level, were similar to those induced by metyrapone given alone. Chlorisondamine-mediated pharmacological ganglionic blockade increased metyrapone-induced adrenal hypertrophy and adrenal DA storage but prevented metyrapone-induced depletion of adrenal E as well as increase of the adrenal NPY mRNA level and NPY content. Chlorisondamine-induced elevation of plasma NPY level was not observed under metyrapone treatment. Present data suggest that the increase in adrenal NPY synthesis in response to metyrapone treatment is mediated by transsynaptic cholinergic activation and implies nicotinic receptors. On the other hand, adrenal TH may be regulated by additional or different mechanisms, which possibly involve nonnicotinic transmission. Present work also suggests that the suppression of the glucocorticoid feedback inhibition of hypothalamic CRH neurons could stimulate sympathoneuronal outflow and consequently elicit transsynaptic cholinergic activation of adrenal neuropeptide Y gene expression.

Regulation of neuropeptide Y and its mRNA by glucocorticoids in the rat adrenal gland.

The effects of glucocorticoids on adrenal neuropeptide Y (NPY) and NPY mRNA levels have been investigated on adult male rats of the Wistar strain subcutaneously injected twice a day with dexamethasone (5 mg/day), metyrapone (66 mg/day) or solvent (NaCl 0.9%) for 2.5 days and sacrificed 2 h after the last injection. Dexamethasone induced a significant decrease in both the adrenal weight (30%) and the plasma corticosterone concentration (85%) but a significant increase of the adrenal NPY content (about 25%) as well as of its mRNA (0.8 kb) (> 100%), revealed by Northern blot analysis and by in situ hybridization. Dexamethasone was unable to affect significantly dopamine (DA), norepinephrine (NE) and epinephrine (E) content of the adrenals; in contrast, it induced a significant decrease (30%) of the plasma NE level. Metyrapone elicited a drop of the plasma corticosterone level (80%), but a rise (near 150%) of the plasma ACTH concentration associated with an increase (19%) of the adrenal weight, a significant increase (30%) in the amount of adrenal NPY as well as a rise (> 200%) of NPY mRNA content of the adrenal. Such treatment increased DA and NE (40%), and decreased E (> 30%) content of the adrenals. Metyrapone-induced changes of catecholamine concentrations in the plasma were similar to that observed in the adrenal gland. Data suggest that the increase in adrenal NPY mRNA content induced by dexamethasone is more likely due to humoral effect rather than nervous effect of this synthetic glucocorticoid on the adrenal medulla. A neural mechanism as splanchnic nerve activation in response to severe corticosterone deficiency is a reasonable hypothesis to explain the increase in adrenal NPY mRNA induced by metyrapone, although there are probably other, nonneural mechanisms by which metyrapone could stimulate adrenal NPY. Present data are consistent with glucocorticoid regulation of NPY gene expression and/or steady-state level of NPY mRNA in the adrenal gland.

Neuropeptide Y (NPY)- and vasoactive intestinal peptide (VIP)-induced aldosterone secretion by rat capsule/glomerular zone could be mediated by catecholamines via beta 1 adrenergic receptors.

The effects of two Neuropeptide Y (NPY) analogs (Y1- and Y2-type) and vasoactive intestinal peptide (VIP) on both catecholamine (adrenaline and noradrenaline) release and aldosterone production by rat adrenal capsule/glomerular zone, have been investigated in vitro. The adrenal capsule/glomerular zones, collected from adult male rats, were incubated in a medium (Krebs-Ringer bicarbonate buffer supplemented with glucose and bovine serum albumin) containing or not one of the following synthetic peptides: human Leu31,Pro34-NPY (an agonist of the Y1-type receptors), human/porcine NPY18-36 (an agonist of the Y2-type receptors) and VIP at the concentration of 10(-7) M, associated or not with 10(-7) M atenolol (a beta 1 adrenergic antagonist) or ICI-118,551 hydrochloride (a beta 2 adrenergic antagonist). The two NPY analogs as well as the VIP stimulated the release of catecholamines and of aldosterone. The beta 1 adrenergic antagonist, but not the beta 2 one, which failed to affect basal aldosterone production when given alone, prevented NPY18-36-, Leu31,Pro34-NPY- or VlP-induced aldosterone secretion. Present data support the hypothesis that adrenaline and/or noradrenaline could mediate the effects of both NPY and VIP on aldosterone secretion via beta 1 adrenergic receptors; alternatively, the steroidogenic effect of NPY or VIP could be related to direct interaction between NPY- or VIP-specific binding sites, present on the capsule/glomerular zone of the rat adrenal cortex, and beta 1 adrenergic receptors. Then the NPYergic, VIPergic and catecholaminergic innervation of the adrenal cortex, previously characterized by immunohistochemistry, may be a potent stimulatory element in the nervous control of the aldosterone secretion.

Effects of dexamethasone, calcium and 1,25-dihydroxycholecalciferol on calcitonin and calcitonin gene-related peptide mRNA levels from the CA-77 C cell line.

We used the CA-77 cell, a murine C-cell line derived from a medullary thyroid carcinoma, to study the effects of glucocorticoids, calcium, and vitamin D metabolites on calcitonin (CT) gene expression. Total RNA was isolated, and CT and CT gene-related peptide (CGRP) mRNAs were measured by Northern hybridizations using specific probes. A control mRNA probe (cyclophilin) was used to quantitate the specificity of the changes in CT and CGRP mRNAs. The CA-77 C cell line cultured in basal conditions with a medium deprived of fetal calf serum, but was supplemented by insulin, expressed mainly the CGRP mRNA. Dexamethasone was found to increase both CT and CGRP mRNAs in a time- and dose-dependent way without changing the alternative splicing. A slight but significant increase in the steady-state CT mRNA level was found 3 days after addition of 10(-10) M dexamethasone; the same dose slightly decreased the CGRP mRNA level; concentrations of dexamethasone > or = 10(-9) M elevated both mRNAs. A twelve-fold increase for CT mRNA, and an eightfold increase in CGRP mRNA occurred 3 days after administration of 10(-6) M dexamethasone. Kinetic data revealed inductions of both mRNAs 24 hours after exposure to 10(-7) M dexamethasone, and highest CT and CGRP mRNAs levels were observed after 72 hours of treatment. Calcium from 1-4 mM in short-term (1 hour and 4 hour) or long-term stimulations (1 day and 4 days), with or without dexamethasone cotreatment was ineffective. CT and CGRP mRNAs levels were both half-reduced 48 hours after addition of 10(-7) M 1,25-dihydroxycholecalciferol; this effect was transient, as it disappeared 2 days later.

Plasma C-peptide levels and clinical remissions in recent-onset type I diabetic patients treated with cyclosporin A and insulin.

Remission from insulin dependency in insulin-treated recent-onset type I (insulin-dependent) diabetic patients can result from a partial recovery of insulin secretion, an improvement in tissue sensitivity to insulin, or both. The same hypothesis must be analyzed when remission occurs in cyclosporin A (CsA)-treated patients. In this study, plasma C-peptide levels were serially measured in the basal state and after stimulation in 219 recent-onset type I diabetic patients; 129 received CsA, and all patients were similarly monitored and insulin treated. The results were analyzed in view of the occurrence of remission. Remission was defined as good metabolic control in the absence of hypoglycemic treatment for greater than or equal to 1 mo. Remission occurred in 44% of the CsA-treated group and lasted for mean +/- SE 10.0 +/- 0.9 mo vs. 21.6% in the non-CsA-treated group with a duration of 4.4 +/- 0.8 mo. Plasma C-peptide levels were initially dramatically lower than normal in both groups in the basal and stimulated states. C-peptide levels increased significantly later, at 3 and 6 mo, in both groups. C-peptide values were proportional to the rates of remission in both groups. In the non-CsA-treated group, C-peptide levels later decreased, and these patients inexorably relapsed to insulin dependency. In contrast, in the CsA-treated group, the initial recovery in insulin secretory capacity was maintained over the 18-24 mo of the study. Furthermore, higher remission rates and longer-lasting remission were obtained in patients who reached higher C-peptide levels at the 3rd mo of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon secretion is essential for aminoacid-induced hyperfiltration in man.

The role of glucagon as a mediator of aminoacid-induced alteration of renal haemodynamics was evaluated in man in three different protocols. In the first it was shown that the increase in glomerular filtration rate (GFR) and renal plasma flow (RPF) observed during an aminoacid infusion was prevented by the additional infusion of somatostatin (SRIF), but reproduced by a glucagon infusion in the presence of SRIF. In the second protocol it was shown that, at variance with normal subjects, six totally pancreatectomised patients, thus deprived of pancreatic glucagon secretion, did not increase their GFR and RPF when infused with amino-acids, whereas they exhibited the expected hyperfiltration when infused with glucagon. In the third protocol it was shown that glucagon infusion in a renal artery did not alter the homolateral renal haemodynamics. It is concluded that glucagon secretion is a mandatory step in the cascade of events linking the infusion of aminoacids to the renal hyperfiltration. Other steps beyond glucagon secretion are necessarily involved because glucagon has no direct renal effect.

Reproducible high yields of rat islets of Langerhans.

Isolation of islets of Langerhans from the pancreas by action of collagenase, a major breakthrough for physiological studies in vitro, has long appeared empirical, and the results were sometimes unpredictable. Isolation yields (number of islets obtained per pancreas) and their reproducibility, purification from exocrine remnants and vitality of the islets obtained, can improve owing to precise techniques, adapted to the architecture of collagen in the pancreas. We have tested four isolation-purification techniques in the rat pancreas. The best results were obtained by combining intra-ductal collagenase injection, with complete but moderate distension of the gland, avoiding leakage, multistep digestion (in situ and then in vitro), followed by purification on a discontinuous bovine serum albumin (BSA) gradient. Average yields were 670 +/- 40 islets per pancreas (range 570-800), versus 170 +/- 9 (range 40-350) with the technique used initially. The use of BSA discontinuous gradient improved the purification yield: 90-96% of islets obtained were concentrated in the 26/29% BSA interface. Furthermore, this technique shortened the duration of purification step: 1 hr (centrifugation gradient) vs 3 hrs (handpicked). It was verified that islets were morphologically free of exocrine tissue. Islet structure was well preserved either in conventional histology or insulin and glucagon immunoperoxidase staining. Islet vitality, as assessed by trypan blue exclusion test, was 100% of freshly isolated islets, and 89% after 24 hrs culture. Insulin secretory responses to a given stimulus were stronger than in the case of islets isolated by former techniques: 10-12 times the basal release (vs 5 times) with clear dose-response proportionality.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of islet cell antibodies in diabetic and nondiabetic BB rats.

The BB rat develops a spontaneous type I diabetic syndrome with anti-islet autoimmunity. Sera from diabetic and nondiabetic BB rats (from diabetes-prone litters), nondiabetic BB rats (from low-risk lines), and nondiabetes-prone Sprague-Dawley rats were collected twice a week from age 40 days to 160 days. Sera were tested for: (1) complement-dependent toxicity to 51Cr-labeled islet cells in vitro; (2) immunoglobulin binding to RIN-5 F insulinoma cells; and (3) ability to selectively suppress insulin secretion from normal islets in vitro. All sera from rats that subsequently became diabetic or glucose-intolerant were toxic to islet cells from various rat strains in the presence of complement. They were toxic neither to hepatocytes nor to fibroblasts. The toxic potency was associated with the globulin fraction. It was, in most cases, maximal either before or immediately after the onset of the disease. Sera from the nondiabetes-susceptible BB rats and the rats which, in diabetes-prone litters, died too early to be classified tended toward greater toxicity to islets. Immunoglobulins from diabetic sera bound to RIN-5 F cells more than did the serum globulins from other groups, their maximal binding capacity occurring after the onset of diabetes. Furthermore, BB diabetic sera were capable of selectively inhibiting the insulin secretion from normal rat islets in vitro either in the presence or, in some cases, in the absence of complement. The A- and D-cell functions were not suppressed. The combination of such results suggests the presence of one or more antibodies capable of binding to beta cells, inhibiting their function, and inducing their lysis.(ABSTRACT TRUNCATED AT 250 WORDS)