Bernard Billaudel

Vitamin D and pancreatic islet function I. Time course for changes in insulin secretion and content during vitamin D deprivation and repletion

After weaning, rats were given free access to a control or vitamin D-deprived diet for 2 to 5 weeks. In the vitamin D deficient rats, plasma concentrations of 25-(OH)D3, 1, 25-(OH)2D3,24,25-(OH)2D3 calcium, glucose and insulin were all decreased. After an overnight fast, the plasma insulin concentration was also decreased even when the plasma glucose concentration was not significantly affected. The food intake and body growth was also impaired in vitamin D-deficient rats. Administration of vitamin D3 in oil for 3 to 6 days to vitamin D-deficient rats increased the plasma concentration of vitamin D metabolites, calcium and insulin, but not that of glucose, and stimulated food intake and body growth to a larger extent than in rats treated by oil alone. Vitamin D deprivation decreased and vitamin D treatment increased the insulin content of the whole pancreas or isolated islets and the secretory response of the islets to D-glucose. The changes in insulin release remained significant when the hormonal output was expressed relative to the insulin content of the islets. These findings confirm that vitamin D deficiency causes alterations of pancreatic B-cell function. Moreover, the time course for changes in biological variables during vitamin D deprivation and treatment suggests that such an alteration cannot be solely accounted for by concomitant abnormalities in either plasma calcium or glucose concentrations.

Modulatory role of 1,25 dihydroxyvitamin D3 on pancreatic islet insulin release via the cyclic AMP pathway in the rat

Previous studies have shown that vitamin D3 deficiency impairs the insulin response to glucose via an alteration of signal transduction pathways, such as Ca2+ handling and the phosphoinositide pathway. In the present study the adenylyl cyclase pathway was examined in islets from 3 independent groups: normal rats, 4 weeks‐vitamin D3 deficient rats and one week‐1,25 dihydroxyvitamin D3 (1,25(OH)2D3) treated rats. We found that the very low rate of insulin release observed in vitamin D3 deficient rats could be restored in vitamin D3 deficient islets only with high concentrations of dioctanoyl‐cyclic AMP (DO‐cyclic AMP), whereas 1,25(OH)2D3 improved the sensitivity of the islets to this exogenous cyclic AMP analogue. The beneficial effect of 1,25(OH)2D3 observed with or without DO‐cyclic AMP was protein kinase A‐dependent, since the addition of N‐[2‐(p‐bromocinnamylamino) ethyl]‐5‐isoquinolinesulphonamide (H‐89), a specific inhibitor of cyclic AMP‐dependent protein kinases, decreased the insulin release of treated rats back to the level seen in vitamin D3 deficient islets. The low rate of insulin release could not be consistently related to an alteration in cyclic AMP content of the islets. Indeed, low insulin response to a barium + theophylline stimulus observed in vitamin D3 deficient islets was paradoxically associated with a supranormal cyclic AMP content in the islets. This paradoxical increase in cyclic AMP observed in these conditions could not be attributed to a lower total phosphodiesterase (PDE) activity, although the portion of Ca2+‐calmodulin‐independent PDE was predominant in islets from vitamin D3 deficient rats. On the other hand, the higher cyclic AMP content of vitamin D3 deficient islets could be related to an increase in glucagon‐induced cyclic AMP synthesis in relation to the hyperglucagonaemia previously observed in vitamin D3 deficient rats. Since higher concentrations of exogenous glucagon and higher endogenous cyclic AMP concentrations were required in vitro to restore insulin release to normal values, the cyclic AMP‐dependent pathways that usually potentiate insulin secretion appeared to be less efficient in relation to an alteration in the post cyclic AMP effector system. 1,25(OH)2D3 exerted a stimulating effect on insulin release via protein kinase A activation but reduced the supranormal cyclic AMP synthesis, thus exerting a differential modulatory influence on biochemical disturbances in islets induced by vitamin D3 deficiency.

Cholinergic stimulation of ion fluxes in pancreatic islets

Cholinergic agents are known to stimulate the hydrolysis of polyphosphoinositides in pancreatic islets. The effect of carbamylcholine upon ion fluxes in the islet cells was investigated. Carbamylcholine provoked a rapid but poorly sustained increase in 45Ca and 86Rb outflow from perifused islets. Such a cationic response was observed at different glucose concentrations (zero to 16.7 mM), at three concentrations of carbamylcholine (10 microM, 100 microM and 1.0 mM), and in the absence or presence of extracellular Ca2+. It coincided with a biphasic stimulation of insulin release, both the cationic and secretory responses being abolished in the presence of atropine (10 microM). At variance with nutrient secretagogues, carbamylcholine failed to affect the net production of cyclic AMP and caused a transient decrease in 32P outflow from islets prelabelled with [32P]phosphate. It is proposed that cholinergic agents mobilize Ca2+ from intracellular sites, possibly through generation of inositol, 1,4,5-triphosphate from phosphatidylinositol 4,5-bisphosphate. The intracellular redistribution of Ca2+ does not appear sufficient, however, to account fully for the secretory response, which may also involve activation of protein kinase C by diacylglycerol.

Rat fertility and embryo fetal development: Influence of exposure to the Wi-Fi signal

In recent decades, concern has been growing about decreasing fecundity and fertility in the human population. Exposure to non-ionizing electromagnetic fields (EMF), especially radiofrequency (RF) fields used in wireless communications has been suggested as a potential risk factor. For the first time, we evaluated the effects of exposure to the 2450MHz Wi-Fi signal (1h/day, 6days/week) on the reproductive system of male and female Wistar rats, pre-exposed to Wi-Fi during sexual maturation. Exposure lasted 3 weeks (males) or 2 weeks (females), then animals were mated and couples exposed for 3 more weeks. On the day before delivery, the fetuses were observed for lethality, abnormalities, and clinical signs. In our experiment, no deleterious effects of Wi-Fi exposure on rat male and female reproductive organs and fertility were observed for 1h per days. No macroscopic abnormalities in fetuses were noted, even at the critical level of 4W/kg.

Effects of Head-Only Exposure of Rats to GSM-900 on Blood-Brain Barrier Permeability and Neuronal Degeneration

Abstract Salford et al. reported in 2003 that a single 2-h exposure to GSM-900 mobile telephony signals induced brain damage (increased permeability of the blood-brain barrier and presence of dark neurons) 50 days after exposure. In our study, 16 Fischer 344 rats (14 weeks old) were exposed head-only to the GSM-900 signal for 2 h at various brain-averaged SARs (0, 0.14 and 2.0 W/kg) or were used as cage or positive controls. Albumin leakage and neuron degeneration were evaluated 14 and 50 days after exposure. No apoptotic neurons were found 14 days after the last exposure using the TUNEL method. No statistically significant albumin leakage was observed. Neuronal degeneration, assessed using cresyl violet or the more specific marker Fluoro-Jade B, was not significantly different among the tested groups. No apoptotic neurons were detected. The findings of our study did not confirm the previous results of Salford et al.

Effect of In Utero Wi-Fi Exposure on the Pre- and Postnatal Development of Rats

BACKGROUND The increase in exposure to the Wireless Fidelity (Wi-Fi) wireless communication signal has raised public health concerns especially for young people. Animal studies looking at the effects of early life and prenatal exposure to this source of electromagnetic fields, in the radiofrequency (RF) range, on development and behavior have been considered as high priority research needs by the World Health Organization. METHODS For the first time, our study assessed the effects of in utero exposure to a 2450 MHz Wi-Fi signal (2 hr/day, 6 days/week for 18 days) on pregnant rats and their pups. Three levels in terms of whole-body specific absorption rate were used: 0.08, 0.4, and 4 W/kg. The prenatal study on fetuses delivered by caesarean (P20) concerned five females/group. The dams and their offspring were observed for 28 days after delivery (15 females/group). RESULTS For all test conditions, no abnormalities were noted in the pregnant rats and no significant signs of toxicity were observed in the pre- and postnatal development of the pups, even at the highest level of 4 W/kg. CONCLUSIONS In the present study, no teratogenic effect of repeated exposures to the Wi-Fi wireless communication signal was demonstrated even at the highest level of 4 W/kg. The results from this screening study aimed at investigating Wi-Fi effects, strengthen the previous conclusions that teratology and development studies have not detected any noxious effects of exposures to mobile telephony-related RF fields at exposure levels below standard limits.

Human skin cell stress response to GSM-900 mobile phone signals. In vitro study on isolated primary cells and reconstructed epidermis

In recent years, possible health hazards due to radiofrequency radiation (RFR) emitted by mobile phones have been investigated. Because several publications have suggested that RFR is stressful, we explored the potential biological effects of Global System for Mobile phone communication at 900 MHz (GSM‐900) exposure on cultures of isolated human skin cells and human reconstructed epidermis (hRE) using human keratinocytes. As cell stress markers, we studied Hsc70, Hsp27 and Hsp70 heat shock protein (HSP) expression and epidermis thickness, as well as cell proliferation and apoptosis. Cells were exposed to GSM‐900 under optimal culture conditions, for 48 h, using a specific absorption rate (SAR) of 2 W·kg−1. This SAR level represents the recommended limit for local exposure to a mobile phone. The various biological parameters were analysed immediately after exposure. Apoptosis was not induced in isolated cells and there was no alteration in hRE thickness or proliferation. No change in HSP expression was observed in isolated keratinocytes. By contrast, a slight but significant increase in Hsp70 expression was observed in hREs after 3 and 5 weeks of culture. Moreover, fibroblasts showed a significant decrease in Hsc70, depending on the culture conditions. These results suggest that adaptive cell behaviour in response to RFR exposure, depending on the cell type and culture conditions, is unlikely to have deleterious effects at the skin level.

Stimulatory effect of 1,25-dihydroxyvitamin D3 on calcium handling and insulin secretion by islets from vitamin D3-deficient rats.

Among the various vitamin D3 metabolites, 1,25-(OH)2D3 is the specific secosteroid hormone that can enhance, in vitro, the weak insulin response to glucose of islets from vitamin D3-deficient rats. Because this potentiating effect is preceded by an increase in Ca2+ handling, several putative sites of action were studied by measuring 45Ca2+ and 86Rb+ (as K+ tracer) efflux during perifusions in the presence of various stimuli known to affect Ca2+ movements in different ways: high glucose without calcium, high calcium without glucose, high potassium, or barium-theophylline without calcium or glucose. The present results show that 1,25-(OH)2D3 may activate Ca2+ handling by at least two mechanisms: (1) an increase of Ca2+ entry via voltage-dependent Ca2+ channels in the experiments in which extracellular Ca2+ was present and where Ca2+ channels were opened; this 1,25-(OH)2D3 influence on Ca2+ channels was not mediated by a possible indirect influence on K+ channels because 86Rb+ fluxes were never observed to be affected by the steroid; (2) an enhancement of 45Ca2+ mobilization from intracellular stores as suggested by barium-theophylline stimulation and probably also via the Ca2+ stimulus. Both of these 1,25-(OH)2D3 influences tended to provide more calcium to the B cell of vitamin D3-deficient rats. But this prerequisite was not sufficient in itself to potentiate the insulin response; indeed, experiments with barium-theophylline suggested that 1,25-(OH)2D3 may also activate a cAMP-dependent exocytosis process.

Regulatory effect of 1,25-dihydroxyvitamin D3 on insulin release and calcium handling via the phospholipid pathway in islets from vitamin D-deficient rats.

The effect of 10−8 M 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3] on the phosphoinositide pathway, was studied on [3H] inositol and 45Ca2+ efflux and on insulin release of islets from vitamin D-deficient rats, during an acetylcholine (Ach) stimulus in perifusion. The insulin release, which was low in vitamin D-deficient rats, was enhanced by this treatment. The 3H flux, reflecting phosphoinositide breakdown, was also increased. The 45Ca2+ flux was stimulated both during the first 14 min peak (mobilization of IP3-sensitive reticular Ca2+ stores) and during the following sustained small elevation of 45Ca2+ flux, reflecting protein kinase C (PKC) activation and consequently increased phosphorylation of Ca2+ channel proteins. These effects were larger during perifusions performed in the presence of glucose which is known to open Ca2+ channels, suggesting a synergistic influence of glucose and 1,25(OH)2D3. This positive influence of 1,25(OH)2D3 on Ca2+ entry by Ca2+ channels was confirmed by the use of nifedipine — a Ca2+ channel blocker-which suppressed the 45Ca2+ flux and lowered insulin secretion. Moreover, the sustained 45Ca2+ flux also disappeared in islets from vitamin D-deficient rats supplemented by 1,25(OH)2D3 but perifused without extracellular Ca2+, supporting the hypothesis of 1,25(OH)2D3-induced activation of PKC. Thus, 1,25(OH)2D3 may provide supplementary calcium to the B cell by regulating the intracellular signalling processes involving phospholipid metabolism, PKC induction, Ca2+ mobilization and Ca2+ entry by Ca2+ channels.

Vitamin D and pancreatic islet function II. Dynamics of insulin release and cationic fluxes

Pancreatic islets were prepared from control and vitamin D-deprived rats 2 or 5 weeks after weaning and, in the latter case, after 3 or 6 days treatment with exogenous vitamin D3 (60 nmol per day). The islets were prelabelled with both 86Rb and 45Ca and placed in a perifusion chamber. Vitamin D deprivation or administration failed to affect 86Rb outflow whether prior or after stimulation of the islets by a rise in either extracellular D-glucose or Ca2+ concentration. However, vitamin D deprivation decreased and vitamin D administration enhanced the basal 45Ca fractional outflow rate, as well as the magnitude of changes in both 45Ca and insulin release evoked by the rise in either D-glucose or extracellular Ca2+. It is proposed that the alteration in 45Ca fluxes and insulin release attributable to changes in the supply of vitamin D are, to a large extent, independent of the changes in nutrient catabolism conceivably associated with vitamin D deprivation and administration.