Marielle Gascon-Barré

Calcium is essential in normalizing intolerance to glucose that accompanies vitamin D depletion in vivo.

Vitamin D is essential for normal insulin secretion in vivo and in vitro. The differential effect of calcium and of the vitamin D endocrine system in the insulin response to secretagogues is still a subject of debate. To study the roles of calcium and the vitamin D system in the in v vitamin D depletion and hypocalcemia or vitamin D depletion supplemented with calcium alone for 3, 7, or 14 days, vitamin D3 (6.5 nmol/day × 7 days), or 1,25(OH)2D3 (28 pmol/day × 7 days). Serum calcium was 1.28 ± 0.04 mM in hypocalcemic vitamin D-depleted rats, 1.47 ± 0.06 (NS), 1.8 ± 0.2 (P <0.0002), and 2.04 ± 0.07 (P < 0.0001) mM after 3, 7, or 14 days, respectively, of calcium supplementation; vitamin D3- or 1,25(OH)2D3- supplemented animals had serum calcium of 2.61 ± 0.04 or 2.48 ± 0.05 mM (P 67 < 0.0001 vs. hypocalcemic vitamin D-depleted rats). Rats with hypocalcemia and vitamin D depletion had significantly higher glucose concentrations (P < 0.0005) and lower insulin response during GTT than all other groups (P < 0.001). Differences in insulin sensitivity could not account for differences in response because exogenous insulin administration led to a similar drop in glucose concentrations in all groups, with the nadir averaging 51.7 ± 2.6% of initial values. To distinguish between calcium and the vitamin D system in the GTT response, rats were treated with a nonhypercalcemic analogue of 1,25(OH)2D3, OCT (28 pmol/day × 4-7days) with or without dietary calcium. Serum calcium was 1.23 ± 0.04 vs. 2.09 ± 0.02 mM in the absence or presence of dietary calcium (P < 0.0001), but normalization of GTT only happened in the presence of calcium. A time course of the calcium effect in vitamin D-depleted rats indicated that 7 days of high dietary calcium intake was needed to normalize GTT, with a significant correlation coefficient being observed between serum calcium and the maximum insulin response (r = 0.5172, P < 0.004). Our data indicate that vitamin D depletion with hypocalcemia is associated with normal insulin sensitivity but glucose intolerance caused by inadequate insulin secretion in response to glucose. Calcium alone contributes in normalizing GTT, whereas the 1,25(OH)2D3 analogue OCT is unable to normalize GTT at a dose equimolar to that of 1,25(OH)2D3 in the absence of calcium, suggesting that hypocalcemia predominates over vitamin D depletion in the glucose intolerance of vitamin D deficiency.

Estimation of collagen content of liver specimens. Variation among animals and among hepatic lobes in cirrhotic rats

We undertook a study to evaluate the correlation between morphometric evaluation and colorimetric determination of hepatic collagen content, and to analyze the variation among animals as well as among lobes of the same liver in hepatic collagen content after CCl4-induced micronodular cirrhosis. The results revealed a significant correlation (r = 0.9458; p less than 0.001) between the morphometric and colorimetric methods of collagen evaluation of liver specimens; both methods also significantly distinguished data obtained from controls and from cirrhotic rats (p less than 0.0005). After induction of micronodular cirrhosis by chronic CCl4 administration, a highly significant variation in hepatic collagen content was observed among animals (p less than 0.0001). By contrast, no significant difference in collagen content was observed (p less than 0.05) among hepatic lobes of a given animal. These results indicate that in this animal model of liver cirrhosis, interpretation of biochemical data would benefit by being related to the severity of the hepatic collagen infiltration of each animal. Our data also show that representative values for total hepatic collagen infiltration can be obtained from a single liver specimen; we suggest, however, that the specimen be taken from a major lobe of the liver and that a sufficiently large number of animals be used to avoid occasional sampling errors.

Reduced Hepatic Synthesis of Calcidiol in Uremia

Calcidiol insufficiency is highly prevalent in chronic kidney disease (CKD), but the reasons for this are incompletely understood. CKD associates with a decrease in liver cytochrome P450 (CYP450) enzymes, and specific CYP450 isoforms mediate vitamin D(3) C-25-hydroxylation, which forms calcidiol. Abnormal levels of parathyroid hormone (PTH), which also modulates liver CYP450, could also contribute to the decrease in liver CYP450 associated with CKD. Here, we evaluated the effects of PTH and uremia on liver CYP450 isoforms involved in calcidiol synthesis in rats. Uremic rats had 52% lower concentrations of serum calcidiol than control rats (P < 0.002). Compared with controls, uremic rats produced 71% less calcidiol and 48% less calcitriol after the administration of vitamin D(3) or 1alpha-hydroxyvitamin D(3), respectively, suggesting impaired C-25-hydroxylation of vitamin D(3). Furthermore, uremia associated with a reduction of liver CYP2C11, 2J3, 3A2, and 27A1. Parathyroidectomy prevented the uremia-associated decreases in calcidiol and liver CYP450 isoforms. In conclusion, these data suggest that uremia decreases calcidiol synthesis secondary to a PTH-mediated reduction in liver CYP450 isoforms.

Calcitriol regulates the expression of the genes encoding the three key vitamin D3 hydroxylases and the drug‐metabolizing enzyme CYP3A4 in the human fetal intestine

background and aims The human fetal jejunum has been shown to harbour the vitamin D3 (D3) nuclear receptor (VDRn) and to be responsive to calcitriol/1,25‐dihydroxyvitamin D3[1,25(OH)2D3] through modulation of proliferation and differentiation processes. The aim of the study was to evaluate the presence as well as the effect of 1,25(OH)2D3 exposure on the expression levels of the three key D3‐hydroxylase gene transcripts (25‐hydroxylase, CYP27A; 24‐hydroxylase, CYP24; 1α‐hydroxylase, CYP27B1) as well as that of the 1,25(OH)2D3‐responsive endobiotic/xenobiotic metabolizing enzyme CYP3A4 (which is also considered a major detoxifiying enzyme) in the human proximal and distal intestine.methods Specimens from normal fetuses ranging from 15 to 20 weeks of gestation were obtained following elective termination of normal pregnancies. Intestinal explants were cultured for a period of 24 h or 48 h with 10−7 m 1,25(OH)2D3. All data were compared to paired‐control cultures without 1,25(OH)2D3. Total RNA was extracted and cDNA synthesized by RT‐PCR. The cDNA obtained was amplified by radioactive PCR, the signal intensity evaluated by densitometric analyses and expressed in relation to the levels of GAPDH.

Interrelationships between vitamin D3 and 25-Hydroxyvitamin D3 during chronic ethanol administration in the rat☆

Vitamin D [D] depleted female Sprague-Dawley rats were fed for a period of 4 wk a D deficient diet containing 36% of total calories as ethanol while control animals received an isocaloric regimen where ethanol was substituted for by dextrins. In conjunction with the ethanol feeding 92 I.U. of [14C]-vitamin D3 [(14C)-D3] were administered by intragastric gavage 3 times 1 wk for 3 2/3 wk. At the end of the experiment, [14C]-D3 and [14C]-25-hydroxyvitamin D3 [(14C)-25(OH)D3] concentrations were analyzed in plasma, liver, striated muscle and adipose tissue. Body reserves in unchanged [14C]-D3 were significantly reduced by ethanol treatment as seen by 24%, 26%, and 59% lower plasma (p less than 0.02), muscle (p less than 0.001) and adipose tissue (p less than 0.001) [14C]-D3 concentrations in ethanol-treated compared to control rats. In contrast total plasma and liver [14C]-25(OH)D3 content were increased by 30% (p less than 0.05) and 55% (p less than 0.001) respectively. This increased liver and plasma [14C]-25(OH)D3 following ethanol treatment was not accompanied by a proportional [14C]-25(OH)D3 incorporation into muscle and adipose tissue. These results suggest that during steady state conditions 25(OH)D3 production is increased during chronic ethanol administration while the body pool in unchanged D3 is significantly lowered. These results also point out that in the rat plasma 25(OH)D concentrations are not a reliable guide for the determination of vitamin D status during chronic ethanol administration.

Influence of calcium or 1,25-dihydroxyvitamin D3 supplementation on the hepatic microsomal and in vivo metabolism of vitamin D3 in vitamin D-depleted rats.

Hypocalcemic vitamin D (D)-depleted rats were supplemented with calcium or 1,25(OH)2D3, and the metabolism of D3 to 25(OH)D3 was studied. Infusion with 7 or 65 pmol 1,25(OH)2D3 X 24 h-1 led to normal or slight hypercalcemia associated with physiological and supraphysiological plasma concentrations of the hormone while calcium supplementation normalized plasma calcium despite 1,25(OH)2D3 concentrations as low as those observed in hypocalcemic controls. Constant administrations of [14C]D3 during the supplementation regimens uncovered a stimulation of the in vivo 25(OH)D3 production by calcium supplementation; this was further confirmed in vitro by an increase in the hepatic microsomal D3-25 hydroxylase. The group supplemented with pharmacological doses of the hormone displayed lower circulating concentrations of both D3 and 25(OH)D3 while the in vitro 25(OH)D3 production remained unaffected by 1,25(OH)2D3. Investigation of the kinetics of intravenous 25(OH)[3H]D3 revealed similar elimination constants in all groups. The data indicate that calcium supplementation of hypocalcemic D-depleted rats results in an increased transformation of D3 into 25(OH)D3 while supplementation with 1,25(OH)2D3 does not affect the in vitro D3-25 hydroxylase but seems to influence the in vivo handling of the vitamin by accelerating its metabolism.

Chronic hypocalcemia of vitamin D deficiency leads to lower intracellular calcium concentrations in rat hepatocytes.

Several lines of evidence indicate that calcium deficiency is associated with cellular defects in many tissues and organs. Owing to the large in vivo gradient between ionized extra- and intracellular Ca2+ concentrations ([Ca2+]i), it is generally recognized that the prevailing circulating Ca2+ does not significantly affect resting cytosolic Ca2+. To probe the consequences of hypocalcemia on [Ca2+]i, a model of chronic hypocalcemia secondary to vitamin D (D) deficiency was used. Hepatocytes were isolated from livers of hypocalcemic D-deficient, of normocalcemic D3-repleted, or of normal control rats presenting serum Ca2+ of 0.78 +/- 0.02, 1.24 +/- 0.03, or 1.25 +/- 0.01 mM, respectively (P < 0.0001). [Ca2+]i was measured in cell couplets using the fluorescent probe Fura-2. Hepatocytes of normocalcemic D3-repleted and of normal controls exhibited similar [Ca2+]i of 227 +/- 10 and 242 +/- 9 nM, respectively (NS), whereas those of hypocalcemic rats had significantly lower resting [Ca2+]i (172 +/- 10 nM; P < 0.0003). Stimulation of hepatocytes with the alpha 1-adrenoreceptor agonist phenylephrine illicited increases in cytosolic Ca2+ leading to similar [Ca2+]i and phosphorylase a (a Ca(2+)-dependent enzyme) activity in all groups but in contrast to normocalcemia, low extracellular Ca2+ was often accompanied by a rapid decay in the sustained phase of the [Ca2+]i response. When stimulated with the powerful hepatic mitogen epidermal growth factor (EGF), hepatocytes isolated from hypocalcemic rat livers responded with a blunted maximal [Ca2+]i of 237.6 +/- 18.7 compared with 605.2 +/- 89.9 nM (P < 0.0001) for their normal counterparts, while the EGF-mediated DNA synthesis response was reduced by 50% by the hypocalcemic condition (P < 0.03). Further studies on the possible mechanisms involved in the perturbed [Ca2+]i homeostasis associated with chronic hypocalcemia revealed the presence of an unchanged plasma membrane Ca2+ ATPase but of a significant decrease in agonist-stimulated Ca2+ entry as indicated using Mn2+ as surrogate ion (P < 0.03). Our data, thus indicate that, in rat hepatocytes, the in vivo calcium status significantly affects resting [Ca2+]i, and from this we raise the hypothesis that this lower than normal [Ca2+]i may be linked, in calcium disorders, to inappropriate cell responses mediated through the calcium signaling pathway as illustrated by the response to phenylephrine and EGF.

Implication of caspases and subcellular compartments in tert-butylhydroperoxide induced apoptosis

Oxidative stress has been implicated in many physiopathologies including neurodegenerative diseases, cancer, cardiovascular and respiratory diseases, and in mechanisms of action of environmental toxicants. tert-butylhydroperoxide (t-BHP) is an organic lipid hydroperoxide analogue, which is commonly used as a pro-oxidant for evaluating mechanisms involving oxidative stress in cells and tissues. This study investigates mechanisms of apoptosis induced by oxidative stress in hepatocytes, in particular, the involvement of caspases and subcellular compartments. Freshly isolated hepatocytes were exposed to 0.4 mM t-BHP during 1 h. A general caspase inhibitor, Boc-D-FMK, reduced t-BHP-induced apoptosis (chromatin condensation), confirming the involvement of caspases in apoptosis. A caspase-9 inhibitor, Z-LEHD-FMK, also reduced t-BHP-induced apoptosis, suggesting that caspase-9 plays a critical role in this process. Procaspase-9 underwent cleavage in mitochondria and translocation to the nucleus, where increased caspase-9 activity was detected. The caspase-9 substrates, caspase-3 and caspase-7, were not activated. Caspase-7 was translocated from the cytosol to the endoplasmic reticulum (ER), where it underwent processing; however, enzymatic activity of caspase-7 was inhibited by t-BHP. t-BHP caused cleavage of procaspase-12 at the ER and its subsequent translocation to the nucleus, where increased caspase-12 activity was found. t-BHP caused translocation of calpain from the cytosol to the ER. Calpain inhibition reduced chromatin condensation and caspase-12 activity in the nucleus, suggesting that calpain is involved in caspase-12 activation and apoptosis. This study demonstrates that caspase-9 and caspase-12 are activated in t-BHP-induced apoptosis in hepatocytes. We highlight the importance of subcellular compartments such as mitochondria, ER and nuclei in the apoptotic process.

Low calcium diet in dogs causes a greater increase in parathyroid function measured with an intact hormone than with a carboxylterminal assay

The influence of low calcium diets (0.08%) with or without a deficiency in vitamin D (D) on the parathyroid function was studied in two groups of six dogs. The animals were first studied on a normal diet and then again after 3 weeks of the experimental diet. Blood tests, urine tests and a functional evaluation of the parathyroid glands via i.v. infusions of CaCl2 and NaEDTA were performed on both occasions. PTH was measured with an intact hormone assay (I) and with a carboxylterminal assay (C). Since similar results were observed on the D deficient and D normal diets at 3 weeks, data were combined for final analysis. We observed an increase in fasting serum PTH (I, 3.2 +/- 2.0 vs. 4.3 +/- 3.3 pmol/l, P less than 0.05; C, 23.4 +/- 13.9 vs. 30.7 +/- 15.5 pmol/l, P less than 0.005) and in stimulated serum PTH (I, 11.7 +/- 2.7 vs. 18.3 +/- 4.5 pmol/l, P less than 0.0005; C, 67.7 +/- 22 vs. 90.4 +/- 31.1 pmol/l, P less than 0.0005) after 3 weeks of a low calcium diet. Fasting ionized calcium concentrations (1.36 +/- 0.03 vs. 1.36 +/- 0.02 mmol/l), 25(OH)D concentrations (94.8 +/- 28 vs. 86.7 +/- 23.1 nmol/l) and 1,25(OH)2D concentrations (101.1 +/- 19.3 vs. 110.9 +/- 27.6 pmol/l) did not change. The increase in parathyroid function measured with I (60.4 +/- 39%) was greater than that measured with C (33.7 +/- 14.2%, P less than 0.05) and the ratio of maximum carboxylterminal PTH to maximum intact PTH decreased from 5.98 +/- 2.17 to 4.95 +/- 1.21 (P less than 0.05) at 3 weeks. These results suggest that reduced catabolism of intact PTH was involved in the increased parathyroid function. The stimulus responsible for the increased parathyroid function remains to be identified.

Folate deficiency among institutionalized elderly. Public health impact.

All patients 65 years and older who had been living in an institution for at least six months had a measurement of red blood cell folate (RBCF), serum folate (SF), vitamin B12, hemoglobin, and mean corpuscular volume. Twenty‐two percent of the 97 studied subjects were found deficient in RBCF, although only 6% had anemia. Length of stay at the institution was significantly associated with deficiency in RBCF and this association was uncon‐founded by age. The proportion of RBCF deficiency among the studied subjects attributable to length of stay is 80%. The prophylactic administration of folate among long‐term nursing home type of patients should be considered.