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Les vitamines D et K sont des micronutriments liposolubles qui participent au bon fonctionnement de l’organisme. Elles jouent des rôles clés dans la prévention de trouble de l'hémostase et de la coagulation, des pathologies osseuses, métaboliques et cardiovasculaires. Cependant, même si ces vitamines sont apportées en quantités suffisantes par notre alimentation, leurs effets bénéfiques sont étroitement conditionnés par leur biodisponibilité. Or, mieux connaitre les mécanismes d’absorption permettrait d’appréhender leur biodisponibilité. Nous avons tout d’abord montré que l’absorption de la vitamine K implique des transporteurs du cholestérol, SR-B1 et CD36. Nous avons également montré que l’entérocyte est non seulement capable d’effluer les vitamines D et K néo-absorbées mais également d’excréter ces vitamines du compartiment sanguin vers la lumière intestinale. Ce phénomène bien connu pour le cholestérol (excrétion transintestinale du cholestérol) implique des transporteurs communs, dont ABCB1 et ABCG5/G8. Dans un second temps, dans le cadre de la relance de la consommation des légumineuses, nous avons mis en évidence que la présence de légumineuses dans un repas limite la biodisponibilité de ces vitamines. En effet, les fibres, phytates, saponines et tanins diminuent leur bioaccessibilité et/ou leur captage. La méthode de cuisson des légumineuses, en affectant leur composition nutritionnelle, peut moduler l’incorporation des vitamines D et K au sein des micelles mixtes et donc affecter leur biodisponibilité. Ces données soulignent ainsi le fait que les légumineuses doivent être cuites de manière appropriée et consommés dans des repas riches en micronutriments. / Vitamin D and K are fat-soluble micronutrients that participate to the proper functioning of the organism. They are essential to prevent bleeding, bone, metabolic and cardiovascular disorders. However, even if those vitamins are provided in sufficient quantities in our diet, their health effects are closely linked to their bioavailability. A better knowledge of their absorption mechanisms would help to optimize their bioavailability.Firstly, we showed that vitamin K absorption involves the cholesterol transporters SR-B1 and CD36. We also showed that enterocytes can not only efflux newly absorbed vitamins D and K but also excrete vitamin D and K from the blood compartment to the intestinal lumen. This phenomenon of transintestinal excretioninvolves the cholesterol membrane transporters ABCB1 and ABCG5/G8.Secondly, we showed that the presence of pulses within a meal limits vitamin D and K bioavailability. Indeed, fibers, phytates, saponins and tannins can decrease bioaccessibility and/or uptake of vitamin K. By modulating the nutritional profile of pulses, the cooking method can impact on fat-soluble vitamin transfer to mixed micelles, and in turn affect their bioavailability. These data suggest that pulses must be cooked in an appropriate manner and consumed in micronutrient-rich meals.Keywords: vitamin D, vitamin K, bioaccessibility, intestinal absorption, pulses.

Two trials were conducted using 48 infant pigs of both sexes. Pigs were taken from the sow when three to seven days old and placed in wire-bottomed metal cages by pairs. Animals in the first trial received synthetic diets containing 100 or 1000 international units of ergocalciferol and 1000 or 4000 international units of retinyl acetate or four or sixteen milligrams of beta carotene per kilogram of feed. Baby pigs in the second trial received the same synthetic diet plus 1000 international units of ergocalciferol (D2) or cholecalciferol (D3) and 2000 international units of vitamin A or eight milligrams of beta carotene per kilogram of feed. Neither growth nor feed efficiency were influenced by diet, but in Trial II, pigs receiving vitamin D2 showed higher gains per kilogram of diet than pigs fed vitamin D3. The weight and density of ribs and femurs did not show any differences due to diet. Different sources and levels of vitamins A and D did not effect strength characteristics of femurs. However, trends indicated 1000 IU of vitamin D in Trial I and vitamin D2 in Trial II slightly increased the bone ash in comparison with beta carotene and in Trial II retinyl acetate with vitamin D2 resulted in the maximum amount of mineral deposition in bone. Liver as a percent of body weight was higher in pigs treated with beta carotene than in those fed retinyl acetate either with vitamin D2 or D3. Serum total sulfur was increased in pigs receiving vitamin D3 in Trial II. Inorganic sulfur in Trial I was lowered significantly by high levels of vitamin D and either low beta carotene, low retinyl acetate, or high beta carotene. There were not significant effects of level and source of vitamin A or D on uronic acid or total sulfur concentration in cartilage. However, on the high level of dietary vitamin D, uronic acid in cartilage was slightly increased (Trial I). Trends indicated that beta carotene increased and high levels of vitamin D lowered total sulfur in cartilage (Trial I).

Vitamin D has been connected with increased intramyocellular lipid (IMCL) mitochondrial function in skeletal muscle. It is also shown to prevent lipotoxicity in several tissues, but this has not yet been examined in skeletal muscle. Perilipin 2 (PLIN2), a lipid droplet protein upregulated with vitamin D treatment, is integral to managing IMCL capacity and lipid oxidation in skeletal muscle. Increased lipid storage and oxidation is associated with increased tolerance to a hyperlipidic environment and resistance to lipotoxicity. Therefore, I hypothesized that vitamin D increases β-oxidation and lipid turnover though a PLIN2 mediated mechanism, thereby preventing lipotoxicity. This hypothesis was divided into two specific aims: 1) Characterize the effect of vitamin D and PLIN2 on lipid turnover and β-oxidation in mature myotubes, and 2) Determine the role of vitamin D and PLIN2 in regulating key markers of lipotoxicity. To address these aims, cells were treated with or without vitamin D, palmitate, and PLIN2 siRNA in an eight group, 2x2x2 design. Key experiments included quantitative real time polymerase chain reaction for markers of lipid accumulation, lipolysis, and lipotoxicity; Seahorse oxygen consumption assay; 14C-palmitate oxidation assay; and analyses of lipid accumulation and profile. Failure of the palmitate treatment to produce a reliable model for lipotoxicity resulted in negative data for Aim 2 of this dissertation and a focus on vitamin D and PLIN2 knockdown treatments as a four group, 2x2 model. Aim 1 showed that vitamin D reliably increases markers of lipolysis and lipid accumulation. Most of these markers were in turn decreased after PLIN2 knockdown, and DGAT2 exhibited an interaction effect between the two treatments. Contrary to our hypothesis and some published research, PLIN2 knockdown did not prevent lipid accumulation. Vitamin D increased oxygen consumption, especially consumption driven by mitochondrial complex II. PLIN2 knockdown decreased oxygen consumption and demonstrated an interaction effect specific to mitochondrial complex II. Data in this dissertation show that vitamin D increases mitochondrial function, and these effects are at least in part accomplished through a PLIN2 mediated mechanism. However, this work lacks the data required to make specific claims regarding β-oxidation and lipid turnover. This research is some of the first to show that PLIN2 knockdown carries negative impacts for skeletal muscle mitochondria and makes valuable contributions to general knowledge of how vitamin D and lipid storage impact muscle health and function. This ultimately provides additional evidence to advocate for vitamin D supplementation as a means of improving musculoskeletal health and function. Future research should investigate how vitamin D and PLIN2 impact markers of lipotoxicity in skeletal muscle.

Vitamin D is hydroxylated to form several active metabolites, of these, 1,25- dihydroxyvitamin D3 [1,25(OH)2D3] is the most studied stimulatory product. It is now accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate homeostasis through its membrane receptor 1,25D3-MARRS (membrane associated rapid response steroid binding) protein. Another metabolite, 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] has been reported to be inhibitory with respect to calcium and phosphate absorption in intestine. Previous work in this laboratory has indicated that 24,25(OH)2D3 inhibits phosphate uptake in isolated intestinal cells and perfused duodenal loops and in vivo. This thesis further tested the hypothesis that the actions of 24,25(OH)2D3 on phosphate homeostasis are physiologically important. Catalase has been identified as a binding protein for 24,25(OH)2D3. We determined the localization of catalase in the presence and absence of steroid, monitored catalase mRNA levels related to gene 24,25(OH)2D3 gene transcription regulation. We studied the effects of the two isomers of 24,25(OH)2D3 on localization of catalase in chicken enterocytes over a time course of 15 sec to 60 min. It was demonstrated that 24R,25(OH)2D3 is the effective metabolite for catalase redistribution in vitro. We also studied the effects of vitamin D on catalase and phosphate uptake in chicken intestinal cells. It was once again demonstrated that 24R,25(OH)2D3 is the effective metabolite for decreasing phosphate uptake and catalase gene expression. These combined results lead us to conclude that 24,25(OH)2D3 is an important hormone in phosphate homeostasis in chick intestinal epithelial cells.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to mediate the rapid, non-nuclear stimulation of calcium and phosphate transport in chick intestine through binding to a receptor localized in the basal lateral membrane. By using an antibody to the N-terminus of the membrane receptor, studies were undertaken to determine whether a comparable protein exists in kidney and brain, and whether it is present in a particular subcellular fraction. The first step was to establish fractionation protocols to separate subcellular organelles as judged by marker enzyme analyses. Differential centrifugation and Percoll gradient fractions were prepared from chick kidney and brain whole homogenates by two methods (method 1 and method 2). Protein and marker enzymes were analyzed in each fraction to determine the distribution of organelles. By method 1, the organelles were not adequately separated. By method 2, chick kidney and brain were found to have the same order of organelle distribution: In the post-nuclear pellet (P2), fraction 1 was found to be enriched for the lysosomal marker acid phosphatase; fractions 2-5 were found to be enriched for the mitochondrial marker succinate dehydrogenase; fraction 8 was found to be enriched for the Golgi marker α-D-mannosidase; and fraction 9 was found to be enriched for the plasma membrane marker Na+,K+ ATPase. In Percoll gradients of microsomal membranes prepared from the 100,000xg pellet (P3), fractions 1-3 contained the endoplasmic reticulum marker enzyme activity glucose-6-phosphatase. Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and Western blots were performed using the antibody to 1,25(OH)2D3 receptors in chick intestinal basal lateral membrane. The areas of the bands were scanned by computer, and analyzed quantitatively. After establishing a suitable protein concentration for Western analysis, differential centrifugation and Percoll gradient fractions were analyzed. Finally, the specific binding of [3H]1,25(OH)2D3 was determined in Percoll gradient fractions to assess whether the receptor is functional. Plasma membrane 1,25(OH)2D3 receptors were found in both chick kidney and brain cells. Golgi membranes also were found to have receptor activity, perhaps since this organelle packages proteins for delivery to other membranes. In kidney, fraction P27 demonstrated a very high receptor activity, and [3H]1,25(OH)2D3 specific binding assays showed these membrane receptors are functional. Although this fraction lacks traditional marker enzyme activity, it may contain endocytic vesicles. The physiological function and the mechanism of action of plasma membrane receptors in these two tissues remain to be determined.

1,25-dihydroxyvitamin D3 (1,25VD), the active form of vitamin D (VD), suppresses the growth of numerous human cancer cell lines by inhibiting cell cycle progression and inducing cell death. Genes that mediate each of these activities remain largely unidentified and there are no preclinical data for 1,25VD analogues in ovarian cancer (OCa). We hypothesize that 1,25VD and its analogues inhibit the development of OCa. In this study, we demonstrated, (a) 1,25VD causes cell cycle arrest at the G1/S and G2/M transition and induces apoptosis in OCa cells. (b) We also found that gadd45 is one of primary target genes for 1,25VD-mediated G2/M arrest. A direct repeat 3 (DR3) vitamin D response element (VDRE) is identified in the fourth exon of gadd45. This exonic VDRE forms a complex with the vitamin D receptor (VDR)/retinoid X receptor (RXR) heterodimer in vitro and mediates the induction of reporter activity by 1,25VD in vivo. VDR is recruited in a ligand-dependent manner to the exonic enhancer but not to the gadd45 promoter regions. In OCa cells expressing GADD45 anti-sense cDNA or GADD45-null mouse embryo fibroblasts, 1,25VD fails to induce G2/M arrest, suggesting that G2/M arrest induced by 1,25VD is mediated through GADD45. Further study showed that GADD45 mediates the effect of 1,25VD by decreasing cdc2 kinase activity. (c) hTERT, the catalytic subunit of telomerase, is identified as a primary target for 1,25VD. 1,25VD decreases telomerase activity and hTERT mRNA expression. The down-regulation of hTERT mRNA is due to decreased mRNA stability by 1,25VD, rather than decreased transcription of hTERT through VDRE. Clones stably transfected with hTERT showed higher telomerase activity and longer telomere length than parental cells. Moreover, hTERT clones resist 1,25VD-induced apoptosis and growth inhibition. In contrast to parental cells which do not recover from prolonged treatment with 1,25VD, hTERT clones re-grew rapidly after 1,25VD withdrawal. (d) We demonstrated that the 1,25VD analogue EB1089 inhibits OCa cells in vitro and OCa xenograft in vivo without inducing hypercalcemia. We also demonstrated precursors for epithelial OCa express VDR and human primary ovarian surface epithelial cells respond to 1,25VD. Taken together, these results strongly suggest that 1,25VD analogues may be effective in the chemoprevention and chemotherapy of OCa.

Le cancer de la prostate est la deuxième cause de décès masculins par cancer dans les pays industrialisés. PTEN est le gène suppresseur de tumeur le plus souvent muté ou délété dans les cancers de la prostate. Notre étude montre que la perte de Pten induit la prolifération des PEC menant à la formation de PIN. L’hyperprolifération des PEC engendre une réponse de dommages à l’ADN suivie de l’entrée en sénescence des PEC. Des études épidémiologiques ont montré que de faibles taux de Vitamine D sont associés à des cancers agressifs. Nos résultats montrent que Vdr et le Gemini-72, un analogue hypocalcémique de la Vitamine D, a des activités anti-inflammatoires et anti-prolifératives pendant la formation des PIN. De plus, le Gemini-72 induit l’apoptose des PEC sénescentes, module la réponse immunitaire et ainsi réduit le nombre de PIN de haut grade et la réaction stromale. Ainsi, notre étude démontre l’importance de l’axe Vitamine D/VDR dans la carcinogenèse prostatique. / Prostate cancer is the 2nd leading cause of cancer-related deaths in males of western societies. Mutations or deletion of the PTEN locus are common in prostate cancer, and are associated with metastasis and resistance to therapeutic castration. Our results show that Pten-loss induces the proliferation of PEC leading to the formation of PIN. The hyperproliferation of PEC induces DDR followed by senescence entry of PEC. Epidemiological studies highlighted that low Vitamin D levels correlate with aggressive prostate cancer. We show that Vdr and Gemini-72, an hypocalcemic Vitamin D analog, have anti-proliferative and anti-inflammatory activities during PIN formation. Moreover, the Gemini-72 induces apoptosis in senescent cells, modulates the immune response and consequently decreases the number of High Grade PIN and reduces the stromal reaction. Thus, our study demonstrate the major role of Vitamin D signaling in prostate carcinogenesis.

Inherited rickets of Corriedale sheep is a newly discovered skeletal disease of sheep with simple autosomal recessive inheritance. The clinical signs resemble rickets in other species and include decreased growth rate, thoracic lordosis and angular limb deformities. Radiographic features include physeal thickening, blurred metaphyseal trabeculae and thickened porous cortices. Computed tomography scanning of long bones reveals increased bone mineral content and cortical area, but decreased bone mineral density. Gross lesions include segmental thickening of physes, growth arrest lines, collapse of subchondral bone of the humeral head, thickened cortices and enthesophytes around distal limb joints. Microscopically there is persistence of hypertrophic chondrocytes at sites of endochondral ossification, inappropriate and excessive osteoclastic resorption, microfractures and wide, unmineralised osteoid seams lining trabeculae and filling secondary osteons. Affected sheep are persistently hypophosphataemic and hypocalcaemic. Normal serum 25-hydroxyvitamin D3 concentration accompanied by a two-fold elevation in 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) suggested a defect in endorgan responsiveness to vitamin D as a likely mechanism, but this was not supported by in vitro studies using cultured skin fibroblasts. These studies revealed normal vitamin D receptor function and the presence of 24- hydroxylase mRNA in cells from affected sheep, even without induction by 1,25(OH)2D3. Inappropriate overexpression of 25-hydroxyvitamin D3-24- hydroxylase, the enzyme that breaks down active vitamin D, is therefore considered the probable cause of inherited rickets in Corriedale sheep. Such a mechanism has not previously been described as a cause of inherited rickets in humans or other animal species. Treatment of affected sheep with high oral doses of vitamin D3 weekly for 3 months showed a trend towards increased bone mineral density, thus supporting an intact vitamin D receptor. Preliminary studies on immune function revealed reduced numbers of CD4+ and CD8+ lymphocytes and reduced interferon-? production by lymphocytes stimulated with parasite antigen. This new form of inherited rickets may be widespread in

The activation of 1,25D-dihydroxyvitamin D3 (1,25D) is catalysed by the enzyme 25-hydroxyvitamin D-1ƒhydroxylase (CYP27B1) in the kidney, which is the primary producer of 1,25D in the body. Although the synthesis of 1,25D by CYP27B1 and the catabolism of 1,25D by 25-hydroxyvitamin D-24-hydroxylase (CYP24) also take place in the bone, the significance of the bone cell-specific metabolism of vitamin D remains largely unknown. This thesis investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone. Furthermore, CYP27B1, CYP24 and VDR mRNA expression were also identified in specific regions of the rat femur and in a number of bone cell lines, with the aim to identify the bone cell types that have the capacity to metabolise and/or to respond to vitamin D. The age-related decrease in the circulating levels of 1,25D detected in animals ranging in age from 3 weeks to 2 years old, was a direct result of a reduction in the expression of CYP27B1 mRNA and an increase in the expression of CYP24 and VDR mRNA in the kidney. In contrast, the expression of CYP27B1 and CYP24 mRNA in the bone is high from 3 to 15 weeks of age, which is the period of rapid growth and development. The expression of CYP27B1 mRNA in the bone was positively correlated with the circulating levels of calcium throughout aging, which suggests that the 1,25D produced in the bone may be involved in the mineralisation process. The positive correlation found between the expression of CYP27B1 and CYP24 mRNA in the bone was in contrast with the negative correlation found between the expression of these two enzymes in the kidney. This suggests that the 1,25D produced locally in the bone, rather than the 1,25D produced in the kidney, is the primary determinant of the CYP24 activity in the bone. In vitamin D-deplete animals, fed a 0.1% calcium diet (D(-)/LC), the expression of CYP27B1 mRNA was induced and the expression of CYP24 mRNA was suppressed in the kidney. In contrast, both the expression of CYP27B1 and CYP24 mRNA were low in the bones of these D(-)/LC animals. When vitamin D-deplete animals were fed a 1% calcium diet (D(-)/HC), the expression of both CYP27B1 and CYP24 mRNA was high in the bone, which was in direct contrast with the low expression of these genes detected in the kidney. Besides this, a positive correlation was found between the expression of CYP27B1 mRNA in the bone, serum calcium levels and bone mineral volume (BV/TV) in the epiphysis, which supports the findings for the age study that the locally produced 1,25D may be involved in the promotion of bone mineralisation. Although serum PTH levels was positively correlated with the expression of CYP27B1 mRNA in the kidneys of hypocalcaemic animals, there was no such relationship detected between the levels of serum PTH and the expression of CYP27B1 mRNA in the bone. This finding suggests that the regulation of the expression of CYP27B1 mRNA in the bone is different from the regulation found in the kidney. The identification of CYP27B1 mRNA in osteoblasts-like cells, taken together with the associations between serum calcium and CYP27B1 mRNA expression in the previous studies, suggests that 1,25D produced in osteoblasts may play a significant role in the bone mineralisation process. The detection of CYP27B1 mRNA expression in a number of bone marrow cells suggests that locally produced 1,25D may also play a role in the growth and differentiation of hematopoietic cells. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2003.