The results of the current study demonstrate 1α-hydroxylase gene expression in extra-renal tissues that contribute to energy balance in rats. We found that the 1α-hydroxylase gene is expressed in liver, skeletal muscle and adipose tissue. In addition, 1α-hydroxylase gene expression was increased in the liver of rats fed a high fat diet, but not by dietary vitamin D or calcium. It is also not regulated by dietary vitamin D or calcium in adipose tissue. Finally, our results demonstrate that the 1α-hydroxylase gene is expressed and active in 3T3 L1 preadipocyte cells. To our knowledge, this is the first report showing 1α-hydroxylase expression in adipose tissue and that the enzyme is functional in adipocytes.
Adipose tissue harvested from animals is comprised of a heterogeneous population including adipocytes as well as other cell types such as macrophages. This is particularly relevant as activated macrophages express functional 1α-hydroxylase [17
]. Our studies in 3T3 L1 preadipocytes suggest that 1α-hydroxylase gene expression in adipose tissue of rats is likely due to adipocytes as well as activated macrophages. In addition, our studies support that the 1α-hydroxylase in adipocytes is functional, shown by induction of CYP24 gene expression following 25OHD treatment, as well as the production of radiolabeled 1,25(OH)2
D from 3
H 25OHD. The activity measured in the adipocytes in our study (1.16±0.07 pmol/mg protein/hour) is comparable to that in other cell lines, including prostate (0.07-3.08 pmol/mg protein/hour, 20), vascular endothelial cells (318 fmol/mg protein/hour, 23) and in human renal tissue (0.20 pmoles/mg protein/20 minutes, 37). The lower induction of CYP24 with 100 nM compared to 10 nM 1,25(OH)2
D is likely due to the increased degradation of the metabolite by 24-hydroxylase. This conclusion is supported by the results shown in in which co-treatment with the p450 inhibitor, ketoconazole, increases the induction of CYP24 by 1,25(OH)2
D. Although 25OHD binds the VDR approximately 1000 fold less well than 1,25(OH)2
], only a 10 fold higher dose of 25OHD (10 nM), within a physiological range for this metabolite, induced CYP24 expression to a similar level compared to 1 nM 1,25(OH)2
D. In addition, co-treatment with the p450 inhibitor, ketoconazole, significantly reduced the induction of the CYP24 expression by 25OHD, supporting that the activity of the 1α-hydroxyalse enzyme is required, at least in part, for the induction. Therefore, the 1α-hydroxylase is expressed in adipose tissue and is functional in cultured adipocytes.
Gene regulation of the extra-renal 1α-hydroxylase is currently under investigation. It has been shown to be independent of PTH in a primary culture of normal prostate epithelial cells and HPV18 DNA transformed normal prostate epithelial cell line, PZ-HPV-7 [33
], although PTH suppressed 1α-hydroxylase reporter construct in ROS 17/2.8 osteoblast [38
]. Several cytokines, such as interferon (IFN)γ [39
], cytokine interleukin (IL)-1β [40
], and epidermal growth factor (EGF) [41
] have been shown to induce extra-renal 1α-hydroxylase expression; while transforming growth factor-beta (TGF-beta) [38
] or insulin-like growth factor-1 (IGF-1) [38
], growth factor independent-1 (GFI1) [42
] decreased its expression of the 1α-hydroxylase. One of the few studies which have investigated the role of diet in regulation of 1-hydroxylae shows that phytoestrogens upregulate 1α-hydroxylase in the colon [43
]. The current study investigated the effect of high calcium and vitamin D diets consumption on 1α-hydroxylase regulation. There were no differences in 1α-hydroxylase expression in liver, skeletal muscle and adipose tissue between intake of high calcium and vitamin D compared to low intakes in the rats demonstrating a lack of dietary regulation by vitamin D and calcium.
On the other hand, in the current study, 1α-hydroxylase expression was higher in liver tissue when rats were fed with high fat diet compared to high sucrose diet. It is important to consider that the mRNA is isolated from liver tissue, which contains a variety of cell types. A high fat diet may induce fat accumulation in the liver, leading to a fatty liver. This high fat diet-induced change may lead to macrophage infiltration. Macrophages express 1α-hydroxylase, which is induced when the macrophages are activated [17
]. Therefore, it is critical to determine if the increase in 1-hydroxylase in liver tissue is due to macrophage infiltration or a specific effect on hepatocytes.
The function of 1,25(OH)2
D in regulating cellular decisions in the adipocyte is not clear. Evidence suggests that 1,25(OH)2
D may increase fat synthesis in human adipocytes [44
] however, other studies demonstrate that 1,25(OH)2
D increases the expression of Insig-2, whose protein product is involved in down regulating fat synthesis [45
]. Our studies demonstrate that the 1α-hydroxylase is functional in adipocytes and capable of localized 1,25(OH)2
D production. The autocrine and paracrine activity of extrarenal 1,25(OH)2
D production and localized concentrations are not yet known but it is possible that localized 1,25(OH)2
D may stimulate different cellular signals compared with exogenously produced hormone. Addressing the role of the locally produced hormone in adipocyte function is critical to a more complete understanding of the role of vitamin D in controlling adiposity and body composition.