Vitamin D Metabolism Revised: Fall of Dogmas

Vitamin D is one of the most frequently used medicinal products around the world. The dietary intake is irregular because few food items contain vitamin D and is usually well below human requirements, so that its synthesis in the skin is the most important source of vitamin D. The global supply of vitamin D is usually considered as a passive series of events, not controlled by enzymes or hormones. In the nearly 100 years after its discovery, we learned that vitamin D has a complex metabolism and steroidlike hormonal action. Vitamin D is totally inactive and requires a complex metabolism, first in the liver (mostly but not exclusively by CYP2R1) into 25-hydroxyvitamin D (25OHD), followed by a second hydroxylation by CYP27B1 into 1,25-dihydroxyvitamin D (1,25(OH)2D). CYP2R1 is usually considered to be constitutively expressed. Therefore, the production of 25OHD is considered to be mostly substrate dependent so that serum 25OHD reflects the global supply of vitamin D. CYP27B1 in the kidney is the unique source of circulating 1,25D and is tightly regulated by different ions and hormones so that it behaves as a classical feedback regulated hormonal system. CYP27B1 is also widely expressed in many extra renal tissues so that 1,25(OH)2D also behaves in a paracrine/autocrine fashion. In these tissues its activity is regulated by a variety of mechanisms different from what happens in the kidney. Although there are probably around 50 known metabolites of vitamin D, measurement of serum 25OHD is clinically used to define the vitamin D status, whereas serum 1,25(OH)2D is used to assess the biological activity of the vitamin D endocrine system. All metabolites of vitamin D in serum are bound with relatively high affinity to a specific binding protein, vitamin D binding protein (DBP). This protein is highly polymorphic and circulates in serum in high concentrations so that the free concentrations of all vitamin D metabolites are very low. 1,25(OH)2D binds to the vitamin D receptor (VDR), present in most cells. This hormonal system functions as most steroid and thyroid hormones and regulates a very large number of genes involved in calcium and phosphate transport but also regulates a very large number of genes (up to 10% of all genes of the some organisms such as the zebrafish) not involved in ion transport or bone metabolism (reviewed in 1). However, a few recent publications have challenged some aspects of our understanding of vitamin D metabolism including the concept of stable expression of the hepatic 25-hydroxylases.2, 3 We review these new data regarding CYP2R1, discuss their potential implications, and extend this review to examine the overall metabolism of vitamin D to explore whether old dogmas still hold today. Obesity and the metabolic syndrome are associated with low vitamin D status.1 Prospective studies suggest that low non-epimeric 25OHD or increased 3-epi-25OHD concentrations are associated with higher risk for type 2 diabetes.4 The causality (in whatever direction) between obesity/diabetes and low vitamin D status is, however, not proven. In a recent JBMR article, Roizen and colleagues clearly demonstrated that the serum concentration of 25OHD is substantially lower (~−20%) in serum of obese mice (fed a high-fat diet) compared with normal-weight mice, whereas serum concentrations of vitamin D3 itself were similar in both groups.2 This is not a surprise because serum 25OHD concentrations in overweight or obese humans are virtually systematically lower than in normal subjects in many different areas of the world having different sun exposure or dietary habits.5 Their novel finding, however, was that the mRNA of major vitamin D-25-hydroxylase (CYP2R1) is markedly (~40%) lower in livers of obese mice (fed a high-fat diet) compared with livers from normal mice. They confirm that by finding lower protein expression (~50% decrease) of CYP2R1. The gene expression of some other potential 25-hydroxylases (CYP27A1 and CYP3A4) as well as the major catabolizing enzyme (CYP24A1) were not changed by diet-induced obesity. Finally, the authors measured the 25-hydroxylase activity by incubating mouse liver homogenates with vitamin D2 and found a ~70% reduction in the overall enzymatic activity. As the substrate concentration was in the millimolar range, such an assay is not specific for the high-affinity, low-capacity CYP2R1 but represents a combined activity of all 25-hydroxylases including those that hydrolyze vitamin D2 less well than D3. They also used the ratio of serum 25OHD to serum vitamin D concentrations as a marker of 25-hydroxylase activity and found a strong positive correlation between this ratio and liver mRNA expression of CYP2R1. Aatsinki and colleagues addressed a similar question about the origin of fairly systematic low serum 25OHD concentrations in diabetic subjects compared with their euglycemic controls, by studying high-fat-diet-induced obesity and type 2 diabetes in mice.3 In addition, they also studied the effect of 24-hour fasting and of streptozotocin-induced type 1 diabetes. All these metabolic situations decreased the hepatic mRNA and protein concentration of CYP2R1. Fasting for 24 hours, type 1 diabetes, or type 2 diabetes decreased the mRNA of CYP2R1 in liver by 80%, 43%, and 45%, respectively, and generated a decrease of about 30% in protein concentration (estimated by Western blot). In vitro measurement of total 25-hydroxylase activity indicated a more than 50% decrease during 12- to 24-hour fasting. In addition, these authors demonstrated that the decrease in CYP2R1 was mediated by PPARγ-coactivator-1α (PGC1α), the key control enzyme induced by metabolic diseases such as fasting or type 1 or type 2 diabetes. By using several in vitro and in vivo gene KO and overexpression experiments, they showed that the control of CYP2R1 gene expression by PGC1α required the presence of another nuclear receptor, estrogen-related receptor α (ERRα), known to bind tightly to this and other nuclear receptors (such as VDR and glucocorticoid receptor GR). Activation of the GR receptor by dexamethasone also decreased hepatic CYP2R1 mRNA and protein concentrations (by 50% and 26%, respectively), again mediated by induction of PGC1α. PGC1α also induced hepatic and renal expression of CYP24A1 several-fold, again mediated by the GR-PGC1α-ERRα pathway (but much less than the 100-fold induction by 1,25(OH)2D). Other major fat-regulating nuclear receptors are less likely involved, as the ENCODE project did not find consensus sequences for nuclear receptor (VDR) binding sites in promoters of genes involved in fat metabolism in the liver, such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), or peroxisome proliferator-activated receptor (PPAR) binding sites in the proximal promoter of mouse or human CYP2R1 (http://www.cbrc.jp/htbin/nph-tfsearch), but several binding sites for NFkB were identified.2 Both studies thus clearly demonstrate that the major (CYP2R1) and global hepatic 25-hydroxylase activity is under tight control of metabolic signals induced by fasting, diabetes, or exposure to high-dose glucocorticoids. PGC1α and ERRα as well as the GR are involved in the regulation of CYP2R1, but additional mechanisms may also be involved. A recent abstract demonstrates that a high-fat diet induces an epigenetic downregulation of CYP2R1 in the mouse liver, thereby causing decreased serum 25OHD, whereas CYP24A1 was upregulated.6 These observations are fully in line with previous studies cited above. However, these authors now add another mechanism by hypermethylation of the promotor regions of these CYP2R1 and CYP27B1 genes and hypomethylation of the CYP24A1 promoter. In addition, they observed a decreased expression of glutathione, and treatment of such mice with glutathione precursors could partially correct the abnormal expression of vitamin D regulatory genes. They concluded that high-fat diet caused glutathione deficiency, changing the methylation pattern of vitamin D regulatory genes and causing low serum 25OHD concentrations. All these studies failed to report blood glucose levels in their obese animals so that the separate effects of obesity and diabetes cannot be fully estimated. There are many other remaining questions such as: 1) Are CYP2R1 and CYP24A1 expression in other tissues also under the same metabolic control? 2) Is DBP, the major transport protein of all D metabolites, also regulated by metabolic factors? DBP is indeed decreased in diabetic subjects or animals.7, 8 Apart from obesity, fasting, and diabetes, many other diseases are associated with poor vitamin D status compared with healthy controls. Therefore, the question arises whether patients with chronic renal failure, liver cirrhosis, and acute illness also have low serum 25OHD due to metabolic control of CYP2R1. If so, it could at least partially explain why such patients and especially patients admitted to intensive care units require so much vitamin D (10 to 100 times the normal doses) to generate serum 25OHD concentrations above 20 ng/mL.9, 10 On the other hand, DBP levels often drop in these circumstances as an acute phase reactant, and this is associated with reduced 25OHD concentrations. Finally, the short- and long-term effects (harm or benefit) of this metabolic regulation of CYPs involved in vitamin D metabolism are not known. The PGC1α-ERRα pathway is known to play a major role in hepatic gluconeogenesis, in energy homeostasis in general and in fat tissue in particular. Indeed, because PGC1α is a strong positive regulator of mitochondrial function (and energy production), one might wonder whether the new observations are linked to overall energy balance and may help to clarify why VDR or CYP27B1 null mice are resistant to high-fat-diet-induced obesity (by activating energy expenditure),11, 12 whereas in humans a low vitamin D status is strongly associated with obesity. Knockdown of CYP2R1 in zebrafish did not affect bone homeostasis but generated a phenotype of abnormal visceral fat accumulation.13 A similar phenotype of increased visceral and subcutaneous fat accumulation was observed in zebrafish raised on a vitamin D–deficient diet, probably related to the increased expression of adipogenic and lipid processing markers in their liver.14 These data clearly indicate that the link between vitamin D metabolism and energy homeostasis already occurred early in the evolution of vertebrates.15 Variations in hepatic (or extra-hepatic) CYP2R1 expression may also play a role in the great variability of serum concentrations of 25OHD in healthy populations with similar food and lifestyle attitudes. Indeed, there is widespread variability in the response of serum 25OHD to comparable amounts of dietary vitamin D and/or vitamin D supplementation16-18 and no compelling mechanism has been able to explain this variability. The studies just reviewed dealt with mice and need to be confirmed in humans. Human and mouse CYP2R1 are structurally and functionally very similar19 and in both species, serum 25OHD is lower in case of type 1 or type 2 diabetes. Therefore, these studies clearly demonstrate that the general belief of constitutive expression of liver 25-hydroxylase activity no longer holds true (Fig. 1). Indeed, the major 25-hydroxylase, CYP2R1, is highly regulated by a variety of “clinical” conditions (obesity, starvation, type 1 or type 2 diabetes) and a number of regulatory factors are now clearly identified, albeit there are still major missing links. Genetic silencing mutations in CYP2R1 can cause rickets or osteomalacia,20, 21 but no activating mutations are so far described. Null mutations of the same gene cause the same phenotype in cats.22 Polymorphisms in CYP2R1 have the greatest effect on interindividual variations in serum 25OHD when comparing with other known polymorphisms.23 If confirmed in humans, serum 25OHD is not only reflecting access to vitamin D of nutritional and skin-produced vitamin D but is also reflecting a complex metabolic regulation of its hepatic synthesis and the likely involvement of many hormones. These studies may also have practical implications for correcting a poor vitamin D status in obese or diabetic subjects. Intervention studies have shown that obese subjects need more vitamin D than normal-weight subjects to achieve similar serum 25OHD concentrations as based on a comparison between an overview of such studies.5, 16 However, vitamin D supplementation of vitamin D–replete prediabetic subjects did not decrease their risk of progression to type 2 diabetes.24 Whether supplementation of more vitamin D–deficient subjects may generate better results is yet unclear. Now that the dogma of a nonregulated CYP2R1 has been challenged, one may also question other “dogmas” regarding vitamin D metabolism (Fig. 1). DHC-7a-reductase (DHCR7) is a key determinant of the amount of the vitamin D precursor, 7-dehydrocholesterol (7-DHC), in the skin. Most reviews mention that older subjects may have lower 7-DHC concentrations in the skin but do not include regulation of DHCR7 as an important regulator of vitamin D status. DHCR7 is the last step in the Kandutsch-Russell pathway of cholesterol synthesis, converting 7DHC to cholesterol. As such, DHCR7 is essential for the presence or absence of 7-DHC in skin cells. In case of overexpression of this enzyme, as in the skin of the members of the feline species (including cats and dogs), the near absence of 7-DHC makes these animals unable to synthesize vitamin D, so that vitamin D is a true vitamin in these species.1 The opposite condition, genetic absence of DHCR7, causes Lemli-Smith-Opitz disease,25 mainly characterized by the consequences of too little cholesterol, steroids, or bile acids. However, this disease the accumulation of 7DHC and thereby the effect of on the synthesis of vitamin D. these patients usually have higher serum 25OHD concentrations than normal In humans, in DHCR7 have been associated with or 25OHD However, the of these on enzyme function has not been The regulation of DHCR7 is and vitamin D (but not of DHCR7, as to increased vitamin D a key and regulator of energy and protein A are of DHCR7, whereas has a lower Most and reviews clearly that the production of vitamin D in the skin is a Although this recent data suggest that the activity of DHCR7 is under metabolic and genetic By substrate these factors thus can interindividual variations in of vitamin D. what this has implications for the vitamin D status of humans is, however, and first the kidney as the source of 1,25(OH)2D in it was to be the However, can a case report of a with chronic kidney disease showed an in serum 1,25(OH)2D during and the human was shown to be of 1,25(OH)2D in and levels of 1,25(OH)2D were found at and could be increased with vitamin D or 25OHD A report by of an with with clearly 1,25(OH)2D levels demonstrated a disease in production The source was to be the from the involved about the same a number of were finding 1,25(OH)2D production by bone and in and many other and the of the in by the that there is only one gene and protein such that the renal and enzyme is the 50 The the of and to the of its expression in many other However, it that the regulation of CYP27B1 activity in tissues from that in the kidney. This in regulation is clearly demonstrated in diseases such as and other that to in circulating 1,25(OH)2D and of CYP27B1 regulation in tissues a of its regulation in the kidney. CYP27B1 in the renal proximal is controlled by having a positive effect and as well as 1,25(OH)2D itself having an at least in to in calcium and phosphate levels can CYP27B1 activity in the proximal and by binding to their receptors and activating their a in the of CYP27B1 in renal that was to and 1,25(OH)2D However, this was not to such regulation in the tissues they including skin and cells. In these a different of the CYP27B1 was regulated by with different regulatory mechanisms in tissues by the and may also CYP27B1 but probably mainly by its effect on These feedback very tight regulation of 1,25(OH)2D production by the of the control that from that of CYP27B1 in other including that of renal has little 1,25(OH)2D has very little effect on CYP27B1 activity in 1,25(OH)2D regulates its levels in the by the enzyme for and on the other hand, are of CYP27B1 activity in the as is 1,25(OH)2D induces the expression and in and of but not (by induces The production of 1,25(OH)2D by is by and but not by and and is by but not by and also CYP27B1 activity in blood whereas is In to and not only but that to decrease CYP27B1 receptors and and to with a reduction in CYP27B1 CYP27B1 in human from bone is by mechanisms both the of acute and the expression of and the of its receptor 25OHD CYP27B1 expression in these but that to be due to a of increased expression of the and as 1,25(OH)2D the expression of CYP27B1 in these However, not all studies have found that CYP27B1 in human The both receptors and the CYP27B1 expression in the Activation of the receptor in the by calcium or also CYP27B1 Both and a link between and CYP27B1 These data clearly that the production of 1,25(OH)2D is much more complex than the dogma of the kidney the source of the vitamin D regulated by key and recent data demonstrate that the renal and especially the production of this is complex and regulated by a variety of The of 1,25(OH)2D production in normal and disease is a of tissues can to the serum concentration of 1,25(OH)2D in case of diseases and but this is in other as 1,25(OH)2D levels can be increased with vitamin D or 25OHD supplementation in or renal the is that 1,25(OH)2D production a function in the tissue it is than an endocrine CYP24A1 is the enzyme for the of all vitamin D metabolites (Fig. 1). a pathway in a large number of metabolites with to also an essential role species in the of 25OHD of this unique (in with results in accumulation of 1,25(OH)2D and This is in mice and In addition, absence of this enzyme may first demonstrate its consequences by or kidney in CYP24A1 null mice also have a with as is able to bind to a and thereby production and Whether this also to humans with mutations however, so far not been of the CYP24A1 gene is for genetic variability of serum 25OHD one of the 8 genes known so far to in higher or lower serum 25OHD CYP24A1 is under control of many hormones but mainly by 1,25(OH)2D strong and or by using the receptor, to be able to Although there be other mechanisms to vitamin D metabolites, as serum 25OHD is only increased in animals or humans with null The most likely are and a variety of enzymes of of all vitamin D This enzyme is well known as the enzyme in steroid synthesis, converting cholesterol to the However, and have demonstrated that also vitamin D3 to with metabolism to a variety of metabolites including have activity comparable in some to 25OHD is not a substrate (Fig. 1). The of production by CYP27B1 on is much lower than that of 1,25(OH)2D production from is expressed in the skin and as well as tissues such as the and this little is known about this enzyme is regulated in the skin and with to its vitamin activity. is the major is expressed in the liver and 1,25(OH)2D induces this enzyme in both liver and in vivo there is probably little induction in the liver the low levels of VDR in that The of 1,25(OH)2D and 25OHD may the levels of more than be based on serum can also function as a for VDR in the can both 25OHD and 1,25(OH)2D as well as other vitamin products such as and These in the 24 and of the as well as the for The induction of by 1,25(OH)2D was at least as great as the induction of CYP24A1 in the is a of and its results in lower levels of 25OHD and This could to The major circulating of activity is can levels comparable to 1,25(OH)2D after (Fig. 1). activity is not known. a has subjects with early of rickets for of the known mutations in the enzymes involved with vitamin D metabolism or VDR could be Both and 1,25(OH)2D levels were whereas levels were The authors used to find the same activating in the The authors this as vitamin rickets type can be with very large of vitamin The enzyme the of This not to be The gene has yet to be The enzymatic activity is and in the of levels of 3-epi-25OHD can be from to of the 25OHD levels in and to in have been to separate the from 25OHD The 3-epi-25OHD can be by CYP27B1 to has in most studies its activity is less than its affinity for the VDR to be substantially its to calcium of or CYP24A1 induction is markedly the of 25OHD and 1,25(OH)2D cannot be but their need amounts of are in serum of normal subjects to of the concentration of concentration a high correlation with serum 25OHD but a lower correlation with The origin or involved have not yet been The of vitamin D into 25OHD is far from on supplementation (reviewed in only one of to 10 of vitamin D is into The same is true for the of 25OHD into The other 25OHD can be by CYP24A1 into and a number of other metabolites (Fig. 1). The of the other vitamin D (or is but is most likely involved as of the This with vitamin vitamin D and 25-hydroxyvitamin or The of vitamin D is already found early in evolution as some of vitamin D are found as in and most vitamin D found in liver is in the of The regulation of these and the potential of vitamin D metabolites by are The serum DBP is for the transport of all vitamin D metabolites due to its high affinity for all metabolites and especially for thereby regulates the free concentration of these metabolites as is demonstrated by the low serum concentrations of 25OHD and 1,25(OH)2D in animals or the human with mutations in the to most considered DBP as expressed by with little or no from the effects of DBP however, are lower in with a similar decrease in total 25OHD concentrations. Polymorphisms in DBP are for of the genetic variability of serum 25OHD concentrations in all populations so DBP in serum can be measured by and and more also by be to of all of DBP concentrations are markedly decreased in liver and in patients with very acute illness or acute due at least in to its Therefore, DBP is not a passive but an in the overall vitamin D homeostasis and is probably under control of metabolic signals (Fig. 1). The origin of vitamin D, about a first into a metabolic of constitutive of vitamin D in the liver to 25OHD, followed by a tightly regulated by a unique CYP27B1 in a unique to generate 1,25(OH)2D as of a nuclear receptor, All these metabolites are by a serum binding protein and are by a unique nearly The present is much more complex with a large number of enzymes expressed in a variety of cells. Most of these genes contain genetic that may their are regulated by hormones and/or metabolic that can in different tissues of the Finally, the vitamin D endocrine system regulates a large number of genes. These recent that the vitamin D endocrine system is much more complex than and still