Differential Frequency of CYP2R1 Variants Across Populations Reveals Pathway Selection for Vitamin D Homeostasis.

CONTEXT: Normal vitamin D homeostasis is necessary to ensure optimal mineral metabolism. Dietary insufficiency of vitamin D and the lack of sunlight each have well understood roles in vitamin D deficiency; however, the extent to which common genetic variations in vitamin D metabolizing enzymes contribute to alterations in vitamin D homeostasis remains uncertain. OBJECTIVE: To examine the possibility that common coding variation in vitamin D metabolizing enzymes alters vitamin D homeostasis we determined the effect of 44 nonsynonymous polymorphisms in CYP2R1, the vitamin D 25-hydroxylase, on enzyme function. RESULTS: Twenty-one of these polymorphisms decreased activity, while 2 variants increased activity. The frequency of CYP2R1 alleles with decreased 25-hydroxylase activity is 3 in every 1000 Caucasians and 7 in every 1000 African Americans. In populations where exposure to sunlight is high, alleles with decreased function occur at a frequency as high as 8%. The pattern of selected variation as compared to nonselected variation is consistent with it being the result of positive selection for nonfunctional alleles closer to the equator. To examine this possibility, we examined the variation pattern in another protein in the vitamin D pathway, the vitamin D binding protein (GC protein). The pattern of selected variation in the GC protein as compared to nonselected variation is also consistent with it being the result of positive selection for nonfunctional alleles closer to the equator. CONCLUSIONS: CYP2R1 polymorphisms have important effects on vitamin D homeostasis, and the geographic variability of CYP2R1 alleles represents an adaptation to differential exposures to UVB irradiation from sunlight.

Obesity Decreases Hepatic 25-Hydroxylase Activity Causing Low Serum 25-Hydroxyvitamin D.

Normal vitamin D homeostasis is critical for optimal health; nevertheless, vitamin D deficiency is a worldwide public health problem. Vitamin D insufficiency is most commonly due to inadequate cutaneous synthesis of cholecalciferol and/or insufficient intake of vitamin D, but can also arise as a consequence of pathological states such as obesity. Serum concentrations of 25(OH)D (calcidiol) are low in obesity, and fail to increase appropriately after vitamin D supplementation. Although sequestration of vitamin D in adipose tissues or dilution of ingested or cutaneously synthesized vitamin D in the large fat mass of obese patients has been proposed to explain these findings, here we investigate the alternative mechanism that reduced capacity to convert parent vitamin D to 25(OH)D due to decreased expression of CYP2R1, the principal hepatic vitamin D 25-hydroxylase. To test this hypothesis, we isolated livers from female mice of 6 to 24 weeks of age, weaned onto either a normal chow diet or a high-fat diet, and determined the abundance of Cyp2r1 mRNA using digital droplet-quantitative PCR. We observed a significant (p < 0.001) decrease in Cyp2r1 mRNA in the liver of high-fat diet-fed mice relative to lean-chow-fed female mice. Moreover, there was a significant (p < 0.01) relationship between levels of Cyp2r1 mRNA and serum 25(OH)D concentrations as well as between Cyp2R1 mRNA and the ratio of circulating 25(OH)D3 to cholecalciferol (p < 0.0001). Using linear regression we determined a curve with 25(OH)D3/cholecalciferol versus normalized Cyp2R1 mRNA abundance with an R2 value of 0.85. Finally, we performed ex vivo activity assays of isolated livers and found that obese mice generated significantly less 25(OH)D3 than lean mice (p < 0.05). Our findings indicate that expression of CYP2R1 is reduced in obesity and accounts in part for the decreased circulating 25(OH)D. © 2019 American Society for Bone and Mineral Research.

Decreased Serum 25-Hydroxyvitamin D in Aging Male Mice Is Associated With Reduced Hepatic Cyp2r1 Abundance.

The prevalence of vitamin D deficiency, as determined by circulating levels of 25-hydroxycalciferol [25(OH)D], is greater in older individuals compared with the young. To examine the hypothesis that altered production or inactivation of 25(OH)D contributes to lower circulating levels of 25(OH)D, we measured the serum levels of parent vitamin D3 (cholecalciferol) and 25(OH)D. We also determined the relative abundance of transcripts encoding hepatic CYP2R1 and CYP27B1, the principal 25-hydroxylases, transcripts encoding enzymes that degrade 25(OH)D in the liver (Cyp3A11) and kidney (Cyp24A1) and transcripts encoding megalin and cubilin, proteins critical to vitamin D resorption in the kidney in mice at three different ages. We observed a significant decline in the relative abundance of Cyp2R1 in the liver with aging (one-way ANOVA, P = 0.0077). Concurrent with the decrease in mRNA, a significant decline in hepatic CYP2R1 protein (one-way ANOVA for trend, P = 0.007) and 25(OH)D (one-way ANOVA for trend, P = 0.002) and in the ratio of 25(OH)D3 to cholecalciferol (one-way ANOVA, P = 0.0003). By contrast, levels of the transcripts encoding Cyp3a11, Cyp24a1, and Cyp27b1 megalin and cubilin were unchanged with aging. A significant positive correlation was found between Cyp2r1 mRNA and 25(OH)D, and a stronger correlation was found between Cyp2r1 mRNA and the ratio of 25(OH)D3 to cholecalciferol. These results indicate that decreased expression of CYP2R1 contributes to the reduced serum levels of 25(OH)D in aging.

CYP3A4 mutation causes vitamin D-dependent rickets type 3.

Genetic forms of vitamin D-dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR; however, using whole exome sequencing analysis, we identified a recurrent de novo missense mutation, c.902T>C (p.I301T), in CYP3A4 in both subjects that alters the conformation of substrate recognition site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than WT CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide insight into vitamin D metabolism and demonstrate that accelerated inactivation of vitamin D metabolites represents a mechanism for vitamin D deficiency.

Ablation of Oxytocin Neurons Causes a Deficit in Cold Stress Response.

The paraventricular nucleus (PVN) is a critical locus of energy balance control. Three sets of neurons in the PVN are involved in regulating energy balance: oxytocin-expressing neurons (OXT-neurons), thyrotropin-releasing hormone-expressing neurons, and corticotrophin-releasing hormone-expressing neurons. To examine the role of OXT-neurons in energy balance, we ablated these neurons in mice by injecting diphtheria toxin into mice possessing both the oxytocin promoter driving cre expression and a cre-inducible diphtheria toxin receptor. Immunohistochemistry and real-time reverse transcriptase polymerase chain reaction confirmed that this injection caused a significant decrease in PVN OXT-neurons and OXT-mRNA abundance. OXT-neuron ablation did not alter food intake, weight, or energy expenditure at room temperature on either chow or a high-fat diet. To further characterize OXT-neuron-ablated mice, we examined their response to 1) intraperitoneal cholecystokinin (CCK) injection and 2) thermogenic stress. OXT-neuron-ablated mice had a blunted decrease in feeding response to CCK. When exposed to the extreme cold (4°C) for 3 hours, OXT-neuron-ablated mice had significant decreases in both rectal and brown adipose tissue temperature relative to controls, which was rescued by OXT treatment. Thermographic imaging revealed that OXT-neuron-ablated mice had increased body surface temperature. Thus, we report that OXT-neuron ablation shows no role for OXT-neurons in energy homeostasis at neutral temperature but reveals a heretofore unappreciated role for OXT-neurons and oxytocin specifically in regulating the thermogenic stress response.