Correlation of CYP2R1 gene promoter methylation with circulating vitamin D levels among healthy adults.

Background & objectives: Despite being a tropical country, vitamin D deficiency is highly prevalent in India with studies indicating 40-99 per cent prevalence. Apart from calcium and phosphate metabolism, vitamin D is involved in cell cycle regulation, cardiovascular, hepatoprotection. The metabolism of vitamin D is regulated by vitamin D tool genes (CYP2R1/CYP27B1/CYP24A1/VDR). The promoter regions of some of these genes have CpG islands, making them prone to methylation induced gene silencing, which may cause a reduction in circulating vitamin D levels. Epigenetic basis of vitamin D deficiency is yet to be studied in India, and hence, this pilot study was aimed to analyze whether methylation levels of CYP2R1 gene were correlated with the levels of 25(OH)D in healthy, adult individuals in Indian population. Methods: In this cross-sectional study, healthy adults of 18-45 yr of age with no history of malabsorption, thyroidectomy, chronic illness or therapeutic vitamin D supplementation were recruited. DNA methylation analysis was carried out by methylation specific quantitative PCR. Serum calcium, phosphate and vitamin D levels were also quantified. Statistical analysis was done by R 4.0.5 software. Results: A total of 61 apparently healthy adults were analyzed. The serum vitamin D levels did not correlate with CYP2R1 methylation levels in our study population. Significant positive correlation was observed between age and serum vitamin D levels. Significant association of gender was found with CYP2R1 methylation levels. Interpretation & conclusions: This study found no significant correlation between levels of CYP2R1 methylation and circulating 25(OH)D deficiency. Further studies on the Indian population having a larger sample size including entire vitamin D tool genes, among different ethnic groups may be conducted to elucidate molecular etiology of circulating 25(OH)D deficiency. The high prevalence of normal serum calcium and phosphate levels among vitamin D deficient subjects in this study coupled with the strikingly high prevalence of the deficiency at the national level, may suggest the need to revise the cut-off criteria for vitamin D deficiency in the Indian population.

Metabolism of Lumisterol2 by CYP27A1.

Lumisterol2 (L2) is a photoproduct of UVB action on the fungal membrane sterol, ergosterol. Like vitamin D2, it is present in edible mushrooms, especially after UV irradiation. Lumisterol3 is similarly produced in human skin from 7-dehydrocholesterol by UVB and can be converted to hydroxy-metabolites by CYP27A1 and CYP11A1. These products are biologically active on human cells with actions that include photoprotection and inhibition of proliferation. The aim of this study was to test the ability of CYP11A1 and CYP27A1 to metabolise L2. Purified CYP27A1 was found to efficiently metabolise L2 to three major products and several minor products, whilst CYP11A1 did not act appreciably on L2. The three major products of CYP27A1 action on L2 were identified by mass spectrometry and NMR as 24-hydroxyL2, 27-hydroxyL2 and 28-hydroxyL2. Minor products included two dihydroxy L2 species, one which was identified as 24,27(OH)2L2, and another metabolite with one oxo and one hydroxyl group added. A comparison on the kinetics of the metabolism of L2 by CYP27A1 with that of the structurally similar compounds, L3 and ergosterol, was carried out with substrates incorporated into phospholipid vesicles. CYP27A1 displayed a 12-fold lower Km with L2 as substrate compared to L3 and a 5-fold lower turnover number (kcat), resulting in a 2.2 fold higher catalytic efficiency (kcat/Km) for L2 metabolism. L2 was a much better substrate for CYP27A1 than its precursor, ergosterol, with a catalytic efficiency 18-fold higher. The major CYP27A1-derived hydroxy-L2 products, 24-hydroxyL2, 27-hydroxyL2 and 28-hydroxyL2, inhibited the proliferation of melanoma and epidermoid cancer cell lines. In conclusion, this study shows that L2 is not metabolized appreciably by CYP11A1, but it is a good substrate for CYP27A1 which hydroxylates its side chain to produce 3 major products that display anti-proliferative activity on skin-cancer cell lines.

Genetic variants in the vitamin D pathway and their association with vitamin D metabolite levels: Detailed studies of an inner-city pediatric population suggest a modest but significant effect in early childhood.

OBJECTIVES: In a large cohort of healthy infants and toddlers 6-36 months of age (n = 776), we have been exploring the potential role of genetic variation in predisposition to vitamin D insufficiency. The genes encoding the key cytochrome P450 hydroxylases (CYP2R1, CYP24A1, and CYP27B1) harbour recurrent mutations of uncertain effect. This study was undertaken to look for biochemically relevant associations of these variants with inter-individual differences in vitamin D metabolism in an at-risk pediatric population. METHODS: Genotyping for CYP2R1-CT (c.-1127 C>T, rs10741657), CYP24A1-AG (c.-686A>G, rs111622401), and CYP27B1-CA (c.-1261 C>A, rs10877012) mutations were performed using SNaPshot assay, followed by Sanger sequencing confirmation. Vitamin D metabolites and vitamin D binding protein (DBP) were measured by established methods. RESULTS: In a multivariate regression model, with corrections for co-variates, subjects with the homozygous CYP2R1-TT variant had significantly higher concentrations of 25(OH)D, free 25(OH)D, and 24,25(OH)2D levels. In subjects with the CYP24A1-AG mutation, concentrations of 25(OH)D were significantly higher. CONCLUSIONS: The CYP2R1-TT and CYP24A1-AG variants have measurable effects on the vitamin D pathway. It seems unlikely that they will be clinically relevant in isolation, but they may be members of the large pool of infrequent mutations contributing to different risks for the vitamin D deficiency phenotype.

Cloning and Characterization of Cyp7a1 and Cyp27a1 Genes from the Non-Parasitic Japanese Lamprey Lethenteron reissneri.

Two cytochrome P450 genes homologous to human CYP7A1 and CYP27A1 were cloned from the non-parasitic Japanese lamprey Lethenteron reissneri. Lamprey cyp7a1 mRNA had varied expression levels among individuals: about four orders of magnitude differences in larval liver and nearly three orders of magnitude differences in male adult liver. Overexpressed Cyp7a1 protein tagged with green fluorescent protein (GFP) was localized to the endoplasmic reticulum. Lamprey cyp27a1 mRNA had relatively constant expression levels: within two orders of magnitude differences in larvae and adult liver and intestine. GFP-tagged Cyp27a1 protein was localized to mitochondria. The expression profiles of lamprey cyp7a1 and cyp27a1 genes and the cellular localizations of their products were in good agreement with their counterparts in mammals, where these two P450s catalyze initial hydroxylation reactions of cholesterol in classical and alternative pathways of bile acid synthesis, respectively. The cyp7a1 mRNA levels in adult male liver showed significant negative correlations to both body weight and total length of the animal, implying the involvement of the gene in the production of female-attractive pheromones in sexually matured male livers. The lamprey Cyp7a1 contains a long extension of 116 amino acids between helices D and E of the protein. Possible roles of this extension in regulating the enzymatic activity of lamprey Cyp7a1 are discussed.

CYP27A1 deficiency promoted osteoclast differentiation.

Background: The elevating osteoclast differentiation can lead to an imbalance in bone homeostasis, which was responsible for bone loss and bone diseases, such as osteoporosis. Multiple pathways and molecules have been involved in osteoclast formation, but the role of CYP27A1 in osteoclast differentiation has never been explored. Methods: CYP27A1 deficient mice were constructed using CRISPR-Cas9 system. Osteoclast differentiation was detected by TRAP staining. Differentially expressed genes (DEGs) were identified using RNA-seq analysis and were confirmed by qRT-PCR and Western blot. Results: The results showed that CYP27A1 knockout (KO) promoted osteoclast differentiation and bone loss. The transcriptomic analysis revealed that CYP27A1 KO led to differential expression of multiple genes, including ELANE, LY6C2, S100A9, GM20708, BGN, SPARC, and COL1A2, which were confirmed by qRT-PCR and Western blot. Enrichment analysis indicated that these differential genes were significantly associated with osteogenesis-related pathways, such as PPAR signaling, IL-17 signaling, and PI3K/AKT signaling, which were confirmed by qRT-PCR and Western blot. Conclusions: These results suggested that CYP27A1 was involved in osteoclast differentiation, providing a novel therapeutic target for osteoclast-related diseases.

Analysis of the ability of vitamin D3-metabolizing cytochromes P450 to act on vitamin D3 sulfate and 25-hydroxyvitamin D3 3-sulfate.

25-Hydroxyvitamin D3 (25(OH)D3) is present in the human circulation esterified to sulfate with some studies showing that 25(OH)D3 3-sulfate levels are almost as high as unconjugated 25(OH)D3. Vitamin D3 is also present in human serum in the sulfated form as are other metabolites. Our aim was to determine whether sulfated forms of vitamin D3 and vitamin D3 metabolites can be acted on by vitamin D-metabolizing cytochromes P450 (CYPs), one of which (CYP11A1) is known to act on cholesterol sulfate. We used purified, bacterially expressed CYPs to test if they could act on the sulfated forms of their natural substrates. Purified CYP27A1 converted vitamin D3 sulfate to 25(OH)D3 3-sulfate with a catalytic efficiency (kcat/Km) approximately half that for the conversion of vitamin D3 to 25(OH)D3. Similarly, the rate of metabolism of vitamin D3 sulfate was half that of vitamin D3 for CYP27A1 in rat liver mitochondria. CYP2R1 which is also a vitamin D 25-hydroxylase did not act on vitamin D3 sulfate. CYP11A1 was able to convert vitamin D3 sulfate to 20(OH)D3 3-sulfate but at a considerably lower rate than for conversion of vitamin D3 to 20(OH)D3. 25(OH)D3 3-sulfate was not metabolized by the activating enzyme, CYP27B1, nor by the inactivating enzyme, CYP24A1. Thus, we conclude that 25(OH)D3 3-sulfate in the circulation may act as a pool of metabolically inactive vitamin D3 to be released by hydrolysis at times of need whereas vitamin D3 sulfate can be metabolized in a similar manner to free vitamin D3 by CYP27A1 and to a lesser degree by CYP11A1.

Clinical presentation and molecular genetic analysis of a Sudanese family with a novel mutation in the CYP2R1 gene.

The aim of this study was to identify the genetic basis of two female siblings - born to consanguineous Sudanese parents - diagnosed clinically as having the rare condition of 25-hydroxylase deficiency (vitamin D-dependent rickets type 1B). The initial diagnosis was established based on clinical data, laboratory and radiological findings retrospectively. Primers for all exons (5) of human CYP2R1 (NM_024514) were generated followed by Sanger sequencing on exons 1-5 for both girls and their parents. Homozygosity for a point mutation (c.85C > T) was detected, leading to a nonsynonymous variant at position 29 in exon 1, resulting in a premature stop codon (p.Q29X). This is a previously unknown variant that leads to a severely truncated protein and predicted to be among the 0.1 % most deleterious genomic variants(CADD score 36). To our knowledge, this family represents the first case series from Sudan with a confirmed CYP2R1 gene mutation and the 6th world-wide. With the lack of genetic facilities, diagnosis should be suspected by the persistently low 25 hydroxyvitamin D level in spite of proper treatment and after ruling out liver disease and malabsorption. Patients in this case series showed healing of rickets when treated with high doses of 1,25-dihydroxyvitamin D3 (1,25(OH)D3; calcitriol) and oral calcium.

The tumor-repressing effect of CYP27A1 on renal cell carcinoma by 27-HC arising from cholesterol metabolism.

As a key approach to mediate cholesterol metabolism, the role of the CYP27A1/27-HC axis in renal cell carcinoma (RCC) remains unclear. Analysis of CYP27A1 expression from public databases and metastatic cases in our center suggested that CYP27A1 was obviously downregulated in RCC tissues, and survival analysis further showed its correlation with favorable clinicopathological features and prognosis. In vitro, up and downregulation of CYP27A1 expression in RCC cell lines could definitely illustrate its anticipation involving apoptosis, proliferation, invasion, migration, and clonality. This could be achieved through upregulation of 27-HC concentration, which mediates the activation of signaling pathways of apoptosis and cell cycle arrest. Further, recovery of CYP27A1 expression could definitely inhibit the proliferation of RCC tumors in vivo. This is the first study to explore the role of the CYP27A1/27-HC axis in RCC. Attempts to maintain the normal function of the axis may be a potential strategy in the treatment of RCC, and the predictive value of CYP27A1 detection on the efficacy of targeted therapy in metastatic RCC is also worthy of attention.

CYP27A1 inhibits proliferation and migration of clear cell renal cell carcinoma via activation of LXRs/ABCA1.

Cholesterol homeostasis plays an important role in the maintenance of normal body functions. CYP27A1 is a key enzyme known to regulate cholesterol homeostasis, which catalyzes the conversion of cholesterol to 27-HC and has been implicated in the occurrence and metastasis of various cancer types. The present study aimed to explore the regulatory role of CYP27A1 in the development of clear cell renal cell carcinoma (ccRCC). In particular, the effect of CYP27A1 on the proliferation and migration of ccRCC cells was investigated. The construction of a stable 786-O cell line overexpressing CYP27A1/pLVX was mediated by lentiviral infection. The proliferative capacity was assessed using MTT and colony formation. Wound healing assay was used to measure cell migration. Production of intracellular cholesterol and 27-HC was detected by enzyme-linked immunosorbent assay. The LXRs/ABCA1 pathway of cholesterol metabolism regulation was studied by RT-qPCR and Western blotting analysis after cells were treated with stimulation agents of 27-HC or T0901317 and inhibition agents of siRNA or GSK2033. The results revealed that overexpression of CYP27A1 could increase the intracellular production of 27-HC and inhibit the proliferation and migration of 786-O cells. And the treatment of 786-O cells with 27-HC induced a similar effect. CYP27A1/27HC mediated activation of the liver X receptors (LXRs) could up-regulate the expression of ATP-binding cassette transporter A1 (ABCA1), further resulting in the reduction of intracellular cholesterol contents. All of these findings indicated a regulatory role of CYP27A1 in the proliferation and migration of ccRCC, via activating LXRs/ABCA1 to regulate cholesterol homeostasis.

Streptozotocin-induced Diabetes Represses Hepatic CYP2R1 Expression but Induces Vitamin D 25-Hydroxylation in Male Mice.

Vitamin D deficiency [ie, low plasma 25-hydroxyvitamin D (25-OH-D)] associates with the prevalence of metabolic diseases including type 1 diabetes; however, the molecular mechanisms are incompletely understood. Recent studies have indicated that both fasting and metabolic diseases suppress the cytochrome P450 (CYP) 2R1, the major hepatic vitamin D 25-hydroxylase. We specifically studied the effect of a mouse model of type 1 diabetes on the regulation of Cyp2r1 and vitamin D status. We show that streptozotocin-induced diabetes in mice suppresses the expression of the Cyp2r1 in the liver. While insulin therapy normalized the blood glucose levels in the diabetic mice, it did not rescue the diabetes-induced suppression of Cyp2r1. Similar regulation of Cyp2r1 was observed also in the kidney. Plasma 25-OH-D level was not decreased and was, in contrast, higher after 4 and 8 weeks of diabetes. Furthermore, the vitamin D 25-hydroxylase activity was increased in the livers of the diabetic mice, suggesting compensation of the Cyp2r1 repression by other vitamin D 25-hydroxylase enzymes. Cyp27b1, the vitamin D 1α-hydroxylase, expression in the kidney and the plasma 1α,25-dihydroxyvitamin D level were higher after 4 weeks of diabetes, while both were normalized after 13 weeks. In summary, these results indicate that in the mouse model of type 1 diabetes suppression of hepatic Cyp2r1 expression does not result in reduced hepatic vitamin D 25-hydroxylase activity and vitamin D deficiency. This may be due to induction of other vitamin D 25-hydroxylase enzymes in response to diabetes.

Role of 27-hydroxycholesterol and its metabolism in cancer progression: Human studies.

Direct translation of findings achieved in experimental cell or animal models to humans is quite a difficult task. We focused here only on the epidemiological and ex vivo human studies so far available about the role of 27-hydroxycholesterol (27OHC) and related metabolism in cancer development. Some studies point to an adverse effect of 27OHC in breast cancer, based on the oxysterol's recognized ability to bind to and modulate estrogen receptors. The detrimental role of this side chain oxysterol would be evident in cancer progression, mainly in post-menopausal women and in an advanced stage of the disease. Other human researches, however, would rather correlate 27OHC intra-tumoral levels to a better prognosis. The analyses on human prostate cancer specimens performed to date are all against a detrimental contribution of 27OHC, rather suggesting interesting anti-prostate cancer effects exerted by this oxysterol. Finally, an increased 27OHC synthesis on the contrary seems to favour progression of late stage cancers in colon, brain and thyroid tissues, as found for breast cancer, possibly due to pro-inflammatory and pro-survival signalling triggered by disproportionate amounts of this oxysterol.

Our evolving understanding of how 27-hydroxycholesterol influences cancer.

Cholesterol has been implicated in the pathophysiology and progression of several cancers now, although the mechanisms by which it influences cancer biology are just emerging. Two likely contributing mechanisms are the ability for cholesterol to directly regulate signaling molecules within the membrane, and certain metabolites acting as signaling molecules. One such metabolite is the oxysterol 27-hydroxycholesterol (27HC), which is a primary metabolite of cholesterol synthesized by the enzyme Cytochrome P450 27A1 (CYP27A1). Physiologically, 27HC is involved in the regulation of cholesterol homeostasis and contributes to cholesterol efflux through liver X receptor (LXR) and inhibition of de novo cholesterol synthesis through the insulin-induced proteins (INSIGs). 27HC is also a selective modulator of the estrogen receptors. An increasing number of studies have identified its importance in cancer progression of various origins, especially in breast cancer. In this review, we discuss the physiological roles of 27HC targeting these two nuclear receptors and the subsequent contribution to cancer progression. We describe how 27HC promotes tumor growth directly through cancer-intrinsic factors, and indirectly through its immunomodulatory roles which lead to decreased immune surveillance and increased tumor invasion. This review underscores the importance of the cholesterol metabolic pathway in cancer progression and the potential therapeutic utility of targeting this metabolic pathway.

Vitamin D Metabolic Pathway Genes Polymorphisms and Their Methylation Levels in Association With Rheumatoid Arthritis.

Abnormal vitamin D metabolism is involved in the pathogenesis of rheumatoid arthritis (RA). In this study, we evaluated the association of single nucleotide polymorphisms (SNPs) and methylation levels in vitamin D metabolic pathway genes with RA susceptibility. Ten SNPs in vitamin D metabolic pathway genes (CYP2R1, CYP24A1, VDR, CYP27B1) were genotyped in 477 RA patients and 496 controls by improved multiple ligase detection reaction (iMLDR). The methylation levels of the promoter regions of these genes were detected in 122 RA patients and 123 controls using Illumina Hiseq platform. We found that the CYP2R1 rs1993116 GA genotype, CYP27B1 rs4646536 GA genotype, rs4646536 A allele frequencies were significantly increased in RA patients when compared to controls. The decreased risk of rs1993116, rs4646536 was found under the dominant mode in RA patients. However, no significant association was found between CYP2R1 rs7936142, rs12794714, CYP24A1 rs2762934, rs6068816, rs2296239, rs2296241, VDR rs11574129, rs3847987 polymorphism, and RA susceptibility. The VDR, CYP27B1 methylation levels in RA patients were significantly lower than those in controls, while CYP2R1, CYP24A1 methylation levels were not associated with RA. There were no statistical associations between CYP2R1, CYP24A1, VDR, CYP27B1 methylation levels and their respective genotype in RA patients. In addition, plasma 25OHD level in RA patients was significantly lower than that in healthy controls. In summary, our results showed that CYP2R1, CYP27B1 genetic variations were associated with the genetic background of RA, while altered VDR, CYP27B1 methylation levels were related to the risk of RA.

New Variants of the Cytochrome P450 2R1 (CYP2R1) Gene in Individuals with Severe Vitamin D-Activating Enzyme 25(OH)D Deficiency.

BACKGROUND: Vitamin D is a fat-soluble cholesterol derivative found in two forms, vitamin D2, and vitamin D3. Cytochrome P450 2R1 (CYP2R1) encoded by the CYP2R1 gene is the major hydroxylase that activates vitamin D by catalyzing the formation of 25-hydroxyvitamin D (25(OH)D). METHODS: We collected 89 (100%) subjects, 46 of which (51.69%) had a documented severe deficiency of 25(OH)D (<10 ng/mL) and 43 (48.31%) in the control group with documented optimum levels of 25(OH)D (>30 ng/mL). We performed Sanger sequencing of three selected fragments of the CYP2R1 gene (Ch11: 14878000-14878499; Ch11: 14880058-14880883 and Ch11: 14885321-14886113) that affect the binding of substrates to this enzyme and analyzed the possible involvement of genetic variation in these regions with an increased risk of vitamin D deficiency in healthy Polish individuals. RESULTS: Two substitutions were found within the three fragments. Bioinformatic analysis suggested that one of these (NC_000011.10: g.14878291G>A) may influence the structure and function of CYP2R1. CONCLUSIONS: Variant NC_000011.10: g.14878291G>A may have a perturbing effect on heme binding in the active site of CYP2R1 and on the function of 25-hydroxylase and probably affects the concentration of 25(OH)D in vivo. We intend to perform functional verification in a larger patient population to confirm and extend these results.

Associations between Genetic Variants in the Vitamin D Metabolism Pathway and Severity of COVID-19 among UAE Residents.

Vitamin D has many effects on cells in the immune system. Many studies have linked low vitamin D status with severity of COVID-19. Genetic variants involved in vitamin D metabolism have been implicated as potential risk factors for severe COVID-19 outcomes. This study investigated how genetic variations in humans affected the clinical presentation of COVID-19. In total, 646 patients with SARS-CoV-2 infection were divided into two groups: noncritical COVID-19 (n = 453; 70.12%) and a critical group (n = 193; 29.87%). Genotype data on the GC, NADSYN1, VDR, and CYP2R1 genes along with data on serum 25-hydroxyvitamin D levels were compiled in patients admitted to a major hospital in the United Arab Emirates between April 2020 and January 2021. We identified 12 single-nucleotide polymorphisms associated with the critical COVID-19 condition: rs59241277, rs113574864, rs182901986, rs60349934, and rs113876500; rs4944076, rs4944997, rs4944998, rs4944979, and rs10898210; and rs11574018 and rs11574024. We report significant associations between genetic determinants of vitamin D metabolism and COVID-19 severity in the UAE population. Further research needed to clarify the mechanism of action against viral infection in vitamin D deficiency. These variants could be used with vaccination to manage the spread of SARS-CoV-2 and could be particularly valuable in populations in which vitamin D deficiency is common.

Effect and mechanism of vitamin D activation disorder on liver fibrosis in biliary atresia.

To investigate the mechanism of 25 hydroxyvitamin D (25(OH)D) deficiency in children with biliary atresia (BA) and its effect on liver fibrosis. The serum vitamin D and 25(OH)D, and expression of 25 hydroxylase (CYP2R1 and CYP27A1) in the liver of BA patients were detected and compared with those in the control group. We investigated the effect of differential expression of CYP2R1 in hepatocytes on the expression of genes related to liver fibrosis in primary hepatic stellate cells (HSCs) of BA and animal models of cholestasis. The ratio of 25(OH)D/vitamin D in the BA group was significantly lower than that in the control group. The mRNA and protein expression of CYP2R1 and CYP27A1 in liver tissue of the BA group was significantly lower than that in the control group. Exogenous active vitamin D (calcitriol) inhibited the proliferation and migration of primary HSCs isolated from BA patients, and reduced the expression of fibrosis-related genes in vitro. Downregulation of expression of CYP2R1 in hepatocytes increased expression of transforming growth factor (TGF)-ß1, collagen (Col)-1α1 and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased the expression of matrix metalloproteinase (MMP)-2 in cocultured primary HSCs of BA. Upregulation of expression of CYP2R1 in mice with bile duct ligation significantly increased the level of 25(OH)D, decreased the expression of TGF-ß1, Col-1α1 and TIMP-1, and increased the expression of MMP-2. Children with BA have impaired vitamin D activation due to CYP2R1 deficiency. The dysactivation of vitamin D can promote the proliferation and activation of HSCs and participate in the development of hepatic fibrosis in BA.

High Fat Diet and High Cholesterol Diet Reduce Hepatic Vitamin D-25-Hydroxylase Expression and Serum 25-Hydroxyvitamin D3 Level through Elevating Circulating Cholesterol, Glucose, and Insulin Levels.

SCOPE: Low circulating 25-hydroxyvitamin D (25(OH)D) levels associate with obesity, diabetes, and hyperlipidemia, but the underlying mechanisms remain uncertain. As energy-dense diet contributes to these disorders, this study investigates whether diet could impair vitamin D metabolism. METHODS AND RESULTS: Compared with control chow-fed mice, high fat diet (HFD)-fed mice show lower serum 25(OH)D3 and 1,25(OH)2 D3 levels, lower hepatic vitamin D 25-hydroxylase Cyp2r1 expression but comparable renal vitamin D metabolic enzymes expression. Time course studies show that after HFD feeding, the serum concentrations of cholesterol, triglycerides, fatty acids, glucose, and insulin elevate sequentially and before the reduction of hepatic Cyp2r1 expression and serum 25(OH)D3 levels. Hepatic Cyp2r1 expression also reduces after consuming high fat and high sucrose diet. After high cholesterol diet feeding, serum total cholesterol rises and hepatic Cyp2r1 expression decreases ahead of the reduction of serum 25(OH)D3 . In vitro studies demonstrate that high concentrations of cholesterol, glucose, and insulin significantly inhibit Cyp2r1expression in primary murine hepatocytes. Further studies show that dietary restriction in HFD-fed mice ameliorates hypercholesterolemia, hyperglycemia, and hypertriglyceridemia, and elevates hepatic Cyp2r1 expression and serum 25(OH)D3 level. CONCLUSION: These findings suggest that diet-induced elevation of circulating cholesterol, glucose, and insulin reduces serum 25(OH)D3 level through suppressing hepatic Cyp2r1 expression.

Vitamin D pathway gene polymorphisms, vitamin D level, and cytokines in children with type 1 diabetes.

AIMS: The study aimed to examine genetic polymorphism of vitamin D-related genes and association between those genes and vitamin D and cytokines levels in children with type 1 diabetes (T1D). MATERIALS AND METHODS: This study was conducted among 100 T1D children and 100 controls at Division of Endocrinology and Metabolism, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, during 2016 to 2018. Vitamin D metabolite levels were measured by liquid chromatography-tandem mass spectrometry method, serum cytokine levels of IFN- É£, IL-10, IL-13, IL-17α, IL-2, IL-4, IL-6, and TNF-α by immunoassay, and genetic variations at VDR, CYP2R1, CYP27B1, GC, DHCR7, and CYP24A1 by polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: A relationship between studied single nucleotide polymorphisms and T1D was found in CYP2R1 (rs10741657) (GA, OR: 1.83, 95% CI: 1.01-3.31; p = 0.04). VDR haplotypes were also remarkably different between T1D patients and controls. Controls had higher frequency of haplotype TACT than T1D patients (p = 0.02). Vitamin D and all cytokine levels, except for IL-17α, were significantly increased in T1D compared to controls. The polymorphism of DHCR7 (rs12785878) was positively associated with 25OHD3 and 3epi25OHD3 levels and was negatively associated with 25OHD2 level. On the other hand, polymorphism of CYP27B1 (rs4646536) was negatively associated with 3epi25OHD3 level. Polymorphisms of CYP27B1 (rs4646536) and GC (rs2282679) were positively associated with TNF-α levels. VDR variation of rs1544410, rs731236, and rs7975232 also showed negative association with IL-10 levels. In contrast, the level of IL-10 was positively associated with DHCR7 (rs12785878). CONCLUSION: Relationships between T1D and CYP2R1 polymorphism and VDR haplotype were found. Vitamin D gene-related variations were associated with vitamin D and circulating cytokine levels in children with T1D.

The effect of umbilical cord blood spexin, free 25(OH) vitamin D3 and adipocytokine levels on intrauterine growth and anthropometric measurements in newborns.

Spexin is a newly described peptide and is known to reduce the uptake of long-chain fatty acids into adipocytes. The serum spexin levels of obese children between the ages of 12-18 are lower. The effect of serum spexin and free 25(OH) vitamin D3 levels on intrauterine development in newborns is unknown. Our aims is to evaluate the effects of spexin and adipocytokin levels in the cord blood of term newborn babies on the weight of the baby according to the gestation age (GA) and anthropometric measurement results. Babies who were born in our hospital and whose GA was ≥37 weeks were evaluated in three groups as appropriate for GA (AGA), small for GA (SGA) and large for GA (LGA). A total of 84 babies, including an equal number of infants in AGA, SGA and LGA groups, were included in the study. Spexin, leptin, active ghrelin, free 25(OH) vitamin D3, glucose, and insulin levels in the cord blood of infants were examined at birth. The results were compared according to GA and birth weight (BW). There was no statistically significant difference between groups in terms of mean spexin, active ghrelin, free 25(OH) vitamin D3, and insulin levels. The mean leptin level was significantly higher in LGA group than SGA and AGA groups (p 0.004). The mean spexin and leptin levels were higher in girls than in boys (respectively p value 0.029, 0.003). Although there is a significant positive correlation between BW, head circumference, height, umbilical circumference, umbilical circumference/height ratio and the mean leptin levels (p < 0.001), there was no significant correlation between mean spexin, active ghrelin, free 25 (OH) vitamin D3, insulin, and glucose levels. This study suggests that spexin may not have an effect on intrauterine development.

CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism.

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.