First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism  For ex effects , 15. In  effects , 17, sug effects , 19. Fur effects . In gene effects , 22. Thede novo synthesis of steroids requires conversion of cholesterol to pregnenolone by the cytochrome P450 (CYP) 11A1 enzyme (CYP11A1). Cholesterol is first transported into the mitochondria, mediated by translocator protein 18 kDa (TSPO) and steroid acute regulatory protein (STAR) . This enzyme activity requires one molecule of oxygen and one molecule of NADPH for each reaction. Unlike CYP450 enzymes in the endoplasmic reticulum that use NADPH-CYP reductase (POR) as a redox partner, mitochondrial CYP11A1 activity involves two cofactors ferredoxin (FDX1) and ferredoxin reductase (FDXR), also known as adrenodoxin and adrenodoxin reductase, respectively, similar to other mitochondrial CYP450s  . In the n (STAR)   are expressed at very low levels in glial cells (B and S1B). Enzymes further upstream in the steroidogenesis pathway are more highly expressed. Glial cells express low but detectable amounts of mRNA for CYP11A1, CYP17A1, CYP19A1, 3β-hydroxysteroid dehydrogenases , 17β-hydroxysteroid dehydrogenases , 3α-hydroxysteroid dehydrogenase , 5α-reductase , steroid sulfatase (STS), and sulfotransferase (SULT2B1). No expression for CYP11B1, CYP11B2, CYP21A2, AKR1C4, and SULT2A1 could be detected in glial cells.To determine whether human glial cells express the components of the machinery used for peripheral steroid biosynthesis, we performed quantitative RT-PCR (qRT-PCR) to evaluate expression of genes important for steroidogenesis. In general, enzymes in the classical steroidogenesis pathway A are expal cells B and S1BCYP11A1 mRNA in various regions of the human central nervous system (CNS) using qRT-PCR. The highest expression of CYP11A1 was found in the spinal cord, followed by the occipital pole and the parietal lobe (B). The expression in the temporal lobe, cerebral cortex, cerebellum, and total brain were approximately the same. CYP11A1 expression in CNS tissue is more than 1000 times lower than in the adrenals and more than 100 times lower than in the testes (C).We determined the overall levels of tal lobe B. The exe testes C.in situ hybridization (ISH) analyses on human cerebellum and cerebral cortex tissue slices using the RNAscope technology described by Wang et al. . Interestingly, we found that a larger proportion of Purkinje cells had positive expression for CYP11A1 (E). We could not observe the presence of CYP11A1 mRNA in white matter of the cerebellum (G). Similar to the cerebellum, extremely low levels of CYP11A1 mRNA were seen in the cerebral cortex gray matter with even lower expression in the white matter (F and CYP11A1 mRNA (E and G), a very scarce number of cells showed positive CYP11A1 signals in the cortex white matter of one donor (H). We estimate that the proportion of CYP11A1-positive cells is less than 1% of cells in both brain regions. Each CYP11A1-positive cell showed no more than two molecules of CYP11A1 mRNA in our ISH assay. There was also little colocalization between MBP and CYP11A1, suggesting that oligodendrocytes and myelinated fibers are not likely to express the most CYP11A1 in the cerebellum and cortex.To further investigate the tissue and cell type localization of CYP11A1 in the brain, we performed g et al. . A duplear layer , D and E CYP11A1 E. We courebellum G. Similae matter F and S2.1A1 mRNA , E and Gne donor H. We estCYP11A1 expression in the glial cell lines . We also confirmed that all four glial cell lines express mRNA for the CYP11A1 cofactors, FDX1 and FDXR (B and C). Despite the presence of low CYP11A1 mRNA levels in the glial cells, we could not detect any CYP11A1 protein expression by immunoblotting using four different commercially available antibodies . No specific signal for CYP11A1 could be observed on immunoblots even when up to 80 μg protein was loaded for glial cells (E). When protein bands around 40, 50, and 60 kDa were subjected to shotgun proteomics mass spectrometry (MS) analysis, no CYP11A1 protein/peptide sequences were found, confirming the lack of specific CYP11A1 bands in the immunoblots (dataset S1). Low levels of FDX1 and FDXR protein can be detected in the four glial cell lines (E). We were, however, able to detect low levels of CYP11A1 staining using immunofluorescence . We found that pregnenolone production can be stimulated by the TSPO ligand XBD-173 but not the TSPO ligand FGIN-1-27 at 50 μM . Ho. HoA andat 50 μM . This reA–H and S6A). In the peripheral cells, AMG also significantly inhibited steroid formation in response to 22(R)-HC, 22(S)-HC, and 20α-HC treatment. However, in all four glial cell lines, AMG failed to inhibit steroid production at both the basal level and when induced with hydroxycholesterols (I–X and S6B).To confirm whether an undetectable but small amount of CYP11A1 enzyme in glial cells is responsible for producing pregnenolone, we treated the cells with CYP11A1 inhibitors. The specific CYP11A1 inhibitor DL-aminoglutethimide (AMG)  significA–H). However, similar to AMG, KC failed to inhibit steroid production in the four glial cell lines at basal and hydroxycholesterol-stimulated conditions (I–X). A summary of these results is presented in C and D), suggesting that these drugs stimulate pregnenolone production in glial cells rather than inhibiting it as in peripheral cells. This induced increase in pregnenolone secretion does not appear to be due to upregulation of CYP11A1 expression or upregulation of CYP450 cofactors . KC inhibits multiple CYPs along the steroidogenic pathway—including CYP11A1, CYP17A1, CYP19A1, CYP11B1, and CYP11B2, as well as other CYPs such as CYP3A4 , 42. Thenditions , I–X. A ofactors  but rathOur findings suggest that human brain cells can synthesize pregnenolone independently of CYP11A1 activity, and the apparent desmolase activity that allows synthesis of pregnenolone from hydroxycholesterols is not inhibited by CYP11A1 inhibitors AMG and KC. These findings support our recent discussion on the discrepancy between levels of CYP11A1 and pregnenolone found in the brain, where we suggested that alternative pathway(s) may be used to produce pregnenolone there . Using tWe first examined the involvement of ROS in pregnenolone synthesis by glial cells. Our results showed that high concentrations of the two CYP11A1 inhibitors AMG and KC seemed to increase rather than inhibit pregnenolone production in glial cells both at the basal and hydroxycholesterol-stimulated conditions. AMG and KC have also been reported to increase cellular ROS , 44. FurA) or cellular ROS  overall. The increase in pregnenolone production with treatment of 2 mM Trolox was marginally significant (p = 0.0477). High dose AMG (0.76 mM) and high dose KC (50 μM) treatments led to higher pregnenolone production but no significant changes in cellular ROS . Furthermore, the addition of Trolox had no significant effect on the increased pregnenolone production induced by AMG and KC (B and C). Low doses of Trolox decreased the levels of cellular ROS induced by KC, but this effect was blunted at the highest dose of 2 mM . Trolox also did not induce significant changes in ROS when cells were cotreated with AMG . To confirm these results, we tested the same conditions in MGM-3 cells. Similar to MGM-1 cells, Trolox did not significantly affect pregnenolone synthesis and cellular ROS by itself or in combination with 0.76 mM AMG in MGM-3 cells (F). However, only lower doses of Trolox reduced the increase in pregnenolone when cells were treated with 50 μM KC (C). Therefore, our results indicate that antioxidants do not affect pregnenolone production in the glial cells.First, we examined the effects of the antioxidant (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), an analog of vitamin E, on pregnenolone production and cellular ROS in MGM-1 cells . We alsoFig. 8-3 cells . On the -3 cells F. Howeve50 μM KC C. Theref2O2) and ferrous sulfate (FeSO4). These two oxidants were chosen because they were previously shown to increase pregnenolone production in C6 rat glial cells, with 10 mM H2O2 inducing a twofold increase and 10 mM FeSO4 inducing a 3.6-fold increase (2O2 had no significant effect on pregnenolone production (A), despite significantly increasing cellular ROS starting at 100 μM (B). We also found that FeSO4 did not have any significant effects on pregnenolone synthesis (C). However, after 2 h of FeSO4 treatment at 500 μM, the highest dose, there was a significant increase in cellular ROS, with a nonsignificant increasing trend at lower concentrations (D). In our preliminary studies where we pretreated MGM-1 cells with FeSO4 for 24 h, we had observed a significant increase in ROS starting at 50 μM but there were still no significant changes in pregnenolone production (data not shown). The same results were seen in MGM-3 cells where both H2O2 and FeSO4 failed to induce significant changes in pregnenolone production , despite dose-dependently increasing cellular ROS levels the same as other oxidants we tested (F). Given that there is no correlation between changes in intracellular ROS levels and pregnenolone production, we believe that the inhibitory effects of nitric oxide on pregnenolone synthesis are due to another mechanism, which will be discussed further in a later section. Taken together, our antioxidant and oxidant experimental results provide evidence against our first possible pathway; thus, the alternative pathway for pregnenolone synthesis does not appear to be dependent on ROS.To confirm whether other forms of oxidative stress may be involved in pregnenolone synthesis, we treated MGM-1 cells with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Interestingly, SNAP significantly inhibited pregnenolone production by the cells E, despite tested F. Given i.e., the classical CYP11A1 enzyme), CYP11A1a will be used to denote the protein while CYP11A1 variant 1 will be used to indicate the mRNA transcript Accessed Apr 11, 2022. https://www.ncbi.nlm.nih.gov/gene/1583. Similarly, CYP11A1b will be used to denote the protein for isoform b and CYP11A1 variant 2 will be used to indicate the mRNA transcript. In comparison to isoform a, isoform b is 158 aa shorter from the N terminus. This second isoform could potentially be resistant to the inhibitory effects of AMG and KC due to possible structural differences but may still be able to produce pregnenolone. Therefore, human glial cells may use the second CYP11A1 isoform to synthesize pregnenolone.Next, we examined the roles of different CYP11A1 isoforms in pregnenolone synthesis. A second CYP11A1 isoform has been reported in mouse and humans , but theCYP11A1 variants in the human brain and in brain cells . Although the relative expression of CYP11A1 variant 2 appears to be lower than that of CYP11A1 variant 1 in all CNS tissues and brain cells, the total CYP11A1 expression was higher than the summed expression of the variants except for in MGM-3 and HMC3 cells (B and C). This may suggest an early truncation of the RNA transcript for CYP11A1b, since exon 1 is not translated into the final protein for this isoform. We could not accurately determine the protein levels for CYP11A1b in the human brain cells because CYP11A1b does not have a unique amino acid sequence that can differentiate it from CYP11A1a, and commercial antibodies that only bind CYP11A1a did not produce clear bands on immunoblots for glial cells to allow for subtraction (A and B).We first examined the levels of the in cells . Since tpression , A and CC3 cells , B and Ctraction , A and BA–C). As expected, CYP11A1a is strictly localized to the mitochondria while CYP11A1b has a more diffused localization throughout the cell and limited colocalization with mitochondria . CYP11A1b protein has a molecular weight of approximately 42 kDa on immunoblots while CYP11A1a appears as an approximately 50 kDa protein (C).To characterize the CYP11A1 isoforms better, we transfected MGM-1 cells with a CYP11A1a or CYP11A1b expression vector with a myc-DDK tag. After drug selection and colony expansion, we verified that the transfected MGM-1 cells highly expressed the corresponding CYP11A1 isoforms , A–C. Aschondria . The CYP1 exon 1 , A and B protein C.Figure D and E). Unlike WT MGM-1 cells where AMG had no inhibitory effect (I and J), pregnenolone production in CYP11A1a+ cells was significantly inhibited by AMG under both basal and hydroxycholesterol-stimulated conditions (F and G). These results suggest that overexpressing the classical CYP11A1a in MGM-1 cells significantly increases pregnenolone synthesis. This enhanced production can be inhibited by AMG similar to adrenal H295R-S1 and MA-10 Leydig cells that use the classical steroidogenesis pathway. These data imply that MGM-1 cells are not inherently resistant to the effects of AMG, but rather endogenous pregnenolone production by MGM-1 cells is independent of classical CYP11A1a. On the other hand, overexpression of CYP11A1b did not significantly alter pregnenolone production compared to control cells (D and E), and the effects of AMG on CYP11A1b+ cells were similar to that of WT cells, where AMG failed to inhibit pregnenolone synthesis and instead increased pregnenolone levels at higher doses (H and I). These results indicate that CYP11A1b is not involved in pregnenolone production unlike CYP11A1a and that the second possibility listed previously of another isoform involved in pregnenolone production is unlikely.Cells overexpressing CYP11A1a showed approximately a 27-fold increase in basal pregnenolone production and a threefold increase in 22(R)-HC-stimulated pregnenolone production compared to control cells , D and Ey effect , I and Jnditions , F and Gol cells , D and Eer doses , H and IA and B). In CYP11A1a+ MGM-1 cells, deferoxamine inhibited pregnenolone production in a dose-dependent manner under both basal and 22(R)-HC stimulated conditions, demonstrating effective inhibition of CYP450 activity (C and D). To confirm the effects of iron chelation on pregnenolone synthesis, we treated the cells with another but structurally different iron chelator, deferiprone (E and F). The inhibitory effects of SNAP and iron chelation on pregnenolone secretion by MGM-1 cells suggest that the alternative pathway for pregnenolone synthesis may involve activity of another CYP450.We demonstrated previously that the nitric oxide donor SNAP significantly inhibited pregnenolone synthesis. Given that there was no correlation between changes in intracellular ROS and pregnenolone production, we propose that the effect of SNAP on pregnenolone synthesis may be due to inhibition of CYP activity . Since Cactivity , C and Deriprone . Indeed,eriprone , E and Fvia POR while enzymes in the mitochondria accept electrons via FDXR and FDX1 (A and B), RNA expression of POR in glial cells is higher than that of FDXR (A and 2C), and POR protein is clearly detectable by immunoblot . However, treatment with diphenyleneiodonium, an inhibitor of POR, failed to alter pregnenolone production in glial cells under both basal and 22(R)-HC stimulated conditions (C and D).To further test this possibility, we explored the effects of inhibiting CYP450 cofactors, specifically POR and FDXR. CYP450 enzymes in the endoplasmic reticulum accept electrons from NADPH and FDX1 . Therefoand FDX1 , A and B of FDXR A and 2C,nditions , C and DA–C and E–G). Knockdown of POR failed to alter pregnenolone synthesis (D), supporting the results observed with diphenyleneiodonium. On the other hand, knockdown of FDXR significantly decreased pregnenolone synthesis by MGM-1 cells (H). Altogether, our results suggest that our third possibility is most probable: the alternative pathway for pregnenolone production in the brain involves a CYP450 enzyme activity other than CYP11A1 and the responsible enzyme(s) is likely localized to the mitochondria as they are dependent on FDXR activity.These data appear to suggest that pregnenolone synthesis in glial cells is not dependent on POR and thus not dependent on a CYP450 activity in the endoplasmic reticulum. However, in addition to POR, diphenyleneiodonium also inhibits other enzymes such as NADPH oxidase, nitric oxide synthase, and xanthine oxidase, which have more varied subcellular localizations including the cytoplasm, cytoskeleton, vesicular organelles, nuclei, and other organelles depending on cell type , 50, 51.ynthesis D, suppor-1 cells H. AltogeCYP11A1 mRNA in various parts of the human brain and four human glial cell lines, with levels between 1000 to 10,000 times lower than that in human adrenal cells and tissue. The difference in expression levels agrees with previous studies comparing CYP11A1 mRNA levels between human brain and adrenal tissues -HC, followed by 20α-HC, whereas the stereoisomer 22(S)-HC had no effect as expected , 61. Thi4 above 0.3 mM . Nevertheless, in combination with our H2O2 results, our data suggests that pregnenolone synthesis by human glial cells is not dependent on ROS unlike DHEA synthesis. These results are in contrast to studies in C6 rat glial cells, where 10 mM H2O2 and FeSO4 both increased pregnenolone production by at least twofold  and the adrenal cortical carcinoma cell line NCI-H295R  were purchased from American Type Culture Collection. HMC3 cells were grown in DMEM/F-12, GlutaMAX  with 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 5% CO2. H295R-S1 cells were grown in DMEM/F-12, GlutaMAX with 2.5% Nu-Serum , 100 IU/ml penicillin, 100 μg/ml streptomycin, plus ITS+ Premix Universal Culture Supplement at a concentration recommended by the manufacturer . NHAs were purchased from Lonza (#CC-2565). NHA cells were grown at 37 °C and 5% CO2 in astrocyte growth medium BulletKit : Astrocyte basal medium plus rhEGF, insulin, ascorbic acid, GA-1000, L-glutamine, and 3% FBS. MA-10 cells were a gift from Dr Mario Ascoli (University of Iowa). MA-10 cells were grown in DMEM/F12, GlutaMAX with 5% FBS, 2.5% horse serum , 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37 °C and 3.5% CO2. NHA cells were passaged using ReagentPack Subculture Reagents , and all other cell lines were passaged using trypsin/EDTA . A maximum of 10 passages were used for all cell lines.The human glioma cell lines MGM-1 and MGM-3 were a gift from Dr Hiroaki Kataoka (University of Miyazaki). MGM-1 and MGM-3 cells were grown in Dulbecco’s modified Eagle medium  with 10% heat-inactivated fetal bovine serum  plus 100 IU/ml penicillin and 100 μg/ml streptomycin  at 37 °C and 5% COFGIN-1-27 (#18461) and 22(S)-HC (#21399) were purchased from Cayman Chemicals. Other compounds used to treat cells were purchased from Sigma: XBD173 (#SML1223), AMG (#A9657), KC (#K1003), 22(R)-HC (#H9384), 20α-HC (#H6378), Trolox (#238813), hydrogen peroxide solution (#216763), iron (II) sulfate (#F8633), SNAP (#N3398), deferoxamine mesylate (#1166003), 3-Hydroxy-1,2-dimethyl-4(1H)-pyridone , and diphenyleneiodonium chloride (#D2926).For steroid synthesis experiments, cells were seeded in 24- or 96-well plates and grown to 70% to 80% confluency. Before treatment, cells were washed three times with PBS to remove steroids from the serum in complete media. Drugs used for treating cells were dissolved in dimethyl sulfoxide, PBS, or ethanol depending on solubility to make stock solutions with at least 200X concentration. Stock solutions were diluted in serum-free base media to treatment concentrations immediately before treatment. Appropriate solvent controls were made based on concentration of solvent in the highest concentration treatment condition. Cells were incubated with treatment media for 2 h, after which the media was collected and stored at 80 °C until used for steroid measurement. Cells were then lysed with 0.1 M NaOH solution, and protein quantity was measured using the Bradford assay  to normalize steroid measurements.The levels of major steroids produced by cells were measured by performing ELISA on collected media. Pregnenolone was measured in media from MGM-1, MGM-3, NHA, HMC3, and H295R-S1 cells using pregnenolone ELISA kits . Progesterone was measured in media from MA-10 cells using progesterone ELISA kits . ELISA assays were performed according to protocols provided by manufacturers.MGM-1 cells were seeded into 24-well plates at a density of 50,000 cells/well. The cells were then transfected with 1 μg of pCMV-entry mammalian expression vector , CYP11A1 transcript variant 1  expression vector , or CYP11A1 transcript variant 2  expression vector  using Lipofectamine 3000 , and Opti-MEM , according to manufacturer’s instructions. After 24 h, culture media was replaced. Transfected cells were then selected with 1 mg/ml geneticin  for 7 days. qRT-PCR, immunofluorescence, and Western blot were used to confirm CYP11A1a or CYP11A1b expression. Steroid measurements for transfected cells were performed as described previously.POR and #SR301560 for FDXR) using siTran 2.0 transfection reagents , according to manufacturer’s instructions. Culture media was replaced 18 h after transfection. Transfected cells in 12-well plates were collected 48 h after transfection for RNA analysis by qRT-PCR. After 72 h post-transfection, transfected cells in 6-well plates were washed and treated with serum-free media for 2 h, after which media was collected for steroid measurement by ELISA and the cell pellet collected for protein quantification and Western blotting. Knockdown efficiency was determined by both qRT-PCR and immunoblot analyses.MGM-1 cells were seeded into 12-well plates at a density of 60,000 cells/well or into 6-well plates at a density of 120,000 cells/well. The cells were transfected with 5 nM scrambled negative control siRNA duplexes or gene-specific siRNA duplexes . Human CNS tissue RNA for total brain (#636530), cerebellum (#636535), cerebral cortex (#636561), parietal lobe (#636571), occipital pole (#636570), temporal lobe (#636564), and spinal cord (#636554) were purchased from Takara Bio. RNA (1000 ng) were reverse transcribed into complementary DNA using PrimeScript RT Master Mix . qRT-PCR was then performed in 384-well plates using SYBR Select Master Mix  with 100 nM forward and reverse primers (Integrated DNA Technologies). Primers were designed using the NCBI Primer Blast tool, where primers pairs that spanned exon–exon junctions were preferentially selected. Plates were assayed on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). Sequences of all forward and reverse primers are listed in -ΔCT method except when comparing transfected cells to negative controls, where the 2-ΔΔCT method was used instead. CYP11A1 expression was detected by the CYP11A1 exon 3 to 4 primers listed in Total RNA was extracted from cell pellets consisting of 106 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with 2% protease inhibitor . Fifteen migrograms of total proteins were resolved on 4% to 20% precast polyacrylamide gels  and transferred to polyvinylidene difluoride membranes . After transfer, membranes were blocked for 30 min with blocking solution consisting of 5% bovine serum albumin  dissolved in PBS with Tween-20 (PBST). Membranes were then incubated with specific primary antibodies diluted in blocking solution overnight at 4 °C, washed three times with PBST, incubated with corresponding secondary antibodies for 1 h at room temperature (RT), and washed three times with PBST. Antibodies were detected using Clarity Western ECL Substrate system , which allows detection of signals for >0.3 ng protein recognized by antibodies, and visualized using a Western blot imaging system . The following primary antibodies were used: anti-CYP11A1 rabbit mAb , anti-CYP11A1 rabbit pAb , anti-CYP11A1 rabbit pAb , anti-CYP11A1 rabbit pAb , anti-GAPDH rabbit mAb , anti-FDXR rabbit pAb , anti-ADX rabbit mAb , antibeta actin mouse mAb , anti-CYP reductase rabbit pAb , and anti-Myc-tag rabbit mAb .Cell pellets consisting of 2 × 10Changes in ROS levels were measured using a Cellular ROS Assay Kit , performed according to manufacturer’s instructions. Briefly, cells were seeded in 96-well plates at 70% to 80% confluency and incubated with ROS Red Stain working solution provided in the kit for 1 h prior to treatment. Treatment solutions made at 10× concentration were then added to the wells to achieve the appropriate treatment concentrations. After a 2 h incubation, fluorescence was measured at 520 nm/605 nm using a Biotek Synergy H1 Hybrid Multi-Mode Microplate Reader. ROS measurements were normalized to that of the control condition.Cells were seeded onto glass coverslips in 12-well plates and grown to 50% confluency. For immunocytochemistry, cells were incubated with 250 nM MitoTracker Red CMXRos  in complete media for 40 min. Then, cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X for 10 min, with three PBS washes between each step. Blocking was then performed for 30 min with 5% donkey serum  + 0.5% bovine serum albumin . Cells were incubated overnight at 4 °C with anti-CYP11A1 rabbit pAb  or anti-Myc-tag rabbit mAb . Following primary antibody incubation, cells were washed three times with PBS and incubated with Alexa Fluor 488 goat anti-rabbit IgG (H + L) secondary antibody  for 1 h at RT. After three PBS washes, the coverslips were mounted onto microscope slides with Vectashield Vibrance mounting medium with 4′,6-diamidino-2-phenylindole . The slides were then imaged at 63× magnification using Zeiss LSM 880 with Airyscan Confocal Microscope.CYP11A1 and a second probe targeting MBP from the ACD Bio catalog (#411058). The ISH assay was performed, and images were taken by ACD Bio according to previously published procedures . Eighty micrograms of total proteins were resolved on 12% acrylamide gels . Protein bands were visualized by staining with Coomassie blue  for 1 h. Bands at 40 kDa, 50 kDa, and 60 kDa were excised and shipped to Creative Proteomics for shotgun proteomics. A summary of the protocol was provided by Creative Proteomics. Briefly, the protein bands were digested with trypsin and resuspended in 0.1% formic acid before LC-MS/MS analysis performed on a Nanoflow UPLC system. A full scan was performed between 300 to 1650 m/z at resolution of 60,000 at 200 m/z. Raw MS files were analyzed and searched against human protein database using Maxquant 1.6.2.6 (Max-Planck Institute of Biochemistry) to obtain a list of matching proteins in each band.H295R-S1, MGM-1, and NHA cells were pelleted by centrifuging at 200Steroids in the media were extracted twice with 1-chlorobutane  using media:solvent ratio of 1:2. The top organic layer was collected and dried using a rotary evaporator. Pregnenolone was derivatized with 1-amino-4-methylpiperazine  to increase detection sensitivity based on a published method . BrieflyTo generate a calibration curve, 400 μl of 1 μg/ml pregnenolone standard  were dried, derivatized as described previously, and reconstituted in 200 μl 50% methanol for a final concentration of 2000 ng/ml pregnenolone-AMP. An internal standard (IS) stock solution was prepared in the same way using d4-pregnenolone . Final concentrations for the calibration curve were prepared at 100, 50, 20, 10, 5, 2, 1, 0.5, and 0.25 ng/ml pregnenolone with 50 ng/ml IS in 50% methanol + 0.1% formic acid using serial dilutions.2O, while mobile phase B was 0.1% formic acid in acetonitrile. MS detection was performed in positive mode. Multiple reaction monitoring transitions used were as follows: precursor ion (m/z = 414) → product ion (m/z = 99) for pregnenolone-AMP and precursor ion (m/z = 418) → product ion (m/z = 99) for IS. MS parameters were optimized to achieve optimal sensitivity: ion spray voltage was 4500 V, source temperature was 300 °C, curtain gas/ion gas 1/ion gas 2 were 20 psi, declustering potential was 79.5 V, collision energy was 26.3, and dwell time was 50 ms. The data were acquired and analyzed using Analyst 1.7 (SCIEX) software.The LC-MS/MS measurements were performed with an Agilent Poroshell 120 EC-C18 column  using Agilent Infinity II HPLC connected to a QTRAP 6500+ mass spectrometer (AB Sciex). Mobile phase A was 0.1% formic acid in H2O:ethanol with 0.1% formic acid (aqueous) and transferred to HPLC vials. LC-MS/MS was performed by the Proteomics Service, Research Institute of McGill University Health Centre (RIMUHC) on an AB Sciex 5600 Time of Flight Mass Spectrometer connected to a Nexera XR LC-20 ADXR UHPLC system using a Zorbax (Agilent) Eclipse C18  column and autosampler. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. MS parameters were as followed: ion spray voltage was 5500 V, source temperature was 500 °C, curtain gas 30 L/min, ion source gas 1 and 2 were 50 l/min, collision energy was 10, and declustering potential was 100 V. Product ions scanned were as follows: androstenedione (m/z = 287.2), testosterone (m/z = 289.2), DHEA (m/z = 289.2), progesterone (m/z = 315.2), and pregnenolone . Analytical standards in solution were used to establish linearity and dynamic range. Data acquisition and analysis were done using MultiQuant 3.0.2 and Analyst TF 1.7 (AB Sciex).H295R-S1 and MGM-1 cells were treated for 2 h with 76 mM AMG or 100 μM KC with or without 50 μM 22(R)-HC stimulation in serum-free media. One milliliter of supernatant was collected for each sample and lyophilized using FreeZone 1 l -50C Freeze Dryers . Lyophilized samples were extracted with 200 μl of methyl tert-butyl ether three times and the combined organic layers were dried using a Speed Vac. Samples were reconstituted in 200 μl of 10 + 90 (v/v) mixture of dHStatistical analyses of steroid and ROS measurements were performed using GraphPad Prism 9.2.0 (GraphPad Software). Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test. For multiple comparison tests, each value was compared to the no-drug solvent control group unless otherwise indicated.All data are contained within the article and/or available upon request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


An ionic radiimismatch between V5+ and P5+ substituents ismanifested in broad XRD lines. The sharpening of the XRD lines isobserved with increasing bismuth ions concentration in the Eu3+ codoped YV0.5P0.5O4 matrix.The difference in the number of the Stark components for the 5D0 → 7FJ transitionsindicates changes in the lattice and a number of possible Eu3+ sites. A thorough, systematic spectroscopic analysis of YV0.5P0.5O4: x mol % Eu3+, y mol % Bi3+ was conducted at roomtemperature and 5 K. Metal-to-metal energy transfers occurring betweenEu3+, V5+, and Bi3+ optically activeions have been investigated. Additionally, efficiency of the Bi3+-Eu3+ energy transfer (ET) was calculated.In this study, solid-state solutions of yttrium orthovanadate-phosphatewith varying concentrations of codopants (Eu 3+ and Bi3+ ionswere synthesized using a wet chemical coprecipitation method. X-raydiffractograms revealed the YV0.5P0.5O4 crystal reordering structure in response to increasing Bi3+ concentration. Furthermore, changes in low and room temperaturespectroscopic properties were observed. Increases in Bi3+ and Eu3+ ion concentrations enhanced europium ion emissionthrough MMCT between vanadium, bismuth, and europium ions.Yttrium orthovanadate-phosphate nanoparticlescodoped withvarying concentrations of Eu Thisis due to their potential applications as laser host materials, polarizers,solar cells, light emitting diodes, host materials for optically activeions, etc.4 and YPO4 crystallize in the zircontetragonalsystem, within the space group I41/amd.9 Hence, a solid-statesolution of yttrium orthovanadate-phosphate can be formed.10 Considering YP0.5V0.5O4, its unit cell is composed of 50 mol % vanadium tetrahedraland 50 mol % phosphate tetrahedral groups, statistically substituted.Furthermore, in this work, yttrium ions in the lattice are statisticallysubstituted with europium and bismuth ions.YVO4–YVO4–BiVO4–BiPO4 pseudoquaternarydiagram (more precisely the shaded area of the YP0.5V0.5O4–BiVO4–BiPO4 pseudo ternary subdiagram) is investigated P0.5V0.5O4 for two purposes: to collect informationon the local crystal structure and to investigate energy transferprocesses involving Bi3+. This paper constitutes an extensionof previous reports16 with more systematic, thoroughspectroscopic analysis. Previous works14 focus on parameters affecting the Eu3+ emission intensity in micron-sized14 andnanosized13 systems. It was previously established thatcodoping YVO4:Eu3+ with P5+, Bi3+, and Gd3+ greatly enhances the europium ions’emission intensity. The compositions which maximize Eu3+ emission intensity are typically Y0.9Bi0.05Eu0.05P0.5V0.5O413 or Y0.45Gd0.45Bi0.05Eu0.05P0.5V0.5O4.2 The reasons for this remain obscure.In the present work,the fraction of the YPOstigated 1. The bod in ref . The com23 Furthermore, it has been observed that Bi3+ is an efficientluminescence sensitizer for trivalent lanthanide ions.28 In this study bismuth and europium ions are chosen as codopants,bismuth ions improve the photoluminescence intensity of Eu3+. This phenomenon occurs as a result of UV excitation. This is dueto the CT transitions from the Bi3+ 6s energy level tothe 5d levels of the vanadate and subsequent energy transfer (ET)to Eu3+ ions 4f orbitals.33Trivalent bismuth ion is known as an attractiveactivator in zirconvanadates.0.5P0.5O4. This matrix has a disordered structure as a resultof phosphate and vanadate units being randomly dispersed throughoutthe lattice. This occurs because the vanadate units are ∼8%larger than the phosphates. The extent to which this disorder contributesto the efficiency of Bi3+-Eu3+ ET will be investigatedin this work. To this end, two series of materials were synthesized.The first one was doped with varying amounts of bismuth ions, whilethe second was doped with varying amounts of europium ions. The chemicalcompositions involved doping and codoping YV0.5P0.5O4 with xBi3+, yEu3+ where x = 0, 1, 3, 5, 10, 15 mol% and y = 0.5, 1, 2, 5 mol %. The solid state solutionswere obtained by the wet chemistry synthesis-coprecipitation methodwith additional heat-treatment at 800 °C for 3 h.This work emphasizes characterization of the luminescent propertiesof these two dopants incorporated into YV22.12O3 ,Bi2O3 , Eu2O3 , (NH4)2HPO4  and NH4VO3  were used in this synthesis process.Yttrium orthovanadate-phosphatepowders, codoped with europium and bismuth ions, were obtained bythe coprecipitation method. The concentrations of vanadium and phosphoruswere fixed to 50 mol % each. Two series of materials were obtained:one in a function of bismuth concentration with fixed concentrationof europium and vice versa. First, the concentration of europium ionswas set to 1 mol %, while bismuth ion concentration changed from 0,1, 3, 5, 10 up to 15 mol %. In the second series, the concentrationof bismuth ions was set to 10 mol %, with concentrations of europiumion varying from 0.5, 1, 2, up to 5 mol %. Stoichiometric amountsof analytical grade Y3. Thereafter, the formed lanthanideand bismuth nitrates were recrystallized, and the HNO3 excesswas removed. Using deionized water as a solvent, separate aqueoussolutions of diammonium phosphate and ammonium metavanadate were made.The vanadium and phosphorus ion sources (NH4VO3 and (NH4)2HPO4) were mixed, followedby the nitrates (Y(NO3)3, Bi(NO3)3, Eu(NO3)3). The liquid mixture wasstirred for 1.5 h at approximately 70 °C. Aqueous ammonia wasused to maintain a pH of 9 during the reaction. The as-prepared precipitateswere then washed and centrifuged at least three times, until neutralpH was reached. They were then dried for 24 h at 70 °C. The powderswere finally crystallized by heat-treatment at 800 °C for 3 hin air.The lanthanide andbismuth oxides were converted into nitrate salts through digestionwith an excess of 65% HNO2.24 (no. 78074)and YPO4 (no. 79754) found in the Inorganic Crystal StructureDatabase (ICSD). Microstructural analyses were performed by electron microscopy. SEM was carried out using anFEI Nova NanoSEM 230. High resolution transmission electron microscopy(HR-TEM) was performed using a Philips CM-20 Super Twin microscope.ICP-OES measurements were conducted on Thermo Scientific ICAP 7000SERIES.Thecrystal structure of synthesized materials was characterized by theX-ray Diffraction (XRD) technique using an X’Pert PRO X-raydiffractometer  . MeasuredXRD patterns were compared to standards of YVO2.35D0 → 7F2 transition at 619 nm was monitored at room temperaturefor the excitation spectra measurements. Emission spectra were alsorecorded in response to the 266 nm excitation of a laser diode (CW)at room temperature and detected using the Hamamatsu PMA-12 photonicmultichannel analyzer. The emission decay profiles were measured at300 K using either a Ti:sapphire tunable laser or a Nd:YAG laser,a Hamamatsu R928 photomultiplier, a Jobin-Yvon THR 1000 spectrophotometer,and a digital LeCroy WaveSurfer oscilloscope. Excitation and emissionspectra were collected at low temperature (5 K) using a temperature-controlled,continuous-flow liquid helium cryostat: Oxford Model CF 1204. Lowtemperature excitation spectra were measured with a Dongwoo OptronDM151i monochromator and a 150W ozone free lamp. The low temperatureemission spectra were measured using a Dongwoo Optron DM750 monochromator,an Electro-Optical System INC PbS photodiode, or a Hamamatsu R928photomultiplier.The NicoletiS50 FT-IR from Thermo Scientific was used to collect infrared spectraat 300 K of samples processed in KBr pellets. The room-temperatureemission spectra utilized excitation at 397, 340, and 300 nm. Thesespectra were collected using a FLS1000 photoluminescence spectrometerfrom Edinburgh Instruments. The same apparatus was used to collectthe excitation spectra. The 33.1The chemicalcomposition of all samples, analyzed by ICP-OES, is given in 0.5P0.5O4 doped derivatives  andP5+ , when statistically distributedin YVxPx1–O4 solid solution. The lattice strains are influencedby changes in grain size. This is a result of point defects, vacancies,34 and varying composition35 as well as dislocations near the grain-boundaries36 caused by the incompatibility of phosphorus and vanadiumatoms. The observed changes in the fwhm of the XRD peaks may indicatethe presence of lattice strains.37XRD results confirmed the crystal phase purity ofYVivatives 2. The XR3+ does not impactthe lattice and,by extension, the width of the XRD peaks. In contrast, it is foundthat codoping with Bi3+ contributes to narrowing of theXRD peaks  carried by these cations is given in 5+ creates two additional spheres at 3.14 and 3.89Å (marked as red in 3+ results in a third sphere (marked as blue in 3+ ion in this position is P(x) = 1–(1–x)4 wherein x is the molar percentage of Bi3+, e.g., P(0.1)= 35%. The bismuth ions (1.17 Å at C.N. 8) substitute yttriumions (1.02 Å at C.N. 8) in a statistical manner. Since Bi3+ is about 15% larger than Y3+, its incorporationin the crystal lattice counterbalances (at least partly) the ionicradius mismatch between V5+ and P5+. This inverserelationship between V5+–P5+ and Bi3+–Y3+ ionic radii mismatches reduces thelattice strains in YV0.5P0.5O4 andthereby sharpens the XRD lines as the Bi3+ concentrationis raised . Differencesin particles sizes contribute to the narrowing of the XRD peaks inaddition to the reordering of the crystal structure. This work shedsa light into a complex structure of Eu3+, Bi3+: YV0.5P0.5O4. However, to individuateor quantify the role of each effect, further crystallographic researchneeds to be conducted. Additionally, d-spacing valueswere calculated by the means of FFT processing in ImageJ software.d-Spacing was calculated to be d101 =0.45 nm for YV0.5P0.5O4 samples codopedwith 1 mol % Eu3+ and 1 mol % Bi3+ and 1 mol% Eu3+ and 5 mol % Bi3+, as well as 1 mol %Eu3+ and 15 mol % Bi3+. Also d200 values were calculated. For samples codoped with 1mol % Eu3+ and 1 mol % Bi3+, d200 was 0.35 nm. However, 5 mol % and 15 mol % Bi3+-doped samples had a d200 valueof 0.33 nm. All d-spacing values coincide with d101 and d200 valuesfrom standard patterns of YVO4  and YPO4 .The TEM images in 3.20.5P0.5O4 codoped withEu3+ and Bi3+ are shown in –1. Thepeaks at 524 cm–1 and at 639 cm–1 represent an antisymmetric bending vibration of ν4(PO4)3–. The antisymmetric stretchingvibration of ν3(PO4)3– can be found at 1010 cm–1 and at 1110 cm–1.39 The peak at 836 cm–1 isascribed to the vibration mode of the (VO3)− group. The weak peak detected at 502 cm–1 is relatedto the Y–O vibration.40 This modeis not observed for the samples with more than 10 mol % of codopantions concentration. The Bi–O modes are on the verge of ourmeasurement range.42The FT-IRspectra of YV3+ upon direct 4f-4f excitation at 397 nm  are comparatively much less intense.The excitation spectra for 5D0 → 7F1 transition of Eu3+. In addition tocharacteristic emission lines of Eu3+, broad emission signalsare observed. Upon 340 nm excitation, the broad signal representsthe emission of the Bi–V metal-to-metal CT,15 whereas upon 300 nm excitation, the broad signal is moresurely due to perturbed vanadate groups. Upon 266 nm excitation, thesebandlike emissions possibly overlap. The presence of these emissionbands indicates an incomplete sensitization of Eu3+ luminescence.Two possible sensitization paths are identified. They involve the(VO4)3- units or the Bi–V self-trappedexcitons as energy donors and the Eu3+ ions as energy acceptors.Decay profiles were collected to quantify the efficiency of theseenergy transfers.7F0 → 5L6 Eu3+ transition)or 355 nm excitation (Bi–V MMCT). Corresponding average valuesof the luminescence lifetimes tav, werecalculated as tav = ∫I(t)t dt/(∫I(t) dt). In this case, I(t) represents the emission intensityat time t. These values are provided in Decay profiles are presented in 3+-to-Eu3+ in the codoped compounds. It isobtainedfrom the equationdoped is the timeconstant in the presence of the Eu3+ acceptor, and τundoped is the time constant without Eu3+ .The parameter η in 7F0 → 5L6 excitation of Eu3+. The left-hand side of thevertical dashed line corresponds to 1 mol % Eu3+ dopedsamples with increasing Bi3+ concentrations . These samples  also exhibit loweremission intensity relative to samples with higher Bi3+ concentrations. This suggests a lower radiative probability fromthe 5D0 state in these cases. Raising the Bi3+ amount further contributes to enhance the Eu3+ emission intensity, and it correlates with a shortening of the lifetime.This effect has already been observed in YPO4:Sm3+, Bi3+ and is ascribed to an increased refractive indexof the host lattice.43 In this case, theradiative probability is increased, contributing to a lower emissionlifetime. A synergetic effect with the reordering of the crystal structureis not excluded. This requires further investigation. The right-handside of the figure shows the effect of Eu3+ content fora fixed amount of Bi3+. In this case, the emission lifetimedoes not vary significantly because the medium’s refractiveindex is unchanged. Here, the increase in emission intensity is ascribedto the larger Eu3+ content that remains beyond the quenchingconcentration.457F0 → 5L6 transition  and in compound I . Nevertheless, the emission intensityof compound J amounts to 6.5 times that of compound D, which demonstratesa synergy between Bi3+ and Eu3+ contents inYV0.5P0.5O4. Furthermore, with respectto both 355 and 397 nm excitations, compound J exhibits the highestemission intensity related to direct Eu3+ excitation.Part (b) of ansition 9. In thi3+ and can now be used as a structural probe. In 2008, Pan et. al46 conducted investigations regarding the spectroscopicproperties of Eu3+ in YO4 solid solution by laser-selective excitation. This work identifiedthree symmetry sites in the yttrium orthovanadate-phosphate mixedcompounds due to disorder generated by the distribution of (PO4) and (VO4) tetrahedra. The Judd–Ofelt intensityparameters further confirmed that significant changes in ligand polarizabilitycontribute to differences in local environments experienced by Eu3+.46At sufficiently low temperatures, vibronic interactions can befrozen out. Thus, the luminescence spectra can give more detailedinformation regarding the electronic transitions of Eu3+5D0 → 7F2 transition. Low temperature excitationspectra depict noticeable differences when compared to the room temperaturespectra. These major differences are as follows: (1)7F0 → 5L6 transitions of Eu3+ to the CT excitation bandsin compounds containing low amounts of Bi3+ (<5 mol%). This indicates a less efficient sensitization at 5 K. Furthermore,it demonstrates that in these conditions, the Bi-to-Eu energy transferis efficiently phonon assisted. However, even at 5 K the Bi-to-Euenergy transfer regains efficacy when the Bi3+ concentrationis 10 mol %. This coincides with the increased presence of a broadexcitation band which peaks at ≈360 nm and extends up to ≈400nm, currently attributed to Bi–V MMCT.differences in relative intensityof the intrinsic (2)3+ content in the compound. Thereis a notable red shift in the excitation spectra as Bi3+ concentration increases. This shift of the Bi–V excitationedge with increasing the Bi content has been noted in previous studies,e.g., in ref32.the presence of structures on theBi–V MMCT excitation with maxima identified at ≈350,330, and 307 nm. The relative intensity of these excitation maximastrongly depends on the Bi(3)2– → Eu3+ and O2– → V5+ charge transfers. This suggeststhese transitions are not involved in the sensitization process ofEu3+ at 5 K. Therefore, only the Bi–V MMCT operatesas a sensitizing channel for Eu3+ at 5 K.the absence of excitationfeaturespertaining to O3+, 1 mol % Eu3+: YV0.5P0.5O4 were collected at 5 K. These samples wereexcited in the CT bands and at 395 nm  correspondingto the 5D0 → 7F2 transition were observed. The spectrum in the 5D0 → 7F4 region looks also simpler.This simplification of the spectral signatures is consistent withthe progressive crystal structure reordering of YP0.5V0.5O4 concomitant with an increase in Bi3+ concentration. This is previously observed in XRD results , and its energy barycenter is upshifted. This is due to the presenceof the Bi3+ ion in the second cationic neighborhood ofEu3+, as we have depicted in 3+ by reinforcing the crystal field and softening the nephelauxeticeffect.Data related to the  results 2. Compar4x mol % Bi3+, y mol % Eu3+: YV0.5P0.5O4 was formedusing a coprecipitation synthesismethod. It is observed that codoping with Bi3+ is followedby narrowing of the XRD peaks, but codoping with Eu3+ doesnot significantly affect the widths of the peaks. The insufficientenergy transfer to Eu3+, resulting from the Bi–VMMCT and the 3T1,2 → 1A1 (VO4)3– broad transition bands,is observed. Yet, Eu3+ ion emission is enhanced by increasingBi3+ ion concentration. Further reaffirmed is the presenceof more than one Eu3+ site due to (PO4)3– and (VO4)3– substitution.Additionally, an ordering of the crystal structure of YP0.5V0.5O4 with increasing the Bi3+ contentcan be observed. It is supposed that at low temperature the sensitizingpathway of Eu3+ is less efficient in comparison to theroom temperature, and mainly the Bi–V MMCT contribution isnoticeable. Based on luminescent decay times, the Bi–Eu ETefficiency was found to be the highest for bismuth-rich compounds.The pure crystal phase of


Iron and vitamin D (VD) is essential to health. Previous studies have shown that iron homeostasis has a potential effect on VD metabolism, but the mechanism is not fully understood.To explore the relationship between VD metabolism and iron metabolism, as well as the regulatory mechanism of iron on VD metabolism.40 male rats were fed adaptively for 7 days and randomly divided into control  group and model  by simple randomization, the latter was used to establish iron deficiency anemia (IDA) model. After 6 weeks of feeding, the M group was randomly divided into: iron deficiency group (DFe), low iron group (LFe), medium iron group (MFe) and high iron group (HFe) by block randomization. Different doses of iron dextran (based on iron content (100 g·bw·d)): 0, 1.1, 3.3 and 9.9 mg) were given respectively. After 4 weeks, the rats were anesthetized with 8% chloral hydrate, Blood , liver and kidney tissues were collected. The serum and tissues were separately packed and frozen at -80℃ for testing.3 and 1,25-(OH)2D3 in DFe group were significantly lower than those in C group (P < 0.05). The correlation analysis showed that the levels of 25-(OH)D3 and 1,25-(OH)2D3 were negatively correlated with TIBC, TF and Tfr no correlation with SI. Western blotting, immunofluorescence, and q-PCR results showed that compared with C group, the protein and gene expressions of CYP2R1, CYP27A1, and CYP24A1 in DFe group were down-regulated, and the expression of CYP27B1 protein and gene was up-regulated in DFe group.The results showed that the levels of hemoglobin (Hb), red blood cell (RBC), serum iron (SI), liver iron, and kidney iron in DFe group were lower than those in the other four groups, while the levels of total iron-binding capacity (TIBC), transferrin (TF) and transferrin receptor (Tfr) in DFe group were higher than those in other groups; The serum levels of 25-(OH)D3 by regulating the expression of VD3 hydroxylase, suggesting that appropriate iron supplementation might promote the activation of VD3.Iron may be involved in the metabolism of VDThe online version contains supplementary material available at 10.1186/s12986-022-00681-5. Vitamin D (VD) is an essential micronutrient for the body, which is vital to health. In addition to regulating calcium and phosphorus metabolism, VD with hormone properties can induce cell differentiation and apoptosis, inhibit cell proliferation and other cell signal transduction processes, participate in the regulation process of multiple genes, and involve in the physiological and pathological mechanism of various diseases , 2. VD d1-VD5, among which VD3  and VD2 (ergosterol) have the greatest impact on human health , low iron group (LFe) [1.1 mg/ (100 g·bw·d)], medium iron group (MFe) [3.3 mg/(100 g·bw·d)], high iron group (HFe) [9.9 mg/(100 g·bw·d)] by block randomization, 2 ml for each animal for 4 weeks, C and DFe groups were perfused with 2 ml normal saline. In addition to the different iron content, the other nutritional components of the two diets were the same. Both of them added 1500 IU of VD3 ; Tissue Iron Determination Kit ; hemoglobin (Hb) Kit ; red blood cell (RBC) diluent ; 25-(OH)D3 kit ; 1,25-(OH)D3 kit ; transferrin (TF) ; transferrin receptor (Trf) ; serum iron (SI) ; iron-binding capacity (TIBC) ; CYP2R1 primary antibody ; CYP27A1 primary antibody ; CYP27B1 primary antibody ; CYP24A1 primary antibody ; Cy3-goat anti-rabbit IgG . Microplate reader ; DAPI ; Paraffin embedding machine ; Microtome ; Electron microscope ; PCR instrument .Iron Dextran ; VD3, 1,25-(OH)2D3, TF, and Tfr; Biochemical kit was used to detect Hb, RBC,TIBC, SI, liver iron and kidney iron, and the operation was carried out strictly according to the operation procedures of the kit.ELISA kit was used to detect 25-(OH)DThe expressions of CYP2R1 and CYP27A1 protein in liver, and the expressions of CYP27B1 and CYP24A1 protein in kidney were detected, each sample was taken 20 mg, and Radio Immunoprecipitation Assay (RIPA) lysate was added, The total protein was extracted, protein was prescribed by BCA Assay Kit, the total protein was sampled with 20 μg protein in 96-well plate, gel electrophoresis, transfer membrane, block, 4℃ primary antibody CYP2R1, CYP27A1, CYP27B1, CYP24A1 (1:500), β-actin (1:5000) were incubated overnight at room temperature, The secondary antibody (1:5000) was incubated for 2 h, CYP2R1, CYP27A1, CYP27B1, and CYP24A1 were detected by the luminescent solution, and the gray value was analyzed by Image J software.The liver and kidney tissue specimens fixed with 4% paraformaldehyde, and then dehydrate after fixation, wax embedding, sectioning, dewaxing, hydration, gradient ethanol elution, antigen retrieval, CYP2R1, CYP27A1, CYP27B1, and CYP24A1 (diluted 1:100) primary antibody incubation, incubated with secondary antibody (diluted at 1:200) and counter-stain with diamidino-2-pheny1 indole (DAPI). The immunofluorescence intensity in the liver tissue was observed and photographed under a fluorescence microscope. The CYP2R1, CYP27A1, CYP27B1 and CYP24A1 protein staining were positive in red. The fluorescence intensity of CYP2R1, CYP27A1, CYP27B1 and CYP24A1 protein was detected by Image Pro Plus image analysis software, and the integrated optical density (IOD) was calculated as the final average immunofluorescence intensity.Total RNA was extracted from 20 mg liver and kidney tissues of rats. The cDNA was reverse transcribed and amplified by fluorescent quantitative PCR with β-actin as an internal reference. The upstream primer sequence of β-actin was 5´-TGTGATGGTGGGAATGGGTCAG-3´, downstream primer sequence 5´-TTTGATGTCACGCACGATTTCC -3´. CYP2R1 upstream primer sequence: 5´-TGCTACTACTCGTGCTGGTGGTC-3´, downstream primer sequence 5´-AGGGCCAGGGA GCAGATGTTG-3´. CYP27A1 upstream primer sequence: 5´-TCGCACCAATGTGAATCTGGC TAG-3´, downstream primer 5´-CTTCCACTGCTCCATGCTGTCTC-3´. CYP27B1 upstream primer sequence: 5´-GCACATAAACGGCAAGGCAAGTC-3´, downstream primer sequence 5´-AGCCAAGCCTCTCACCTCCTATG-3´. CYP24A1 upstream primer sequence 5´-GGAACTGT A CGCCGCTGTCAC-3´, downstream primer 5´-GCACGCTCTGAACTTCCTGAAGG-3´. PCR conditions: pre-denaturation 95℃, 30 s; denaturation: 95℃, 5 s, 60℃, 30 s (40 cycles); annealing: 95℃, 5 s, 60℃, 30 s, 95℃, 1 min; extension: 50℃, 30 s. The relative quantitative values were calculated according to the Ct values of the original q-PCR data.3 metabolism index, iron metabolism index, tissue iron and other indicators were compared by one-way ANOVA, LSD-t test or Dunnett's T3 test were used for pairwise comparison, P < 0.05 means the difference was statistically significant.Spss 25.0 software was used for statistical analysis of the data. The HB value and body weight of group C and group M were tested by t test; VDDuring the modeling period, the animals appeared some phenomena, such as fluffy hair, falling off, dull eyes, loose stool, fear of cold, white teeth, and slow growth and development and so on. After 6w of modeling, Hb in group M was detected to be lower than that in group C (63.61 ± 5.39 g/L) vs (125.58 ± 2.90 g/L) to judge the modeling situation , and those in LFe group were significantly lower than those in HFe group (P < 0.05) (Table The contents of liver iron and kidney iron in DFe group were much lower than those in other four groups (5) Table .Table 1TP < 0.05)  Table .Table 2H3 and 1,25-(OH)2D3 in serum of DFe group were much lower than those in C, MFe and HFe groups, and the differences were statistically significant (P < 0.05). The content of 25-(OH)D3 and 1,25-(OH)2D3 gradually increased in the LFe, MFe and HFe groups, with the increase of iron intervention concentration, and the difference between the LFe group and the HFe group was statistically significant (P < 0.05); The content of 25-(OH)D3 in MFe group was significantly different from that in HFe group (P < 0.05) (Table The levels of 25-(OH)DP < 0.05); The level of SI was the lowest in the DFe group, which was significantly lower than that in the C, LFe, MFe and HFe groups (P < 0.05); compared with the HFe group, the level of Tfr was significantly increased in the DFe and LFe groups (P < 0.05)  Table .Table 4T3 level was significantly negatively correlated with TIBC, TF and Tfr indicators; 1,25-(OH)2D3 level was significantly negatively correlated with TIBC, TF and Tfr indicators; However, 25-(OH)D3 and 1,25-(OH)2D3 levels had no correlation with SI DP < 0.05). With the increase of iron intervention concentration, the CYP2R1 protein expression level gradually increased Fig. B; CYP27B.05)Fig. C; compar.05)Fig. D.Fig. 3TP < 0.05); CYP2R1 protein expression in DFe group was significantly lower than that of MFe and HFe groups (P < 0.05). Compared with C group, CYP27A1 protein expression did not change significantly in DFe group, and the difference was not statistically significant; compared with HFe group, CYP27A1 protein expression was significantly lower (P < 0.05); CYP27B1 protein expression was highest in DFe Group, which was statistically significant compared with C, MFe and HFe groups (P < 0.05); compared with C group, CYP24A1 protein expression was significantly down-regulated (P < 0.05), CYP24A1 protein expression in MFe and HFe groups were higher than the DFe group (P < 0.05)  Table , Fig. 4.P < 0.05); the content of CYP27A1mRNA was the lowest in DFe group, which in DFe and LFe groups were significantly lower than that in C group (P < 0.05); the content of CYP27B1mRNA was the highest in DFe group, and the content of CYP24A1mRNA was the lowest in DFe group, there was no statistically significant difference between the groups D3 and 1,25-(OH)2D3.Iron is an essential trace element that is required for the growth and survival of the body, and plays an important role in the whole life process of the body. VD is mainly synthesized by ultraviolet rays, and environmental factors affect the seasonal fluctuation of VD concentration. The most classic role of VD is to regulate calcium and phosphorus metabolism and promote bone development. As research progresses, iron homeostasis also has a potential impact on bone formation, bone resorption and bone development . The sertabolism . Iron detabolism . Our stu3, the serum 25-(OH)D3 concentration of the children was still very low, and the serum 25-(OH)D3 concentration returned to normal after iron supplementation. In our experiment, iron deficiency led to the iron content of liver and kidney of SD rats decreased. The levels of serum 25-(OH)D3 and 1,25-(OH)2D3 in DFe group were significantly lower than those in C group. The levels of serum 25-(OH)D3 and 1,25-(OH)2D3 gradually increased with the increase of iron intervention concentration. It is suggested that iron may promote the metabolic level of VD3 and convert it into an active form of 1,25-(OH)2D3 in the body. In this process, iron is used by the body and the level of VD3 activity increases. Other studies have shown that iron loading also affects the levels of vitamin D3 metabolism. Napoli et al. [3 levels. Otto-Duessel et al. [3 and 1,25-(OH)2D3 levels in the HFe group were not found to be inhibited. It possibly because of the IDA model built in the early stage that the iron consumption in the rat body took too long. Iron only satisfied the needs of pre-consumption and growth and development requirements, and could not accumulate too much in the body, and thus did not inhibit the serum 25-(OH)D3 and 1,25-(OH)2D3 levels.Heldenberg’s  study oni et al.  found thl et al.  observedl et al. . In our 2D3 caused by iron deficiency is low to a certain value, it will cause the result of feedback regulation. This phenomenon suggested that under the condition of sufficient iron, the hydroxylation of VD is the use of this metal enzyme, which may effectively carry out hydroxylation reaction; when iron deficiency, hydroxylase may not rely on iron.Our study found that iron deficiency reduced the iron concentration in the liver and kidney, the SI concentration was increased accordingly, the SI concentration in HFe group is lower than that in LFe group and MFe group, which is consistent with the study obtained by DiJiong et al. . In this3 and 1,25-(OH)2D3 levels were significantly correlated with TIBC, TF and TFR, but such correlation was not found with SI, indicating that there was a some correlation between vitamin D3 metabolism and iron. We think that reasonable iron supplementation to increase the level of VD activity is a simple, safe, cheap and effective method. At the same time, accurate identification of VD hydroxylation cofactors improves the bioavailability of VD, and the participation of iron in the formation of CYP450 enzymes plays a key role in VD metabolism. In our study, iron-deficient feed was used to establish an IDA model, the method reduced the levels of SI and Hb in rats at a stable rate, with no trauma and reliable effects.In this study, serum 25-(OH)DOur study takes the lead in investigate the systematic mechanism of iron on VD metabolism. However, our research also had certain limitations, we only based on animal experiments, the digestibility and absorptivity of iron and VD metabolism in group C and group M were not studied. It is necessary to further explore the effect of iron on VD metabolism in the population, so that proper iron supplementation can promote the expression of VD active products in the VD deficiency patient population. To understand the potential benefits of VD deficiency, provide new ideas for VD deficiency diseases.3 and 1,25-(OH)2D3 by regulating hydroxylase at the translation and transcription levels; It is illustrated that the decrease of iron reserve may be a necessary condition for the control of these hydroxylase enzymes, even in the case of mild iron deficiency, the enzyme function may change.Iron deficiency reduces iron reserves in liver and kidney, which may affect hydroxylase activity; Iron may reduce the levels of 25-(OH) DAdditional file 1: Weight changes before and after intervention.


S-transferase alpha 4 (GSTA4) is a phase II detoxifying enzyme that is overexpressed in colorectal cancer (CRC) and regulated by the oncogenic transcription factor AP-1. However, the role of GSTA4 in these CRC cells remains unclear. In this study, we investigated the roles of GSTA4 in the CRC cells by inactivating GSTA4 in HCT116 human CRC cells (Defined as HCT116ΔGSTA4) using the CRISPR/Cas9 gene editing. Cell proliferation, clonogenicity, and susceptibility to chemotherapeutic drugs were analyzed in vitro and in a xenograft model. The results showed that loss of GSTA4 significantly decreased cell proliferation and clonogenicity, whereas it increased intracellular reactive oxygen species and cell susceptibility to 5-fluorouracil (5-FU) and oxaliplatin. Additionally, exposure of HCT116ΔGSTA4 cells to 5-FU increased the expression of γH2AX, a hallmark of double-stranded DNA breaks. In contrast, no remarkably increased γH2AX was noted in oxaliplatin-treated HCT116ΔGSTA4 cells compared with HCT116 cells. Moreover, loss of GSTA4 blocked the AKT and p38 MAPK pathways, leading to proliferative suppression. Finally, the xenograft model showed decreased tumor size for HCT116ΔGSTA4 cells compared with HCT116 cells, confirming in vitro findings. These findings suggest that GSTA4 is capable of promoting proliferation, tumorigenesis, and chemoresistance and is a potential target for CRC therapy.Glutathione  S-transferases (GSTs) are phase II detoxifying enzymes that are responsible for detoxifying xenobiotics and endogenous metabolites of oxidative stress (via blocking the mitogen-activated protein kinase (MAPK) signaling pathway . HCT116 cells were cultured in McCoy’s 5A medium  supplemented with 10% fetal bovine serum , 100 μg/ml streptomycin, and 100 units/ml penicillin G in 5% COhttps://crispr.dbcls.jp)  as previously reported  . Guide Rbcls.jp) . Selectereported , 26. Thereported Figure 13 cells/well) and incubated overnight at 37°C. Following a 48-hour treatment with 5-FU or oxaliplatin, 10 μl of CCK-8 solution was added to each well and incubated at 37°C for 4 h. The absorbance was measured at 450 nm using a Varioskan LUX microplate reader .Cell viability was determined using the CCK-8 assay kit . Cells were seeded in 96-well plates  at 37°C for 2 h. Cells were then harvested by trypsinization, fixed with 4% paraformaldehyde at room temperature (RT) for 15 min, and permeabilized with 0.3% Triton X-100 in PBS at RT for 10 min. Cells were washed, stained with 500 μl of Click Additive Solution at RT for 30 min, and sorted on a DxFLEX flow cytometer . EdU-positive cells were analyzed using CytExpert software.Cell proliferation was determined using the BeyoClick™ EdU-555 kit (Beyotime Biotechnology) according to the instructions of the manufacturer. Briefly, cells were seeded in 6-well plates at a density of 2.5 × 102 at 37°C for 10 days. The clones were fixed with methanol and stained with 0.1% crystal violet for 30 min. The number of clones was counted.Cells were seeded into 6‐well plates at a density of 1,000 cells/well and cultured in 5% CO5) were resuspended in 100 µl binding buffer and incubated with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) at RT for 10 min. Following staining, 400 µl binding buffer was added and cells were sorted on a DxFLEX flow cytometer (Beckman Coulter). The percentage of total apoptotic cells was calculated.Apoptosis was analyzed using an Annexin-V/PI Staining kit  according to the instructions of the manufacturer. In brief, cells were harvested by trypsinization, washed with PBS, and counted. Cells (1 × 104 cells/well. The fluorescence intensity was measured at 525 nm using an excitation wavelength of 488 nm at the Varioskan LUX microplate reader (Thermo Fisher Scientific). The intensity of fluorescence was normalized to the fluorescence intensity of the PBS control.ROS was detected using a ROS Assay Kit (Beyotime Biotechnology). Briefly, cells were treated with PBS, 5-FU, and oxaliplatin, respectively, for 48 h. Following treatment, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was added to a final concentration of 10 μM and incubated at 37°C for 30 min. Cells were harvested, washed with PBS, and re-seeded into a 96-well plate at a density of 2 × 10Western blotting was performed as described previously . Cells wnuFoxn1/Gpt nude mice were used for tumor xenograft. A total of 30 mice were randomly and equally divided into six groups, with 5 mice per group. HCT116 (3 groups) or GSTA4-deficient HCT116 (HCT116ΔGSTA4) cells (3 groups) were subcutaneously injected into the flanks of the mice (5 × 106 cells/side) for xenograft tumor growth. One week after xenografting, mice were intraperitoneally injected with 5-FU (30 mg/kg), oxaliplatin (3 mg/kg), and saline as control, once a week for 4 weeks. Mouse body weight was recorded and tumor size was measured weekly. The tumor size was calculated as described previously . P <0.05 was considered statistically significant.Data are expressed as means ± SEM from at least three independent experiments. Student’s ΔGSTA4 and was used for the subsequent studies. Because GSTA4 is a detoxifying enzyme for 4-HNE, inactivation of GSTA4 in HCT116ΔGSTA4 was further confirmed by a decreased detoxifying capability for 4-HNE. The viability of HCT116ΔGSTA4 cells significantly decreased compared to the parental HCT116 cells when exposed to various concentrations of 4-HNE for 48 h . Finally, apoptosis was analyzed to determine whether the inactivation of GSTA4 induced apoptosis. FACS analysis showed that, when cells left untreated, there were no significant differences in the proportions of the early, late, and total apoptotic cells for HCT116ΔGSTA4 cells compared to those of HCT116 cells  and oxaliplatin (5 μM) compared with untreated controls (ΔGSTA4) did not increase apoptosis compared with the parental HCT116 cells when treated with 5-FU (5 μM) and oxaliplatin (5 μM), suggesting that loss of GSTA4 augmented chemotherapeutic reduction of cell viability without affecting apoptosis.GSTs, as phase II detoxifying enzymes, are associated with anti-cancer drug resistance . To inveΔGSTA4 cells after treatment with 5-FU and oxaliplatin. Treatment with 5-FU (5 µM) for 48 h resulted in a 56.28 ± 4.93% decreased proportion of EdU-positive cells  for HCT116ΔGSTA4 cells compared to a 19.22 ± 6.82% decrease  for HCT116 cells (P <0.01) for HCT116ΔGSTA4 cells compared to a 11.77 ± 9.79% decrease  for HCT116 cells , suggesting that loss of GSTA4 has no effect on DNA damage in unstressed cells. Following a 48-hour treatment with 5-FU, the proportion of γH2AX-positive cells increased to 12.73 ± 1.06% and 4.23 ± 0.28% for HCT116ΔGSTA4 and HCT116 cells, respectively (P <0.01). In contrast, although the proportion of γH2AX-positive cells increased for both HCT116ΔGSTA4 (1.67 ± 2.42%) and HCT116 cells (3.69 ± 6.00%) after treatment with oxaliplatin for 48 h compared with untreated controls, no significant difference in the proportion of γH2AX-positive cells was seen between HCT116ΔGSTA4 and HCT116 cells (P = 0.618), indicating that the inactivation of GSTA4 has limited effect on low-dose oxaliplatin-induced crosslinking of DNA.Many chemotherapeutic agents act as DNA damage inducers, leading to cancer cell death. Inactivation of GSTs often induces ROS production that causes DNA damage . HoweverΔGSTA4 cells compared with HCT116 cells, indicating inhibition of AKT and p38 signaling pathways. Additionally, treatment of HCT116 cells with 5-FU and oxaliplatin slightly decreased p-AKT and p-p38 pathways in HCT116 cells while being prone to increasing p-AKT and p-p38 in HCT116ΔGSTA4 cells. These results suggest that inactivation of GSTA4 reduces proliferation by blocking PI3K/AKT and p38 MAPK signaling pathways and that activation of AKT and p38 pathways in HCT116 CRC cells occurs in a GSTA4-dependent manner.To further investigate the mechanisms by which inactivation of GSTA4 inhibits proliferation, we examined proliferative signaling pathways including phosphoinositide 3 kinase (PI3K) and MAPK. As shown in 6 cells per side of the flank and treated them with saline, 5-FU, and oxaliplatin, respectively. For saline-treated mice, the average tumor size significantly decreased for HCT116ΔGSTA4-derived xenografts compared with HCT116-derived xenografts (P <0.001), indicating reduced tumor growth for GSTA4-deficient HCT116 cells . Of note, both 5-FU and oxaliplatin reduced tumor size in HCT116-derived xenografts compared with saline control. However, no significant difference was found between 5-FU and oxaliplatin (P = 0.75). In contrast, for HCT116ΔGSTA4-derived xenografts, 5-FU, but not oxaliplatin, remarkably reduced tumor size compared to saline control . This is consistent with the findings that 5-FU, but not oxaliplatin, at the tested doses, induced more γH2AX foci in HCT116ΔGSTA4 cells. These results indicate that GSTA4 promotes in vivo tumorigenesis and contributes to chemoresistance.Finally, we investigated the effect of GSTA4-deficiency on tumorigenesis using a xenograft tumor model. We injected nude mice with 5 × 10in vitro and in a xenograft tumor model. Inactivation of GSTA4 increases intracellular ROS production and blocks proliferative signaling pathways, including AKT and p38 MAPK. These results show that GSTA4 activation by AP-1 in CRC promotes tumor growth via activation of proliferative signaling pathways.GSTA4 is overexpressed in the colon biopsies of human colorectal cancer and the bacteria-induced murine CRC model through activating oncogenic transcription factor activator protein 1 (AP-1) . The impvia upregulation of transforming growth factor, cyclin-dependent kinase 2, and protein kinase C beta II and downregulation of p53  that we have tested in these experiments. Notably, previous studies have demonstrated that exposure of SW480 CRC cells to oxaliplatin at a dose of 10 µM, which is 20-fold of the dose that we used to treat HCT116 cells in this study, induced a remarkably low proportion of γH2AX-positive cells , the National Natural Science Foundation of China (81972783 to XW), the Jiangsu Distinguished Medical Experts Program (to XW), the Jiangsu Health Innovation Team Program (to XM), the Jiangsu 333 Talent Project (to HL), and the Nantong Commission of Health (MA2021029 to LX).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ketone bodies, including 3HBA, are endogenous products of fatty acid oxidation, and Hmgcs2 is the first rate-limiting enzyme of ketogenesis. From database analysis and in vivo and in vitro experiments, we found that adipose tissue and adipocytes express Hmgcs2, and that adipocytes produce and secrete 3HBA. Treatment with 3HBA enhanced the gene expression levels of the antioxidative stress factors, PPARγ, and lipogenic factors in adipose tissue in vivo and in adipocytes in vitro, accompanied by reduced ROS levels. Knockdown of endogenous Hmgcs2 in adipocytes markedly decreased 3HBA levels in adipocytes and decreased the gene expression levels of the antioxidative stress factors, PPARγ, and lipogenic factors with increased ROS levels. Conversely, overexpression of Hmgcs2 in adipocytes increased 3HBA secretion from adipocytes and enhanced the gene expression levels of the antioxidative stress factors, PPARγ, and lipogenic factors. These results demonstrate that 3HBA plays significant roles in enhancing the physiological function of adipocytes. In mammals, 3HBA is predominantly synthesized in the liver from acetyl-CoA derived from β-oxidation of fatty acids2, transported to extrahepatic tissues, taken up in peripheral tissues by monocarboxylate transporter (MCT), and oxidized for ATP production3. 3HBA becomes an important energy source within extrahepatic tissues in various physiological states, such as postexercise, fasting, starvation, pregnancy, the neonatal period, and adherence to low-carbohydrate diets. Circulating total ketone body concentrations in healthy adult humans normally exhibit circadian oscillations between approximately 0.1 and 0.25 mM, rise to 1 mM after prolonged exercise or 24 h of fasting, and can accumulate to as high as 20 mM in pathological states such as diabetic ketoacidosis.The ketone body, including 3-hydroxybutyric acid (3HBA), is an endogenous product of fat metabolism, and a vital alternative metabolic fuel source for all domains of life, eukarya, bacteria, and archaea4. Astrocyte cell lines produce ketone bodies by treatment with lauric acid5. In the small intestine, Hmgcs2 expression is highly enriched in self-renewing Lgr5 + intestinal stem cells (ISCs), which produce ketone bodies to regulate intestinal stemness6. When artificially induced by retroviral overexpression of PRDM16, beige adipocytes produce and secrete 3HBA with upregulation of Hmgcs27, however there were no reports about ketogenesis in white adipocytes.3-Hydroxy-3-methylglutaryl-CoA synthetase 2 (Hmgcs2) is the first rate-limiting enzyme in the production of ketone bodies. Several reports have revealed the extrahepatic expression of hmgcs2, and the production of ketone bodies. Retinal pigment epithelial (RPE) cells express particularly high levels of hmgcs2 and metabolize fatty acids to produce 3HBA, which is transported to the retina for use as a metabolic substrate8. 3HBA inhibits class I HDACs, which increases histone acetylation and thereby induces the expression of genes. Regarding the small intestine, 3HBA mediates intestinal stem cell homeostasis through enhancement of Notch signaling by inhibition of HDAC activity6. 3HBA suppresses oxidative stress by inducing the expression of antioxidative factors, such as forkhead box O3 (Foxo3), metallothionein 2 (Mt2), MnSOD and catalase, with increasing acetylation of lysine residues in histone H3 by inhibition of HDAC9. In cardiomyocytes, 3HBA is produced as a compensatory response against oxidative stress in heart failure through inhibition of HDAC10. Moreover, histone lysine residues are subject to direct epigenetic modification by 3HBA, which is associated with active gene expression11. Related to this study, we reported that 3HBA induces β-hydroxybutyrylation of histone H3 at lysine 9 and upregulation of adiponectin in 3T3-L1 adipocytes independent of their acetylation or methylation12.Although 3HBA has been an energy source, recent evidence demonstrates that 3HBA is also a signaling metaboliteIn the present study, we identified that Hmgcs2 is an upregulated gene in adipose tissues of fasted mice and is expressed in differentiated 3T3-L1 adipocytes. In addition, treatment with 3HBA or overexpression of the Hmgcs2 gene enhanced, but silencing of the Hmgcs2 gene impaired, adipocyte function to regulate the expression of antioxidative stress factors, PPARγ, lipogenic genes, and insulin signaling.Conventionally, production of ketone body, i.e., Ketogenesis occurs primarily in the mitochondria of liver cells. In short, Hmgcs2 converts acetyl-CoA derived from β-oxidation of free fatty acids (FFAs) into 3-hydroxy-3-methylglutaryl-CoA, which is catalyzed into acetoacetate by 3-hydroxy-3-methylglutaryl-CoA lyase (Hmgcl). β-Hydroxybutyrate dehydrogenase Bdh1) then converts acetoacetate to 3HBA  and Hmgcs2 homo knockout mice . Hmgcs2 of epiWAT were deleted in KO mice . EpiWAT of the 3HBA-injected mice exhibited increased transcript abundance of antioxidative stress factors, such as SOD2  production in 3T3-L1 adipocytes  and 3T3-L1-TetON-empty cells (empty-L1). Hmgcs2-L1 cells conditionally expressed ectopic protein and mRNA of Hmgcs2 when treated with doxycycline followed by insulin treatment Fig. a and b. 23. In this dataset, the expression levels of Hmgcs2 and Hmgcl were significantly higher in epiWAT of fasting mice than in feeding mice. Sorting 4,666 upregulated genes in descending order, Hmgcs2 was among the most highly ranked genes  activity and histone acetylation, resulting in nuclear accumulation of PPARα, a metabolic master regulator, in the intestine24. On the other hand, 3HBA acts as a ligand of the G protein-coupled receptor (GPR). 3HBA is a ligand of GPR41 and regulates sympathetic nervous system activity and energy expenditure25. 3HBA is also a ligand of GPR109A and suppresses lipolysis through activation of immune cells27. However, gene expressions of both GPR41 and GPR109A in epiWAT of mice were not detected (GSE46495)23. Thus, ketone bodies, including 3HBA, exert various effects through multiple molecular mechanisms. Nevertheless, few studies have been reported on the relationship between 3HBA and adipocytes. Further studies are required to determine the mechanism by which 3HBA enhances the physiological function of adipocytes.Ketone bodies, including 3HBA, are not only an alternative energy source under starvation but also play multiple roles as signaling molecules28. In the current study, 3HBA did not induce PPARγ or lipogenic genes in the absence of insulin  released from adipocytes by lipolysis. FFAs are converted to ketone bodies mainly in the liver and transported to extrahepatic tissues. Therefore, prolonged fasting is accompanied by low serum insulin levels, elevated serum ketone bodies, and reduced storage of triglycerides in adipocytes. In the initial phase of feeding following prolonged fasting, plasma insulin levels rise quickly, while plasma 3HBA levels fall slowly and remain high in human subjectsIn conclusion, our results revealed for the first time that adipocytes express Hmgcs2 and produce 3HBA and that 3HBA enhances adipocyte function in endocrine and autocrine/paracrine manners. Further investigations are required to reveal the physiological and pathological significance of ketogenesis in adipocytes in vivo by establishing an adipocyte-specific Hmgcs2-deficient mouse model.Seven-week-old male C57BL/6 J mice were obtained from CLEA Japan  and acclimated for more than 1 week before the experiment. Mice were housed individually in sterile cages, maintained in a room under controlled temperature (23 ± 1.5 °C) and humidity (45 ± 15%) on a 12-h dark/12-h light cycle, and had free access to water and normal chow diets . Samples from six tissues were collected, including epididymal fat, subcutaneous fat, mesenteric fat, BAT, liver, and skeletal muscle (gastrocnemius muscle). The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University, Graduate School of Medicine. These mouse studies were approved by the Ethics Review Committee for Animal Experimentation of Osaka University, Graduate School of Medicine, and carried out in accordance with the Institutional Animal Care and Use Committee Guidelines of Osaka University.+/+) mice and KO (Hmgcs2−/−) mice were previously established by Hiroshi Maegawa’s lab31. Briefly, a mouse model harboring a deletion mutation in exon 2 of the mouse Hmgcs2 gene was generated using the CRISPR/Cas9 system. The following sequences were used for single guide RNA (sgRNA) synthesis: sgRNA1, TGGAACGCACAAAGCTGCCG; sgRNA2, GTGCCTGCAGTGGTACAGA. Cas9 mRNA and sgRNAs (Cas9 mRNA: gRNA1gg:gRNA2cc = 2:1:1) were microinjected into fertilized C57BL/6 J mouse embryos. Among these, one mouse line with a 38-bp deletion mutation in exon 2, which led to a frameshift and subsequent complete deletion of Hmgcs2 protein synthesis, was selected. Homozygous Hmgcs2−/− mice were born from heterozygous intercrosses and used for phenotypic analyses in parallel with age- and sex-matched wild-type littermates as a control group. These mice were housed in a temperature-controlled environment (23 °C) with a 12-h light and 12-h dark (20:00–08:00) photoperiod and cared for in facilities operated by the Research Center for Animal Life Science at Shiga University of Medical Science. CLEA Rodent Diet  was used as the standard chow in the center. The experimental protocol was approved by the Gene Recombination Experiment Safety Committee and Research Center for Animal Life Science (RCALS) at Shiga University of Medical Science.WT  mice versus KO (Hmgcs2 −/−) mice experiment, 12- or 13-week-old male mice were subjected to fasting condition for 12 h, followed by feeding for 12 h, and then anesthetized with isoflurane and blood samples were collected from the heart, followed by sacrifice. Body weight, blood glucose, and blood 3HBA were measured at prefasting, postfasting, and postfeeding, and food intake was determined by weighing the metal cage top, including the food at the end of the experiment.In the injection of PBS or 3HBA experiment, 8-week-old male C57BL/6 J mice were subjected to fasting conditions for 12 h before being injected intraperitoneally with PBS or 3HBA (20 mmol/kg of body weight), fed for 12 h, and then anesthetized with isoflurane. Blood samples were collected from the heart, followed by sacrifice. Body weight was measured at prefasting, postfasting, and postfeeding, and food intake was determined by weighing the metal cage top, including the food at the end of the experiment.Blood glucose levels were measured by tail vein sampling before anesthesia using the Glutest Neo alpha . Blood 3HBA levels were measured by tail vein sampling before anesthesia using the PRECISION XCEED (Ketometer) . Plasma concentrations of 3HBA were measured using a beta Hydroxybutyrate (beta HB) Assay Kit (Abcam) according to the instructions provided by the manufacturer.3T3-L1 adipocytes were cultured in DMEM  supplemented with 10% FBS until two days post-confluence. Then (day 0), the cells were treated with or without adipogenic mixture containing 0.5 mM 3-isobutyl-1-methylxanthine , 1 μM dexamethasone , and 1 μM insulin  for 48 h. After 48 h (day 2), medium was replaced with DMEM supplemented with 10% FBS. Until day 7, the cells were maintained in DMEM with 10% FBS.3-HBA abundance in cell lysate and culture supernatant was measured using a beta Hydroxybutyrate (beta HB) Assay Kit (Abcam) according to the instructions provided by the manufacturer.3 , 119 mM NaCl , 4.74 mM KCl , 1.19 mM MgCl2 , 1.19 mM KH2PO4 , 2.54 mM CaCl2 , 10 mM 4-(2-hydroxyethyl)-1-iperazineethanesulfonic acid , and 0.05 mM Bovine Serum Albumin solution (Sigma), supplemented with 10 μM DCFDA. After staining with DCFDA for 1 h at 37 ℃ under 5% CO2, 3T3-L1 adipocytes were wash 3 times with PBS and detected by fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm.Intracellular ROS were measured using the DCFDA Cellular ROS Detection Assay Kit (Abcam) with the instructions provided by the manufacturer. Briefly, on day 9 of treatment of 3HBA on 3T3-L1 adipocytes Fig. , or on dOn day 7 after differentiation, the medium of 3T3-L1 adipocytes was replaced with DMEM composed of 2.5 mM glucose with 10 mM concentrations of 3-hydroxybutyric acid (Sigma–Aldrich). After 24 h (day 8), 3T3-L1 adipocytes were additively stimulated by 1 nM insulin for 24 h, followed by harvesting (day 9).On day 7 after differentiation, the medium of 3T3-L1 adipocytes was replaced with DMEM composed of 25 mM glucose with 1 nM insulin or 1 µM dexamethasone for 24 h, followed by harvesting (day 8).3T3-L1 adipocytes were harvested, and small interfering (si)RNA was introduced by reverse transfection on day 3 after differentiation, followed by forward transfection on day 5, using the Lipofectamine RNAiMAX Reagent (Invitrogen) according to the instructions provided by the manufacturer. On day 7 after differentiation, the 3T3-L1 adipocytes were maintained in serum-free DMEM composed of 25 mM glucose and 1 nM insulin for 24 h, followed by harvest (day 8).Platinum-E cells were transfected with pRetroX-Tet-On Advanced (TaKaRa), and RetroX-Tight-Pur (TaKaRa) harboring the mouse Hmgcs2 gene. The media containing the retroviruses were harvested 48 h after transfection, filtered, and transferred to 3T3-L1 cells. Infected cells were selected with 200 mg/mL G418, 400 mg/mL hygromycin, and 1 mg/mL puromycin.On day 5 after 3T3-L1-TetON-empty and 3T3-L1-TetON-Hmgcs2 adipocytes were differentiated, the adipocytes were treated with 2 µg/mL doxycycline for 48 h. On day 7 after differentiation, these adipocytes were maintained in serum-free DMEM composed of 25 mM glucose and 1 nM insulin for 24 h, followed by harvesting on day 8 after differentiation.Total RNA was isolated with TRI-Reagent (Sigma–Aldrich) based on the method recommended by the manufacturer. cDNA was synthesized using Transcriptor Universal cDNA Master Mix (Roche) and was subjected to RT-PCR using FastStart Essential DNA Green Master Mix (Roche) on a LightCycler® 96 Instrument (Roche) according to the protocol provided by the manufacturer. The primers used are described in Supplementary Table Cell lysates of 3T3-L1 cells were collected. Protein levels were determined by blotting with phospho-Akt  were purchased from Cell Signaling Tschnologies. β-actin (A5441) were purchased from Sigma–Aldrich. Hmgcs2 (ab137043) were purchased from Abcam. Chemiluminescent images were obtained by ChemiDoc Touch imaging system  with Optimal Auto-exposure mode.P value < 0.05 denoted the presence of a statistically significant difference.All data were expressed as the mean ± SEM values. Differences between two groups were examined for statistical significance by Student’s t-test. A Supplementary Information.