1,25-Dihydroxyvitamin D3: A novel agent for enhancing wound healing

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃), has diverse effects in a variety of tissues and cell types, including skin. Since 1,25(OH)₂D₃ affects both fibroblast and keratinocytes, we evalauated the effect of 1,25(OH)₂D₃ or wound healing. We investigated the effect of the topically applied 1,25(OH)₂D₃ or vehicle on the healing of cutaneous wounds in rats in a blinded manner. Wound areas were measured by planimetry technique. Healing was expressed as the percentage of the original wound area that was healed. 1,25(OH)₂D₃ at concentrations between 5 and 50 ng/day caused a dose-dependent acceleration of healing. Time course and specificity studies indicated that 1,25(OH)₂D₃ specifically promoted healing between 1–5 days after wounding as compared with vitamin D (0.5 μg/day), which showed no significant improvement over control. Our results suggest that 1,25(OH)₂D₃ and its analogues may be a new class of compounds that could be developed to enhance wound healing. © 1995 Wiley-Liss, Inc.     

Regulation of the CAMP gene by 1,25(OH)2D3 in various tissues

The induction of antimicrobial peptides such as the human cathelicidin, CAMP/hCAP18, by 1,25(OH)₂D₃ provides a very exciting therapeutic approach in boosting immunity against infectious diseases. To explore the range of cell types and expand the number of cell models for studying the regulation of CAMP gene expression by 1,25(OH)₂D₃, we treated cell lines from various tissue types and determined CAMP gene expression. Also, we tested additional compounds together with 1,25(OH)₂D₃ to look for possible cooperative activation of the gene. We identified 1,25(OH)₂D₃-mediated induction of the CAMP gene in B-cell lymphomas, prostate and endometrial cancer lines and found cooperative activation with the histone deacetylase inhibitor sodium butyrate. The data suggest that regulation of CAMP by 1,25(OH)₂D₃ is potentially important in a wide range of tissues.     

Nasal Levels of Antimicrobial Peptides in Allergic Asthma Patients and Healthy Controls: Differences and Effect of a Short 1,25(OH)2 Vitamin D3 Treatment

Background

Allergy is often accompanied by infections and lower levels of antimicrobial peptides (AMPs). Vitamin D has been shown to increase expression of selected AMPs. In this study we investigated whether antimicrobial peptide levels in nasal secretions of allergic asthma patients are lower than in healthy controls, and whether administration of the active form of vitamin D (1,25(OH)₂D3) affects these antimicrobial peptide levels.

Methods

The levels of antimicrobial peptides in nasal secretions were compared between 19 allergic asthma patients and 23 healthy controls. The effect of seven days daily oral treatment with 2 μg 1,25(OH)₂D3 on antimicrobial peptides in nasal secretions was assessed in a placebo-controlled cross-over clinical study.

Results

Levels of neutrophil α-defensins (human neutrophil peptides 1–3; HNP1-3) and lipocalin 2 (LCN2; also known as NGAL) were significantly lower in asthmatics, but no differences in LL-37 and SLPI were detected. Treatment with a short-term 1,25(OH)₂D3 caused a small increase in HNP1-3, but not when the asthma and control groups were analyzed separately. LL-37, LCN2 and SLPI did not change after treatment with 1,25(OH)₂D3.

Conclusion

Levels of the antimicrobial peptides HNP1-3 and LCN2 are lower in nasal secretions in asthmatics and are not substantially affected by a short-term treatment with active vitamin D.     

The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH₂, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025–4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH₂ now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH₂ to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.     

25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 Promote the Differentiation of Human Subcutaneous Preadipocytes

1,25(OH)₂D₃ inhibits adipogenesis in mouse 3T3-L1 adipocytes, but little is known about its effects or local metabolism in human adipose tissue. We showed that vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates 25(OH)D₃ to 1,25(OH)₂D₃, were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)₂D₃, as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)₂D₃ enhanced adipogenesis as determined by increased expression of adipogenic markers and triglyceride accumulation (50% to 150%). The magnitude of the effect was greater in the absence of thiazolidinediones. 1,25(OH)₂D₃ was equally effective when added after the removal of differentiation cocktail on day 3, but it had no effect when added only during the induction period (day 0–3), suggesting that 1,25(OH)₂D₃ promoted maturation. 25(OH)D₃ also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)₂D₃. Consistent with this possibility, incubation of preadipocytes with 25(OH)D₃ led to 1,25(OH)₂D₃ accumulation in the media. 1,25(OH)₂D₃ also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.     

Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

The most biologically active metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D₃ (25(OH)D₃) can affect osteoblast function via conversion to 1,25(OH)₂D₃, however, it is largely unknown whether 25(OH)D₃ can affect primary osteoblast function on its own. Furthermore, 25(OH)D₃ is not only converted to 1,25(OH)₂D₃, but also to 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D₃ itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)₂D₃ can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D₃ and 1,25(OH)₂D₃ by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)₂D₃ synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D₃. We demonstrated that 24R,25(OH)₂D₃ increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)₂D₃ strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D₃, 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)₂D₃, but also to 24R,25(OH)₂D₃ by enhancing osteoblast differentiation. This suggests that 25(OH)D₃ can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)₂D₃, but also via conversion to 24R,25(OH)₂D₃. Whether 25(OH)D₃ has direct actions on osteoblast function needs further investigation.     

Regulation of cathelicidin antimicrobial peptide expression by an endoplasmic reticulum (ER) stress signaling, vitamin D receptor-independent pathway

Vitamin D receptor (VDR)-dependent mechanisms regulate human cathelicidin antimicrobial peptide (CAMP)/LL-37 in various cell types, but CAMP expression also increases after external perturbations (such as infection, injuries, UV irradiation, and permeability barrier disruption) in parallel with induction of endoplasmic reticulum (ER) stress. We demonstrate that CAMP mRNA and protein expression increase in epithelial cells (human primary keratinocytes, HaCaT keratinocytes, and HeLa cells), but not in myeloid (U937 and HL-60) cells, following ER stress generated by two mechanistically different, pharmacological stressors, thapsigargin or tunicamycin. The mechanism for increased CAMP following exposure to ER stress involves NF-κB activation leading to CCAAT/enhancer-binding protein α (C/EBPα) activation via MAP kinase-mediated phosphorylation. Furthermore, both increased CAMP secretion and its proteolytic processing to LL-37 are required for antimicrobial activities occur following ER stress. In addition, topical thapsigargin also increases production of the murine homologue of CAMP in mouse epidermis. Finally and paradoxically, ER stress instead suppresses the 1,25(OH)(2) vitamin D(3)-induced activation of VDR, but blockade of VDR activity does not alter ER stress-induced CAMP up-regulation. Hence, ER stress increases CAMP expression via NF-κB-C/EBPα activation, independent of VDR, illuminating a novel VDR-independent role for ER stress in stimulating innate immunity.     

Modulation of myelomonocytic U937 cells by vitamin D metabolites

Investigation of the effects of 1,25(OH)₂D₃ and 24,25(OH)₂D₃ on the proliferation and differentiation of the human myelomonocytic cell line U937 has been complemented with studies of the effect of the same metabolites on the number of nuclear receptors for 1,25(OH)₂D₃. Both 1,25(OH)₂D₃ and 24,25(OH)₂D₃ inhibit the proliferation of U937 cells in a dose-dependent manner. The concentrations of 24,25(OH)₂D₃ required to produce this effect were 100-times greater than those of 1,25(OH)₂D₃. Inhibition of proliferation was associated with increased expression of the CD14 and 200 kDa 63D3 antigens thus confirming differentiation of U937 towards a more mature cell type.Studies of the nuclear receptor for 1,25(OH)₂D₃ showed that pre-treatment of the cells with 1,25(OH)₂D₃ resulted in an apparent 40% decrease in the number of detectable 1,25(OH)₂D₃ receptors as compared to control U937 cells. This is due to the fact that the 1,25(OH)₂D₃ binds to U937 cell nuclei during culture and thus blocks the subsequent binding of radiolabelled 1,25(OH)₂D₃ used to measure the number of 1,25(OH)₂D₃ receptors. Measurement of the binding of unlabelled 1,25(OH)₂D₃ by radioimmunoassay indicated that pre-treatment of the cells with 1,25(OH)₂D₃ increased the capacity of U937 to bind the hormone, although measurement of these receptors by whole cell assay was prevented by the binding of 1,25(OH)₂D₃ itself. This effect was not observed with 24,25(OH)₂D₃ which was more easily displaced from binding sites by radiolabelled 1,25(OH)₂D₃ and it appears to act through low affinity binding to the 1,25(OH)₂D₃ receptor.     

Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation

1,25-Dihydroxyvitamin D³ [1,25(OH)₂D₃] has been proposed as a physiologic regulator of keratinocyte growth and differentiation. Utilizing a proliferative serum-free culture system, we have found that at physiologic (picomolar) concentrations this hormone stimulated proliferation of primary mouse epidermal keratinocytes; at higher (nanomolar to micromolar) doses, growth was inhibited by 1,25(OH)₂D₃. We investigated the nature of the signal transduction mechanism underlying the response to 1,25(OH)₂D₃ and observed little or no effect of either low or high concentrations of the hormone on cytosolic calcium levels or Fos expression. Furthermore, the protein kinase C inhibitor, Ro 31-7549, had very little effect on the growth inhibition induced by a high dose (1 μM) of 1,25(OH)₂D₃. This lack of rapid signal transduction events was consistent with the inability of a short (4-hour) exposure to 1,25(OH)₂D₃ to initiate a complete growth-inhibitory response as measured using [³H]thymidine incorporation. Our results indicate that physiologic concentrations of 1,25(OH)₂D₃ are required for optimal keratinocyte growth. Furthermore, we found no evidence of rapid effects of 1,25(OH)₂D₃ and suggest that in mouse epidermal keratinocytes, the response to this hormone is mediated by a slow transduction pathway, such as that activated by the intracellular 1,25(OH)₂D₃ receptor (VDR). © 1995 Wiley-Liss, Inc.     

Increased biological potency of hexafluorinated analogs of 1,25-dihydroxyvitamin D3 on bovine parathyroid cells

1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is known to be involved in regulating the proliferation of parathyroid cells and PTH synthesis through reactions involving its nuclear receptor. We evaluated the effects of 1,25-(OH)₂D₃ and its hexafluorinated analog, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D₃ (26,27-F₆-1,25-(OH)₂D₃), on parathyroid cells. The 1,25-(OH)₂D₃ and 26,27-F₆-1,25-(OH)₂D₃ each inhibited [³H]thymidine incorporation and ornithine decarboxylase (ODC) activity, which is important in cell proliferation, in primary cultured bovine parathyroid cells. The inhibitory effect of 26,27-F₆-1,25-(OH)₂D₃ on PTH secretion from parathyroid cells was significantly more potent than that of 1,25-(OH)₂D ₃ between 10⁻¹¹ M and 10⁻⁸ M. Study of 26,27-F₆-1,25-(OH)₂D₃ metabolism in parathyroid cells in vitro elucidated its slower degradation than that of 1,25-(OH)₂D₃. After 48 h of incubation with [1β-³H]26,27-F₆-1,25-(OH)₂D₃, two HPLC peaks, one for [1β-³H]26,27-F₆-1,25-(OH)₂D₃, and a second larger peak for [1β-³H]26,27-F₆-1,23(S),25-(OH)₃D₃, were detected. No metabolites were detected after the same period of incubation with 1,25-(OH)₂[26,27-³H]D₃. We observed that 26,27-F₆-1,23(S),25-(OH)₃D₃ was as potent as 1,25-(OH)₂D₃ in inhibiting the proliferation of parathyroid cells.Data suggest that the greater biological activity of 26,27-F₆-1,25-(OH)₂D₃ is explained by its slower metabolisms and by the retention of the biological potency of 26,27-F₆-1,25-(OH)₂D₃ even after 23(S)-hydroxylation.     

The Paracrine Feedback Loop Between Vitamin D3 (1,25(OH)2D3) and PTHrP in Prehypertrophic Chondrocytes

The endocrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH‐related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)₂D₃ and PTH. This was investigated in ATDC5 cells treated with 10^?8 M 1,25(OH)₂D₃ or PTHrP, Col2‐pd2EGFP transgenic mice, and primary Col2‐pd2EGFP growth plate chondrocytes isolated by FACS, using RT‐qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)₂D₃ receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)₂D₃ decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α‐ and 24‐hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)₂D₃ treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate. 1,25(OH)₂D₃ decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)₂D₃ by increasing VDR production. In light of the role of 1,25(OH)₂D₃ and PTHrP in modulating chondrocyte differentiation, 1,25(OH)₂D₃ in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 229: 1999–2014, 2014. © 2014 Wiley Periodicals, Inc.

     

Involvement of endogenously produced 1,25-dihydroxyvitamin D-3 in the growth and differentiation of human keratinocytes

In this study, we investigated the possibility that cultured keratinocytes from normal human adult skin produce 1,25-dihydroxyvitamin D-3 (1,25(OH)₂D₃, a biologically active form of vitamin D-3) from 25-hydroxyvitamin D-3 [25(OH)D₃], and that 1,25(OH)₂D₃ endogenously produced by keratinocytes is involved in the self regulation of their growth and differentiation. To determine whether 1,25(OH)₂D₃ is produced from 25(OH)D₃ by skin keratinocytes, 25(OH)[³H]D₃ was added to keratinocyte cultures and incubated for 1 h and 5 h. The intracellular and extracellular metabolites were analyzed by three chromatographic systems. The three chromatograms revealed that the major metabolite produced from 25(OH)₂D₃ was 1,25(OH)₂D₃. Most of the 1,25(OH)₂D₃ endogenously produced from 25(OH)D₃ remained within the cells. To examine the time course of 1,25(OH)₂D₃ production, the amount of 1,25(OH)[³H]D₃ was measured at 15 min, 1 h, 5 h and 10 h, being at a maximum 1 h after the addition of 25(OH)D₃. These data indicate that keratinocytes rapidly convert 25(OH)D₃ to 1,25(OH)₂D₃ and that 1,25(OH)₂D₃ is not released into the medium. To determine whether endogenously produced 1,25(OH)₂D₃ is involved in the regulation of growth and differentiation of normal human keratinocytes, we examined the effects of 1,25(OH)₂D₃ and 25(OH)D₃ on their growth and differentiation. Keratinocyte growth was inhibited to 52.6% and 23.4% by 10^?8 M and 10^?7 M 1,25(OH)₂D₃ and to 80.5% and 23.9% by 10^?8 M and 10^?7 M 25(OH)D₃, respectively. Differentiation of these cells was evaluated by quantifying the number which express involucrin, a precursor protein of cornified envelope. The population of involucrin expressing cells (differentiated cells) increased from 6.2% to 14.5% by 2.5·10^?7 M 1,25(OH)₂D₃, and to 11.8% by 2.5·10^?7 M 25(OH)D₃. These results clearly indicate that 25(OH)D₃ is as effective on human keratinocytes as 1,25(OH)₂D₃ in inhibiting growth and inducing differentiation, although to a slightly lesser extent than 1,25(OH)₂D₃. The possibility that the effect of 25(OH)D₃ is mediated through binding to the 1,25(OH)₂D₃ receptor can be excluded, since a competitive binding assay revealed that the affinity of 25(OH)D₃ for the 1,25(OH)₂D₃ receptor in a cytosolic extract of keratinocytes was 100-times lower than that of 1,25(OH)₂D₃. Thus, these results suggest that 1,25(OH)₂D₃ endogenously produced in keratinocytes from 25(OH)D₃ is involved in the regulation of their growth and differentiation in vitro.     

The Establishment of a HeLa Cell Demonstrating Rapid Mitogen-Activated Protein Kinase Phosphorylation in Response to 1α,25-Dihydroxyvitamin D3 by Stable Transfection of a Chick Skeletal Muscle cDNA Library

1α,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] phosphorylates the extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, within 30 sec in primary cultured chick skeletal muscle cells. MAPK of HeLa cell lines, which had been stably transfected with a cDNA library derived from mRNA of chick skeletal muscle cells, was also rapidly phosphorylated by 1,25-(OH)₂D₃. These cell lines have the potential to be a good tool for further investigation of rapid non-genomic mechanism activated by 1,25-(OH)₂D₃.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

Cathelicidin antimicrobial peptide inhibits fibroblast migration via P2X7 receptor signaling

Fibrosis is one of the most common pathological alterations in heart failure, and fibroblast migration is an essential process in the development of cardiac fibrosis. Experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease characterized by inflammatory cell infiltration followed by healing without residual fibrosis. However, the precise mechanisms mediating termination of inflammation and nonfibrotic healing remain to be elucidated. Microarray analysis of hearts from model mice at multiple time points after EAM induction identified several secreted proteins upregulated during nonfibrotic healing, including the anti-inflammatory cathelicidin antimicrobial peptide (CAMP). Treatment with LL-37, a human homolog of CAMP, activated MAP kinases in fibroblasts but not in cardiomyocytes, indicating that fibroblasts were the target of CAMP activity. In addition, LL-37 decreased fibroblast migration in the in vitro scratch assay. P2X7 receptor (P2X7R), a well-known receptor for LL-37, was involved in LL-37 mediated biological effect on cardiac fibroblasts. Stimulation of BzATP, a P2X7R agonist, activated MAPK in fibroblasts, whereas the P2X7R antagonist, BBG, as well as P2X7R deletion abolished both LL-37-mediated MAPK activation and LL-37-induced reduction in fibroblast migration. These results strongly suggest that CAMP upregulation during myocarditis prevents myocardial fibrosis by restricting fibroblast migration via activation of the P2X7R−MAPK signaling pathway.     

Enhanced effects of guggulsterone plus 1,25(OH)2D3 on 3T3-L1 adipocytes

Guggulsterone (GS) and 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] have been shown to influence adipogenesis in 3T3-L1 cells. We investigated the ability of GS and 1,25(OH)₂D₃, alone and in combination to inhibit adipogenesis and induce apoptosis in 3T3-L1 adipocytes. Maturing preadipocytes were treated with 1,25(OH)₂D₃ in combination with GS for 6 days during differentiation. GS and 1,25(OH)₂D₃ each inhibited lipid accumulation, but the combination potentiated the inhibition of lipid accumulation. Apoptosis was increased by 1,25(OH)₂D₃ while GS had no effect, but GS + 1,25(OH)₂D₃ increased apoptosis more than either compound alone. Furthermore, GS + 1,25(OH)₂D₃ caused a potentiated decrease in the expression of aP2 and farnesoid X receptor expression more than either compound alone. In addition, 1,25(OH)₂D₃ increased vitamin D receptor expression after 6 days, while GS had no effect. GS + 1,25(OH)₂D₃, however, caused a potentiated increase in the expression of VDR. These findings show that GS potentiates 1,25(OH)₂D₃’s anti-adipogenic and pro-apoptotic effects in maturing 3T3-L1 preadipocytes.     

Quercetin exerts an inhibitory effect on cellular bioenergetics of the B164A5 murine melanoma cell line

This study examined the hypothesis that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) upregulates the insulin-independent signaling cascade of glucose metabolism. C2C12 myotubes were treated with high glucose (HG, 25 mM) and 1,25(OH)₂D₃ (0–50 nM). 1,25(OH)₂D₃ supplementation upregulated both insulin-independent (SIRT1) and insulin-dependent (p-IRS) signaling molecules, and stimulated the GLUT4 translocation, and glucose uptake in HG-treated myotubes. The effect of 1,25(OH)₂D₃ on IRS1 phosphorylation, GLUT4 translocation, and glucose uptake was attenuated in SIRT1-knockdown myotubes. Treatment with 1,25(OH)₂D₃, coupled with insulin, enhanced GLUT4 translocation and glucose uptake compared to treatment with either insulin or 1,25(OH)₂D₃ alone in HG-treated myotubes, which suggests that insulin-independent signaling molecules can contribute to the higher glucose metabolism observed in 1,25(OH)₂D₃ and insulin-treated cells. The data, therefore, suggest that 1,25(OH)₂D₃ increases glucose consumption by inducing SIRT1 activation, which in turn increases IRS1 phosphorylation and GLUT4 translocation in myotubes.