Deuterium enrichment of vitamin A at the C20 position slows the formation of detrimental vitamin A dimers in wild-type rodents

Degenerative eye diseases are the most common causes of untreatable blindness. Accumulation of lipofuscin (granular deposits) in the retinal pigment epithelium (RPE) is a hallmark of major degenerative eye diseases such as Stargardt disease, Best disease, and age-related macular degeneration. The intrinsic reactivity of vitamin A leads to its dimerization and to the formation of pigments such as A2E, and is believed to play a key role in the formation of ocular lipofuscin. We sought a clinically pragmatic method to slow vitamin A dimerization as a means to elucidate the pathogenesis of macular degenerations and to develop a therapeutic intervention. We prepared vitamin A enriched with the stable isotope deuterium at carbon twenty (C20-D(3)-vitamin A). Results showed that dimerization of deuterium-enriched vitamin A was considerably slower than that of vitamin A at natural abundance as measured in vitro. Administration of C20-D(3)-vitamin A to wild-type rodents with no obvious genetic defects in vitamin A processing, slowed A2E biosynthesis. This study elucidates the mechanism of A2E biosynthesis and suggests that administration of C20-D(3)-vitamin A may be a viable, long-term approach to retard vitamin A dimerization and by extension, may slow lipofuscin deposition and the progression of common degenerative eye diseases.     

Studies on the mechanism of action of calciferol VII. Localization of 1,25-dihydroxy-vitamin D3 in chick parathyroid glands.

When 1,25(OH)₂-vitamin D₃ was administered to vitamin D-deficient chicks, within two hours the parathyroid glands were observed to accumulate this steroid to a concentration four times that present in the blood and equivalent to levels observed in the target intestine. Similarly, when 25-(OH)-vitamin D₃ was administered, the parathyroid glands had 2.4 times the concentration of the metabolite, 1,25-(OH)₂-vitamin D₃ as that seen in the blood and 60% of that found in the intestine. These results are consistent with the concept that the hormonally active form of vitamin D, 1,25-(OH)₂-vitamin D₃, may interact with the parathyroid glands to effect changes in parathyroid hormone secretion.     

Biotransformations of 25-hydroxyvitamin D3 by kidney microsomes.

Vitamin D₃-deficient chick kidney microsomes $\text{in}\text{vitro}$ metabolize 25-hydroxy-[26(27)-methyl-³H]-vitamin D₃ to yet structurally unidentified polar metabolites previously designated MIC-I and MIC-II. Kidney microsomes of vitamin D₃-repleted chicks could not be demonstrated to produce these metabolites when ³H was the radioactive isotope in positions C-26 and C-27 of the substrate. However, when 25-hydroxy-[26,27-¹⁴C]-vitamin D₃ was the radioactive substrate, MIC-I and MIC-II production was independent of the vitamin D₃ status of the chicks. These results suggest that under conditions of vitamin D₃-sufficiency, there is augmented sequential kidney metabolism of 25-hydroxyvitamin D₃ to products with modified side-chains involving C-26 and/or C-27. It is possible that this metabolism is responsible for the regulation of kidney cellular concentrations of 25-hydroxyvitamin D₃.     

C20-D3-vitamin A slows lipofuscin accumulation and electrophysiological retinal degeneration in a mouse model of Stargardt disease

Stargardt disease, also known as juvenile macular degeneration, occurs in approximately one in 10,000 people and results from genetic defects in the ABCA4 gene. The disease is characterized by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE) of the eye and by vision loss. No cure or treatment is available. Although lipofuscin is considered a hallmark of Stargardt disease, its mechanism of formation and its role in disease pathogenesis are poorly understood. In this work we investigated the effects of long-term administration of deuterium-enriched vitamin A, C20-D(3)-vitamin A, on RPE lipofuscin deposition and eye function in a mouse model of Stargardt's disease. Results support the notion that lipofuscin forms partly as a result of the aberrant reactivity of vitamin A through the formation of vitamin A dimers, provide evidence that preventing vitamin A dimerization may slow disease related, retinal physiological changes and perhaps vision loss and suggest that administration of C20-D(3)-vitamin A may be a potential clinical strategy to ameliorate clinical symptoms resulting from ABCA4 genetic defects.     

Dopamine Promotes Ascorbate Release from Retinal Neurons: Role of D<Subscript>1</Subscript> Receptors and the Exchange Protein Directly Activated by <Emphasis Type="Italic">cAMP type 2</Emphasis> (EPAC2)

Ascorbate, the reduced form of vitamin C, is highly concentrated in the central nervous system (CNS), including the retina, where it plays important physiological functions. In the CNS, the plasma membrane transporter sodium vitamin C co-transporter 2 (SVCT2) is responsible for ascorbate transport in neurons. The neurotransmitter dopamine (DA), acting through D₁- and D₂-like receptor subfamilies and classically coupled to adenylyl cyclase, is known to modulate synaptic transmission in the retina. Here, we reveal that DA controls the release of ascorbate from retinal neurons. Using primary retinal cultures, we show that this DA effect is dose-dependent, occurring by the reversal of the SVCT2, and could be elicited by brief and repetitive pulses of DA. The DA effect in inducing ascorbate release occurs by the activation of D₁R and is independent of PKA. Moreover, the exchange protein directly activated by cAMP type 2 (EPAC2) is present in retinal neurons and its specific knockdown using shRNAs abrogates the D₁R-induced ascorbate release. Confirming the physiological relevance of this pathway, activation of D₁R or EPAC2 also triggered ascorbate release ex vivo in acute preparations of the intact retina. Overall, DA plays pivotal roles in regulating ascorbate homeostasis through an unanticipated signaling pathway involving D₁R/adenylyl cyclase/cAMP/EPAC2, thereby suggesting that vitamin C might fine-tune dopaminergic neurotransmission in the retina.     

Dopamine D1 receptor activation of adenylyl cyclase, not phospholipase C, in the nucleus accumbens promotes maternal behavior onset in rats

A body of evidence supports the idea that the mesolimbic dopamine (DA) system modulates the natural increase in responsiveness female rats show toward offspring (biological or foster) at birth. In the absence of the full hormonal changes associated with pregnancy and birth, female rats do not show immediate responsiveness toward foster offspring. Activation of the mesolimbic DA system can produce an immediate onset of maternal behavior in these females. For example, female rats that are hysterectomized and ovariectomized on day 15 of pregnancy (15HO) and presented with pups 48 hours later normally show maternal behavior after 2-3 days of pup exposure, but will show maternal behavior on day 0 of testing after microinjection of the DA D₁ receptor agonist, SKF 38393, into the nucleus accumbens (NA) at the time of pup presentation. DA D₁ receptor stimulation is known to activate cAMP intracellular signaling cascades via its stimulation of adenylyl cyclase (AC). However, some DA D₁ receptors are also linked to phospholipase C (PLC) and are capable of activating phosphatidylinositol signaling cascades. SKF 38393 stimulates both types of D₁ receptors. Here we provide evidence that the facilitatory effects of DA D₁ receptor stimulation in the NA on maternal behavior are mediated by AC-linked DA D₁ receptors. By examining the effects of intra-NA application of SKF 83822, a drug which selectively binds DA D₁-AC receptors, or SKF 83959, a drug which selectively activates D₁-PLC-linked receptors, we find that only SKF 83822 facilitates maternal behavior onset.     

Vitamin D3 protects against respiratory syncytial virus-induced barrier dysfunction in airway epithelial cells via PKA signaling pathway

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in infants and young children globally and is responsible for hospitalization and mortality in the elderly population. Virus-induced airway epithelial barrier damage is a critical step during RSV infection, and emerging studies suggest that RSV disrupts the tight junctions (TJs) and adherens junctions (AJs) between epithelial cells, increasing the permeability of the airway epithelial barrier. The lack of commercially available vaccines and effective antiviral drugs for RSV emphasizes the need for new management strategies. Vitamin D₃ is a promising intervention for viral infection due to its critical role in modulating innate immune responses. However, there is limited evidence on the effect of vitamin D₃ on RSV pathogenies. Here, we investigated the impact of vitamin D₃ on RSV-induced epithelial barrier dysfunction and the underlying mechanisms. We found that pre-incubation with 1,25(OH)₂D₃, the active form of vitamin D₃, alleviated RSV-induced epithelial barrier disruption in a dose-dependent manner without affecting viability in 16HBE cells. 1,25(OH)₂D₃ induced minor changes in the protein expression level of TJ/AJ proteins in RSV-infected cells. We observed increased CREB phosphorylation at Ser133 during 1,25(OH)₂D₃ exposure, indicating that vitamin D₃ triggered protein kinase A (PKA) activity in 16HBE. PKA inhibitors modified the restoration of barrier function by 1,25(OH)₂D₃ in RSV-infected cells, implying that PKA signaling is responsible for the protective effects of vitamin D₃ against RSV-induced barrier dysfunction in airway epithelial cells. Our findings suggest vitamin D₃ as a prophylactic intervention to protect the respiratory epithelium during RSV infections.     

Metabolism and biological potency of retro-retinyl acetate in the rat

1. retro-Retinyl acetate was shown to exert its biological activity by conversion into vitamin A. 2. When administered orally, retro-retinyl acetate was hydrolysed to retro-retinol in the intestine, isomerized to retinol and esterified before being transported to the liver for storage. 3. Administration of the compound at as high a dose as 4·0mg./day for 4 days led to the accumulation of both vitamin A and retro-vitamin A in the liver. The amount of retro-vitamin A in liver gradually decreased until it was almost completely converted into vitamin A in 18 days. 4. Intraperitoneal administration of the compound led to the accumulation of both vitamin A and retro-vitamin A in liver and other tissues. No vitamin A was detected in any tissue of rats receiving retro-retinyl acetate intraperitoneally after enterectomy. 5. The small intestine is the major site of conversion of retro-vitamin A into vitamin A. The conversion could also be demonstrated by everted intestinal sacs. 6. The biological potency of retro-retinyl acetate determined by the rat-growth assay was 20·5% that of all-trans-retinyl acetate, when given orally.     

1,25-dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: An effect modulated by vitamin D status

Our laboratory has recently demonstrated that 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effects of 1,25(OH)₂D₃ were, however, lost in vitamin D-insufficient rats and restored by the in vivo repletion of 1,25(OH)₂D₃. In the present studies we have examined the ability of 1,25(OH)₂D₃ to stimulate the phosphorylation of colonic membrane proteins in intact D-sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)₂D₃ increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rats, treatment of colonocytes with 1,25(OH)₂D₃ or 12-Otertradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)₂D₃ were both rapid and transient. In addition, PKC_19–36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)₂D₃. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D-sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D-insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)₂D₃-treatment of colonocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)₂D₃ following the in vivo repletion of vitamin D-insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)₂D₃ treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. © 1995 Wiley-Liss, Inc.     

Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,Differentiation, and Myotube Hypertrophy

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)₂D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)₂D₃ and 25(OH)D₃ affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. We show that myoblasts not only responded to 1,25(OH)₂D₃, but also to the precursor 25(OH)D₃ by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)₂D₃ as well as 25(OH)D₃ stimulated VDR mRNA expression and in myotubes 1,25(OH)₂D₃ also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D₃ metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)₂D₃ or 25(OH)D₃ myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D₃ to 24R,25(OH)₂D₃ which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D₃ metabolites, but is also able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Enhanced Dopamine D1 and D2 Receptor Gene Expression in the Hippocampus of Hypoglycaemic and Diabetic Rats

Hypoglycaemic coma and brain injury are potential complications of insulin therapy. Hippocampal neurons are particularly vulnerable to hypoglycaemic stress leading to memory impairment. In the present article, we have investigated the dopamine (DA) content, homovanillic acid (HVA)/DA turnover ratio, DA D₁ and DA D₂ receptors in the hippocampus of insulin-induced hypoglycaemic (IIH) and streptozotocin induced diabetic rats where brain functions are impaired. The DA content decreased significantly in hippocampus of diabetic, diabetic +IIH and control +IIH rats compared to control. The HVA/DA turnover ratio also increased significantly in diabetic, diabetic +IIH and control +IIH rats compared to control. Scatchard analysis using [³H] DA in the hippocampus showed a significant increase in DA receptors of diabetic, diabetic +IIH and control +IIH rats with decreased affinity. Gene expression studies using Real-time PCR showed an increased expression of DA D₁ and DA D₂ receptors in the hippocampus of hypoglycaemic and diabetic rats. Our results indicate that the dopaminergic system is impaired in the hippocampus of hypoglycaemic and hyperglycaemic rats impairing DA related functions of hippocampus. We observed a prominent dopaminergic functional disturbance in the hypoglycaemic condition than in hyperglycaemia compared to control. This dopaminergic dysfunction in hippocampus during hypoglycaemia and hyperglycaemia is suggested to contribute to cognitive and memory deficits. This will have clinical significance in the treatment of diabetes.     

Characterization of the vitamin D endocrine system in human sebocytes in vitro

Sebocytes are sebum-producing cells that form the sebaceous glands. We investigated the role of sebocytes as target cells for vitamin D metabolites and the existence of an enzymatic machinery for the local synthesis and metabolism of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, calcitriol], the biologically active vitamin D metabolite, in these cell types. Expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25OHase), 25-hydroxyvitamin D-1α-hydroxylase (1αOHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24OHase) was detected in SZ95 sebocytes in vitro using real time quantitative polymerase chain reaction. Splice variants of 1αOHase were identified by nested touchdown polymerase chain reaction. We demonstrated that incubation of SZ95 sebocytes with 1,25(OH)₂D₃ resulted in a cell culture condition-, time-, and dose-dependent modulation of cell proliferation, cell cycle regulation, lipid content and interleukin-6/interleukin-8 secretion in vitro. RNA expression of VDR and 24OHase was upregulated along with vitamin D analogue treatment. Although several other splice variants of 1αOHase were detected, our findings indicate that the full length product represents the major 1αOHase gene product in SZ95 cells. In conclusion, SZ95 sebocytes express VDR and the enzymatic machinery to synthesize and metabolize biologically active vitamin D analogues. Sebocytes represent target cells for biologically active metabolites. Our findings indicate that the vitamin D endocrine system is of high importance for sebocyte function and physiology. We conclude that sebaceous glands represent potential targets for therapy with vitamin D analogues or for pharmacological modulation of 1,25(OH)₂D₃ synthesis/metabolism.     

Use of vitamin D4 analogs to investigate differences in hepatic and target cell metabolism of vitamins D2 and D3

In this study, we used molecules with either of the structural differences in the side chains of vitamin D₂ and vitamin D₃ to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems—HepG2 and HPK1A-ras—to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1α-hydroxyvitamin D₂ (1α-OH-D₂) but not for 1α-OH-D₃ is also observed in both 1α-OH-D₄ and Δ²²-1α-OH-D₃ metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22–23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1α,25-(OH)₂D₄, was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D₂ compounds without evidence of side chain cleavage observed for vitamin D₃ derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D₂ and D₃ compounds. Novel vitamin D₄ compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D₄ analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

Incubation of Metanephroi with Vitamin D3 Increases Numbers of Glomeruli

To characterize actions of vitamin D3 on metanephroi transplanted from rat embryos to adult recipients, we incubated metanephroi with or without 0.01, 0.1 or 1 ug/ml vitamin D3, 25-hydroxyvitamin D₃ [25(OH)D₃] or 1, 25-hydroxyvitamin D₃ [1,25(OH)2D₃] prior to implantation. The number of glomeruli in developed metanephroi three weeks post-transplantation that had been incubated with 1.0 ug/ml vitamin D₃ was increased relative to the number in metanephroi that were not incubated with vitamin D₃ (control), an effect that was not recapitulated by administration of vitamin D₃ directly to hosts at the time of transplantation. Incubation of metanephroi with 1.0 ug/ml vitamin D₃ also enhanced inulin clearances of metanephroi measured at 12 weeks post-transplantation. The hydroxylated derivative of vitamin D₃, 25(OH)D₃, increased glomerulus number when applied at 0.01 ug/ml but not at higher concentrations, while the twice-hydroxylated derivative 1,25(OH)₂D₃, failed to increase glomerulus number at any concentration tested. We conclude that incubation with vitamin D₃ prior to implantation enhances inulin clearance possibly by increasing the number of glomeruli that develop post-transplantation.

Our findings suggest the vitamin D₃ effect is mediated locally.