Vitamin D: Beyond Bone

Abstracts

Session I: Non-Classical Effects of Vitamin D

Vitamin D Biology Beyond Bone
Sylvia Christakos, PhD, UMDNJ-New Jersey Medical School

In recent years vitamin D has received increased attention due to the resurgence of vitamin D deficiency and rickets in developed countries together with the identification of extraskeletal effects of vitamin D, suggesting unexpected benefits of vitamin D in health and disease. The possibility of extraskeletal effects was first noted with the discovery of the presence of the vitamin D receptor (VDR) in tissues and cells that are not involved in maintaining calcium homeostasis including skin, placenta, pancreas, breast, prostate and colon cancer cells, and activated T cells. However, the biological significance of the presence of VDR in different tissues is not fully understood and the role of vitamin D in extraskeletal health has been a matter of debate. Evidence in the laboratory, including the use of animal models, indicates that 1,25(OH)₂D₃ generates a number of extraskeletal effects including inhibition of cancer progression, effects on the cardiovascular system and skin, modulation of innate immunity with subsequent killing of bacteria, and inhibition of certain autoimmune diseases. Although, unlike in the case of vitamin D deficiency and rickets, there is not a causal link between vitamin D deficiency and specific diseases related to extraskeletal effects, the evidence in the laboratory of beneficial effects of 1,25(OH)₂D₃ beyond bone is convincing. Findings in animal models may suggest mechanisms involving similar pathways in humans that could lead to the identification of new therapies.
 

Vitamin D in Immune Function and Disease Prevention
Martin Hewison, PhD, University of California Los Angeles

Beyond its role in calcium homeostasis and bone metabolism, vitamin D is also known to exert non-classical effects that include potent immunomodulatory properties. Recent studies have shown that this includes actions that enhance innate immune responses to infection whilst simultaneously preventing over-exuberant inflammatory adaptive immune responses. At the innate immune level, conversion of precursor 25-hydroxyvitamin D (25D) to active 1,25-dihydroxyvitamin D (1,25D) via the enzyme CYP27B1 in cells such as macrophages can trigger vitamin D receptor (VDR)-mediated responses that include increased production of antibacterial proteins and enhanced autophagy. These responses are highly dependent on the availability of 25D for intracrine conversion to 1,25D, indicating that impaired vitamin D (25D) status may lead to dysregulated responses to infection. However, innate immune responses to vitamin D are also influenced by T cells from the adaptive immune system that can fine-tune macrophage vitamin D metabolism, with concomitant effects on innate immunity. Vitamin D-mediated innate immunity has been linked to diseases such as tuberculosis and leprosy, but may also be important for more common infectious diseases. The same intracrine vitamin D system reported in macrophages is also present in dendritic cells (DCs) that present antigen to T cells during the adaptive immune response. In this setting localized synthesis of 1,25D acts to suppress DC maturation and therefore inhibit antigen presentation. However, vitamin D is also known to modulate T cell phenotype, most notably promoting T helper 2 (Th2) and regulatory (Treg) cells, whilst suppressing Th1 and Th17 cells. Thus, the overarching effect of vitamin D on adaptive immunity is tolerogenic and anti-inflammatory, prompting a link between vitamin D and the prevention of autoimmune disease. These features of vitamin D-mediated immunity and human disease, along with new developments and translational applications will be discussed in more detail during the presentation.
 

Role of the Vitamin D Receptor in the Cardiovascular System
David G. Gardner, MD, University of California San Francisco

A variety of basic and clinical research studies suggest that vitamin D plays a significant role in the promotion of cardiovascular health. We have generated a mouse model that allows for selective deletion of the vitamin D receptor (VDR) in different cell types. Deletion of the VDR in the cardiac myocyte leads to hypertrophy of the myocyte, activation of a hypertrophy-sensitive gene transcription program, and increased myocardial mass. This is associated with increased expression of the gene encoding modulatory calcineurin interacting protein1, a protein associated with the calcineurin signaling cascade that has been linked to hypertrophy in other models. Combining VDR deficiency with a model of murine myocyte steatosis serves to amplify the pathological phenotype associated with the latter, suggesting that deficiency of VDR or its ligand, 1,25 dihydroxyvitamin D, creates a setting which is more vulnerable to the influence of pathological stimuli affecting the heart. Finally, we have created a model of VDR deficiency in the endothelial cell of the heart (VDR^ECKO). These mice have normal blood pressure at baseline but show an exaggerated response to chronic angiotensin II infusion. Isolated aortic tissue from the VDR^ECKO mouse contracts normally in response to phenylephrine but demonstrates a reduced vasorelaxant response to acetylcholine, an effect which appears to be related to reduced endothelial nitric oxide synthase gene expression. Collectively, these data support a role for the liganded VDR in suppressing the effects of pathological stimuli and supporting the maintenance of normal structure and function in the cardiovascular system.
 

Vitamin D during Pregnancy and Lactation: Emerging Concepts
Carol L. Wagner, MD, Medical University of South Carolina

Vitamin D as a preprohormone has effects that extend beyond calcium metabolism and homeostasis throughout life, but this is most vivid during pregnancy when vitamin D metabolism is disengaged from its normal constraints. At no other time during the lifecycle is 25(OH)D, the first vitamin D metabolite, linked directly to 1,25(OH)₂D production, which rises more than 2.5 times above nonpregnant levels. This elevation of 1,25(OH)₂D likely serves the purpose of immune regulation, but there have been few studies that have evaluated the effect of vitamin D status on immune function during pregnancy to support this premise. In addition, few randomized controlled trials have been conducted in pregnant women to determine what is optimal vitamin D status. Two recent studies, the NICHD trial and the Thrasher Research Fund Trial, indicate that 400 IU vitamin D, the amount found in most prenatal vitamins, is woefully inadequate and that 4000 IU/day is necessary in order to optimize 1,25(OH)₂D production, achieved when total circulating 25(OH)D is at least 40 ng/mL. Both studies give evidence that 4000 IU/day is not only safe but that adverse events of pregnancy are lower in the 4000 IU group when compared to 400 IU/day and even 2000 IU/day. Comorbidities of pregnancy appear to be directly linked with lower 25(OH)D, and conversely, increasing 25(OH)D appears to afford protection to the mother and her developing fetus. What we have learned about vitamin D requirements during pregnancy extends to lactation. It was thought for decades that human milk was minimally sufficient in vitamin D yet preliminary results of a recently completed NICHD vitamin D supplementation trial during lactation shows that when mother is vitamin D replete, her milk is replete and that her breastfeeding infant has excellent vitamin D status without infant supplementation. The implications of achieving vitamin D sufficiency solely through lactation is just beginning to be understood and will challenge researchers for decades to come.

 

Session II: Early Career Investigator Presentations

The Role of Vitamin D in Regulation of Apoptotic Signaling Pathways in Obesity
Igor N. Sergeev, PhD, DSc, South Dakota State University

Modulation of apoptosis is emerging as a promising strategy for prevention and treatment of obesity because removal of adipocytes through this process will result in reducing body fat and long-lasting maintenance of weight loss. Effects of 1,25(OH)2-vitamin D₃ (1,25D) on apoptotic cell death are mediated via multiple signaling pathways that involve common regulators and effectors converging on cellular Ca²⁺ (Sergeev, 2005, 2009, 2012). However, the 1,25D-regulated, Ca²⁺-dependent apoptotic molecular targets have not been identified in adipose tissue. We investigated the mechanism by which 1,25D regulates apoptosis in adipocytes. The results obtained demonstrated that 1,25D induced, in a concentration- and time-dependent fashion, the apoptotic Ca²⁺ signal (a sustained, prolonged increase in concentration of intracellular Ca²⁺) in mature mouse 3T3-L1 adipocytes. The 1,25D-induced increase in cellular Ca²⁺ was associated with activation of the Ca²⁺-dependent µ-calpain and the Ca²⁺/calpain-dependent caspase-12. The activation of these proteases was sufficient for effecting morphological and biochemical changes attributed to apoptosis. The 1,25D-induced increase in cellular Ca²⁺ was also associated with the reduced lipid accumulation in mature adipocytes. A murine diet-induced obesity (DIO) model was used to evaluate the role of vitamin D in adiposity. The DIO mice (C57BL/6J) fed the high-vitamin D and, especially, high-vitamin D plus high-Ca diet demonstrated the decreased body and fat weight gain and improved markers of adiposity and vitamin D status (plasma concentrations of glucose, insulin, adiponectin, 25(OH)D, 1,25(OH)2D, and PTH). The findings obtained imply that the 1,25D-induced cellular Ca²⁺ signal can act as an apoptotic initiator that directly recruits Ca²⁺-dependent apoptotic effectors capable of executing apoptosis in adipose tissue. Targeting of Ca²⁺ signaling and the vitamin D/Ca²⁺-dependent calpains and caspases in adipocytes can represent an effective approach for chemoprevention and treatment of obesity. These findings also indicate the need to reevaluate the roles of vitamin D and calcium in obesity.
 

Plasma Vitamin D is Independently Associated with Lung Function in Patients with Chronic Obstructive Pulmonary Disease (COPD)
Erica Rutten, PhD, Ciro +, Centre of Expertise for Chronic Organ Failure

Patients with COPD primarily suffer from lung function impairment. Besides this, COPD is more and more believed to be a systemic disease and as such, high prevalence of vitamin D deficiency has been described. An association between lung function parameters and plasma vitamin D levels has been shown previously in the healthy (1), but never in a group of patients with COPD. The objective of the present study was to investigate whether there is an independent association between lung function parameters and plasma vitamin D levels in a group of 157 COPD patients admitted for pulmonary rehabilitation at Ciro+, the Netherlands. Plasma 25(OH)vitamin D levels, body mass index [BMI, weight in kg/(height in m)²] and lung function parameters [forced expiratory volume in 1s (FEV₁), forced vital capacity (FVC) and diffusing capacity of the lung (DLCO)] were measured. There were 57% males, mean age was 65±9y, mean FEV1 was 47.2±17.9% predicted, mean FVC was 94.6±20.6% predicted and mean DLCO was 53.2±19.7% predicted. Pearson correlation coefficient showed significant correlations between plasma vitamin D concentration and FEV1 (r=0.29, p<0.01), FVC (r=0.29, p<0.01) and DLCO (r=0.22, p=0.01). Multivariate regression analyses revealed that, after correction for age, gender and BMI, vitamin D concentration was independently associated with FEV₁ (beta=0.22, p<0.01), FVC (beta=0.24, p<0.01), DLCO (beta=0.25, p<0.01). In conclusion, there is evidence that vitamin D plays a role in the lung pathology of patients with COPD, which needs further investigation.
 

Impact of Cholecal Ciferol Repletion on Erythropoietin Requirements in Vitamin D-Deficient Hemodialysis Patients: Pilot Data from a Randomized Controlled Trial
Lily Li, BA, Mount Sinai School of Medicine

Vitamin D deficiency is common in hemodialysis patients. Uncontrolled studies suggest correction of Vit D deficiency is associated with decreased erythropoietin (EPO) requirements. To characterize this relationship, we examined the impact of D₃ repletion on EPO requirements in 79 Vit D-deficient (25OH-D level <25 ng/mL) hemodialysis patients randomized to receive D₃ (n=51) or standard of care (no repletion, n=28) in a 2:1 ratio. Patients randomized to D₃ received 50,000 IU/wk to a goal 25OH-D of >35ng/mL. Changes in Vit D level, hemoglobin (Hb), and EPO requirements were assessed at 3 months. EPO (Darbepoetin) doses were adjusted by nursing staff as per dialysis unit protocol (target Hb 10-12 g/dL). Baseline characteristics were similar between groups, as were baseline Vit D levels (median 13.5 vs 13.1, p=0.623), baseline Hb (mean Hb 11.8 g/dL vs 11.4 g/dL, p=0.155), and baseline EPO requirements (median Darbepoetin dose 40 units/wk vs 50 units/wk, p=0.262). Patients randomized to D₃ had a rise in 25OH-D levels at 3 months (11.9 to 44.1 ng/mL, p<0.001, n=30), with a corresponding fall in EPO requirements (50.00 to 40.32 units/wk, p=0.029, n=30) despite no change in Hb (11.9 to 11.5 g/dL, p=0.180, n=30). No change in Vit D level, Hb, or EPO dose was observed in control patients at 3 months (n=15). Patients randomized to D₃ did not experience hypercalcemia. Our preliminary data from this ongoing randomized controlled trial suggest that treatment of Vit D-deficient dialysis patients with D₃ is safe, effective, and may result in lower EPO requirements.

 

Session III: Translating Epidemiological Data into Policy and Clinical Applications in Adult and Aging Populations

Vitamin D for Type 2 Diabetes; to D or not to D
Anastassios G. Pittas, MD, Tufts Medical Center

The most well recognized function of vitamin D is to maintain calcium and phosphorus homeostasis and to promote bone health; however, there is evidence to suggest that optimal vitamin D status may also be important for a variety of non-skeletal outcomes, including type 2 diabetes. Cross-sectional studies have reported consistent associations between lower vitamin D status and prevalent diabetes. In most longitudinal observational studies, lower vitamin D status is associated with increased risk of incident type 2 diabetes but the strengths of associations are attenuated compared to cross-sectional studies. Mechanistic trials have shown that vitamin D supplementation may alter the pathophysiology of type 2 diabetes. Vitamin D is a promising new element in the prevention and management of diabetes. However, because vitamin D is an excellent marker of general health status, the positive results reported in the observational studies and post-hoc analyses of trials might reflect confounding. Therefore, the hypothesis that vitamin D may modify diabetes risk needs to be confirmed in trials specifically designed for that purpose.
 

Molecular Aspects of the Role of Vitamin D in Muscle Function
Ricardo Boland, PhD, Universidad Nacional del Sur, Argentina

Various lines of clinical and experimental data, as well as the presence of the VDR in skeletal muscle, support the notion that Vitamin D₃ plays a role in this tissue. There is a wealth of evidence indicating that the hormone 1α,25(OH)₂-vitamin D₃ [1α,25(OH)₂D₃] acts in muscle through membrane initiated mechanisms. The purpose of this talk is to review the regulation of signal transduction pathways in skeletal muscle cells by 1α,25(OH)₂D₃. The hormone induces fast responses, involving transmembrane stimulation of adenylyl cyclase/cAMP/PKA, PLC/DAG + IP3/PKC, Ca²⁺ messenger system and MAPK cascades. 1α,25(OH)₂D₃ rapidly induces reverse translocation of the VDR from the nucleus to the plasma membranes. Accordingly, a complex is formed between the VDR and TRCP3, an integral protein of capacitative Ca²⁺ entry (CCE), suggesting an association between both proteins and a functional role of the VDR in 1α,25(OH)₂D₃ activation of CCE. In addition, the formation of complexes between the VDR and Src has been involved in the activation of tyrosine phosphorylation paths by hormone. Integration of these mechanisms throw light on the regulation of contractility and myogenesis by 1α,25(OH)₂D₃.
 

Level of Vitamin D Affects Physical and Cognitive Function in Older Persons
Luigi Ferrucci, MD, PhD, National Institute on Aging, NIH

Vitamin D, the "sunshine vitamin," is recognized for its role in bone health and calcium metabolism. Vitamin D supplementation is generally considered a critical component for the prevention of osteoporosis. Recent research suggest that vitamin D deficiency might also contribute to the pathogenesis and clinical progression of chronic diseases highly prevalent in older persons, including cardiovascular disease, cancer, chronic pulmonary diseases, depressive symptoms, and autoimmune disease. Observational evidence for a role of vitamin D in these conditions is generally strong and consistent, but data on vitamin D in the prevention of specific or multiple morbidity is still limited and different studies often report conflicting findings. Over the last few decades, geriatricians realized that assessing health in older persons should rely on measures of physical and cognitive function rather than on disease diagnoses. Thus, both preventive and therapeutic interventions that target older patients should be evaluated on their ability to prevent or delay physical and cognitive impairment with aging. Through a series of cross-sectional and longitudinal analyses we have demonstrated a strong association of vitamin D deficiency with lower extremity performance, pulmonary function, muscle strength, pain, mobility disability, transition to physical frailty, cognitive decline, dementia, and cardiovascular and all-cause mortality. The mechanisms for these associations are not always fully understood. Because of multifaceted associations with conditions that span across multiple physiological systems and pertain to factors important for quality of life, vitamin D supplementation may be the ideal intervention to slow down the development of major aging phenotypes.
 

Keynote Address
Vitamin D Dietary Reference Intakes: Navigating the Recommendations
Daniel D. Bikle, MD, PhD, University of California San Francisco and VA Medical Center

Recommendations for the appropriate amount of vitamin D to be taken for optimal health, be it for musculoskeletal strength or for the numerous non skeletal presumptive benefits of vitamin D, remain contentious. There is general consensus that the earlier recommendation of 400IU (10µg) of vitamin D per day was insufficient. Recently, an Institute of Medicine (IOM) expert panel to formulate new guidelines has recommended that 600IU per day would suffice for the general public aged 1–70 years with the dose increased to 800IU per day for those older than 70 years. However, doses up to 4000IU per day were considered safe. The task force compiling the Endocrine Society Clinical Practice Guidelines (ESCPG) reached similar conclusions. However, vitamin D status is best assessed by the circulating levels of the vitamin D metabolite 25 hydroxyvitamin D (25OHD). Individuals vary widely as to the serum level of 25OHD that is achieved with a given dose of oral vitamin D and it is the serum level that counts with respect to biologic effect. On this point these two major guidelines differ. The IOM panel concluded that 20ng/ml (50nM) 25OHD is sufficient for normal musculoskeletal strength/function, that 97.5% of the American public was above that level, and that higher levels were not beneficial and might even be detrimental. In contrast, the ESCPG recommend a higher optimal level, namely 30ng/ml (75mM), and point out that many individuals with a variety of conditions including obesity, dark skin, malabsorption, renal failure, lactose intolerance, ingestion of certain drugs, and the institutionalized are likely not to achieve adequate 25OHD levels ingesting only 600IU per day. If 30ng/ml is deemed the optimal level (and neither set of recommendations considers this level unsafe), then the percent of Americans that maintain this level falls well below 50% and across the developing world, the percentage of vitamin D-deficient individuals is much higher. Moreover, 25OHD assays have not proved consistent, making decisions regarding supplementation all that more difficult.