Molecular actions of vitamin D in reproductive cell biology

Vitamin D (VitD) is an important secosteroid and has attracted attention in several areas of research due to common VitD deficiency in the population, and its potential to regulate molecular pathways related to chronic and inflammatory diseases. VitD metabolites and the VitD receptor (VDR) influence many tissues including those of the reproductive system. VDR expression has been demonstrated in various cell types of the male reproductive tract, including spermatozoa and germ cells, and in female reproductive tissues including the ovaries, placenta and endometrium. However, the molecular role of VitD signalling and metabolism in reproductive function have not been fully established. Consequently, the aim of this work is to review current metabolic and molecular aspects of the VitD­VDR axis in reproductive medicine and to propose the direction of future research. Specifically, the influence of VitD on sperm motility, calcium handling, capacitation, acrosin reaction and lipid metabolism is examined. In addition, we will also discuss the effect of VitD on sex hormone secretion and receptor expression in primary granulosa cells, along with the impact on cytokine production in trophoblast cells. The review concludes with a discussion of the recent developments in VitD­VDR signalling specifically related to altered cellular bioenergetics, which is an emerging concept in the field of reproductive medicine.

The potential role of irisin in the thermoregulatory responses to mild cold exposure in adults.

OBJECTIVES: To determine the acute effect of a mild cold exposure on thermoregulatory thermogenesis and the role of circulating irisin in the process. METHODS: We studied 22 adults (9 males, 13 females) aged 57.7 ± 10.07 years and body mass index 27.8 ± 4.52 kg/m(2) . Participants experienced a 90-min exposure to 20°C and 25°C in a randomized cross-over design. Resting metabolic rate (RMR), forearm to finger-tip gradient (FFG), blood pressure (BP), in-the-ear temperature (IET), and fasting bloods were measured on each occasion. RESULTS: There were significant increases in FFG [mean ± SD: +3.8 ± 3.0°C, P < 0.001], systolic blood pressure (SBP) [+8 ± 13 mm Hg, P = 0.015], and diastolic blood pressure (DBP) [+4 ± 6 mm Hg, P = 0.005] and decreases in IET [-0.24 ± 0.29°C, P = 0.001]. Overall, RMR [+190 ± 570 kJ/d, P = 0.135], irisin, glucose or insulin did not differ between temperatures. There were no significant between-gender differences, but males significantly increased SBP (+12 ± 16, P = 0.02) and DBP (+6 ± 7, P = 0.02) with decreases in heart rate (-4 ± 3, P = 0.002), while females did not. Moreover men had approximately 50% higher thermogenic response while women had approximately 25% greater vasoconstrictor response. Adjusted for age, gender, insulin sensitivity, and body composition, fold changes in irisin were inversely related to respiratory quotient (r= -0.54, P = 0.048), while IET was related to FFG (r= -0.55, P = 0.043). CONCLUSIONS: Mild cold exposure increased vasoconstriction with a drop in IET and these were related. Greater irisin was related to a greater fasting fat oxidation in the absence of shivering. A potential gender bias in thermoregulation was noted. Am. J. Hum. Biol. 28:699-704, 2016. © 2016 Wiley Periodicals, Inc.

The potential regulatory role of vitamin D in the bioenergetics of inflammation.

PURPOSE OF REVIEW: The extraskeletal health benefits of vitamin D still need scientific endorsement. Obesity and related chronic diseases are pathogenically linked by inflammation, which carries a considerable energetic cost. Recent techniques for the determination of the bioenergetic demand of inflammation, offer an avenue to cement the regulatory role of vitamin D in this process. RECENT FINDINGS: Nuclear vitamin D receptors may be translocated into mitochondria of certain cell types, opening up a pathway for direct action on cellular bioenergetics. Classical M1 (inflammatory)/M2(anti-inflammatory) phenotypes can vary with the clinical context. M2 macrophages do not always depend on oxidative metabolism/fatty acid oxidation. Newer methodologies offer real-time bioenergetic measurements that can be used as an index of metabolic health. SUMMARY: Vitamin D may prove to be a therapeutic agent for inflammation of chronic disease and understanding its role in cellular bioenergetics may offer a diagnostic/prognostic indicator of its action.

The impact of cryopreservation on human peripheral blood leucocyte bioenergetics.

Circulating immune cells are considered a source for biomarkers in health and disease, since they are exposed to nutritional, metabolic and immunological stimuli in the vasculature. Cryopreservation of leucocytes is routinely used for long-term storage and determination of phenotypic/functional changes at a later date. Exploring the role of bioenergetics and mitochondrial (dys)function in leucocytes is often examined by using freshly isolated cells. The aim of the pilot study described herein was to assess leucocyte bioenergetics in cryopreserved cells. Leucocytes were isolated from whole blood, counted and frozen in liquid nitrogen (LN2) for a period of 3 months. Cells were thawed at regular intervals and bioenergetic analysis performed using the Seahorse XFe96 flux analyser. Cryogenic storage reduced cell viability by 20%, but cell bioenergetic responses were largely intact for up to 1 month storage in LN2. However, after 1 month storage, mitochondrial function was impaired as reflected by decreasing basal respiration, ATP production, maximum (MAX) respiration, reserve capacity and coupling efficiency. Conversely, glycolytic activity was increased after 1 month, most notably the enhanced glycolytic response to 25 mM glucose without any change in glycolytic capacity. Finally, calculation of bioenergetic health index (BHI) demonstrated that this potential diagnostic parameter was sensitive to cryopreservation. The present study has demonstrated for the first time that cryopreservation of primary immune cells modified their metabolism in a time-dependent fashion, indicated by attenuated aerobic respiration and enhanced glycolytic activity. Taken together, we recommend caution in the interpretation of bioenergetic responses or BHI in cryopreserved samples.

Calcium and vitamin D in the regulation of energy balance: where do we stand?

There is a pandemic of obesity and associated chronic diseases. Dietary calcium and vitamin D have many extra-skeletal roles in human health. In this review we have summarized the current understanding of their influence on human energy balance by examining the epidemiological, clinical, animal, cellular and molecular evidence. We opine that while calcium and vitamin D are functional nutrients in the battle against obesity, there is a need for prospective human trials to tilt the balance of evidence in favour of these nutrients.

The impact of leucine supplementation on body composition and glucose tolerance following energy restriction: an 8-week RCT in adults at risk of the metabolic syndrome.

BACKGROUND: L-Leucine (Leu) supplementation may benefit fat-free mass (FFM) per se and glucose metabolism. OBJECTIVES: To determine whether Leu supplementation during energy restriction blunted the loss of FFM, enhanced the loss of fat mass (FM) and improved glucose tolerance. DESIGN: Thirty-seven adults, aged 20-65 years, with increased waist circumference and at least one other metabolic syndrome (MetS) component, were selected. We employed a two-arm parallel, double blind, randomized control trial (RCT) design. Participants were randomly assigned to an intervention group (leucine - 3 g/d) or placebo (lactose - 2.67 g/d), while following an individualised energy restricted diet for an 8-week period. Detailed body composition (DEXA), oral glucose tolerance test (OGTT), insulin and components of MetS were measured before and after the trial. Analysis of covariance (ANCOVA) assessed the effect of Leu on an intention-to-treat (ITT) principle. Bootstrapping method with 1000 bootstrap samples was used to derive parameter estimates, standard errors, p-values, and 95% confidence intervals for all outcomes. RESULTS: Adjusted for baseline values and other covariates, FFM (p = 0.045) and lean tissue mass (LTM) (p = 0.050) were significantly higher following Leu. These outcomes were modified by a significant treatment x sex interaction that indicated Leu had the greater effect in men. However, on adjustment for body composition changes, there was no difference in insulin sensitivity, oral glucose tolerance, or change in MetS components following Leu. CONCLUSION: Short-term leucine supplementation during energy restriction resulted in a greater preservation of FFM and LTM particularly in men, but did not impact glucose metabolism.

Hypothesized pathways for the association of vitamin D status and insulin sensitivity with resting energy expenditure: a cross sectional mediation analysis in Australian adults of European ancestry.

BACKGROUND: The role of vitamin D in human energy expenditure requires confirmation. We explored whether insulin sensitivity (IS)/insulin resistance (IR) mediated the association of vitamin D status (25OHD) on resting energy expenditure (REE). METHODS: REE, body composition (by DEXA) and clinical biochemistry of 155 Australian men and women were collated. A hypothesized mediation pathway through IS/IR on the direct association between 25OHD and REE was modeled, using three surrogate indices of IS/IR: McAuley's insulin sensitivity index (McA), Quantitative insulin sensitivity check index (QUICKI) and triglyceride to glucose ratio (TYG). The modeling was performed on PROCESS SPSS Macro (version 4.0) based on 5000 bootstrapped samples, with and without the adjustment for covariates. RESULTS: Unadjusted models indicated a sizeable negative mediation by all IS/IR indices but no significant direct effect of 25OHD on REE. On adjustment for covariates, a negative indirect mediation effect of McA [ß coefficient (SE) -2.1(0.821); bootstrapped 95% CI:-3.934, -0.703; p < 0.05] and a similar negative mediation of TYG [-1.935 (0.780); bootstrapped 95% CI: (-3.679, -0.622; p < 0.05] was observed. These models also showed a positive direct effect of 25OHD on REE. In contrast, QUICKI made a smaller contribution to the total effect though in the same direction as the other two measures [-0.783 (0.534); bootstrapped 95% CI: (-1.939, 0.134; P > 0.05]. CONCLUSIONS: A sizeable, partial, negative mediation of IS/IR on the direct relationship between 25OHD and REE, dampened the total effect of vitamin D on REE. Validation of the proposed causal framework would clarify vitamin D's role in human energy metabolism.

Triglycerides and systolic blood pressure negatively mediate the direct relationship of vitamin D status to resting energy expenditure: A cross sectional analysis.

BACKGROUND AND AIMS: We determined whether individual components of metabolic syndrome (MetS) mediated the direct association of vitamin D status (25OHD) on resting energy expenditure (REE). METHODS: Multiple linear regression determined predictors of REE from data on 180 men and women from two ethnic groups. We then modelled a mediation pathway through components of MetS on the direct association between 25OHD and REE. The mediation modelling used the PROCESS SPSS Macro (version 4.0) based on 5000 bootstrapped samples, with the adjustment for different sets of covariates. RESULTS: REE was significantly predicted by age, fat mass (FM), fat free mass (FFM), ethnicity, inverse ln insulin, 25OHD, triglycerides (TG), systolic blood pressure (SBP) and, to some extent, by time of REE measurements (p < 0.094). Adjustment for all these covariates, resulted in a negative indirect mediation effect of TG [ß coefficient (bootstrapped SE): 0.95 (0.519); bootstrapped 95% CI: 2.172, -0.165; p < 0.05] and a concurrent negative mediation of SBP [ß coefficient (bootstrapped SE): 0.72(0.484); bootstrapped 95% CI: 1.851, -0.011; p < 0.05]. There remained a positive direct pathway from 25OHD to REE [ß coefficient (S.E): 4.715 (2.129); p = 0.028], however the total effect of 25OHD was dampened [ß coefficient (S.E): 3.04 (2.126); p = 0.154]. CONCLUSIONS: Independent of insulin sensitivity, a negative mediation by TG and SBP dampened the overall effect of 25OHD on REE.

Winter to summer change in vitamin D status reduces systemic inflammation and bioenergetic activity of human peripheral blood mononuclear cells.

BACKGROUND: Vitamin D status [25(OH)D] has recently been reported to be associated with altered cellular bioenergetic profiles of peripheral blood mononuclear cells (PBMCs). No study has tracked the seasonal variation of 25(OH)D and its putative influence on whole body energy metabolism, cellular bioenergetic profiles, inflammatory markers and clinical chemistry. MATERIAL AND METHODS: Whole body energy metabolism and substrate utilisation were measured by indirect calorimetry. PBMCs obtained from the same subjects were isolated from whole blood, counted and freshly seeded. Bioenergetic analysis (mitochondrial stress test and glycolysis stress test) was performed using the Seahorse XFe96 flux analyser. 25(OH)D was assessed using the Architect immunoassay method. RESULTS: 25(OH)D increased by a median (IQR) of 14.40 (20.13)nmol/L (p<0.001) from winter to summer and was accompanied by significant improvements in indices of insulin sensitivity, McAuley's index (p=0.019) and quantitative insulin sensitivity check index (p=0.028). PBMC mitochondrial parameters basal respiration, non-mitochondrial respiration, ATP production, proton leak, and maximal respiration decreased in summer compared to winter. Similarly, PBMC glycolytic parameters glycolytic activity, glucose response, and glycolytic capacity were all reduced in summer compared to winter. There was also a trend for absolute resting metabolic rate (RMR) to decrease (p=0.066). Markers of systemic inflammation MCP-1, IL-6, IL-8, IL-10, and IL-12p70 decreased significantly in summer compared to winter. Participants who entered winter with a low 25(OH)D (<50nmol/L), had the greatest alteration in bioenergetic parameters in summer, relative to those with winter 25(OH)D concentrations of 50-75nmol/L or >75nmol/L. The absolute change in 25(OH)D was not associated with altered bioenergetics. CONCLUSION: Seasonal improvements in 25(OH)D was associated with reduced systemic inflammation, PBMC bioenergetic profiles and whole body energy metabolism. These observational changes in PBMC bioenergetics were most pronounced in those who had insufficient 25(OH)D in winter. The data warrants confirmation through cause and effect study designs.

Calcium and Vitamin D in Obesity and Related Chronic Disease.

There is a pandemic of lifestyle-related diseases. In both developed and lesser developed countries of the world, an inadequacy of calcium intake and low vitamin D status is common. In this chapter, we explore a mechanistic framework that links calcium and vitamin D status to chronic conditions including obesity, systemic inflammation, endothelial dysfunction, dyslipidemia and cardiovascular disease, and type 2 diabetes mellitus. We also update the available clinical evidence, mainly from randomized controlled trials, to provide a synthesis of evidence in favor or against these hypotheses. There is consistent data to support calcium increasing whole body fat oxidation and increasing fecal fat excretion, while there is good cellular evidence for vitamin D reducing inflammation. Clinical trials support a marginal reduction in circulating lipids and some meta-analysis support an increase in insulin sensitivity following vitamin D. However, these mechanistic pathways and intermediate biomarkers of disease do not consistently transcribe into measurable health outcomes. Cementing the benefits of calcium and vitamin D for extraskeletal health needs a reexamination of the target 25(OH)D level to be achieved and the minimum duration of future trials.

Prevailing vitamin D status influences mitochondrial and glycolytic bioenergetics in peripheral blood mononuclear cells obtained from adults.

BACKGROUND: Circulating peripheral blood mononuclear cells (PBMCs) are exposed to metabolic and immunological stimuli that influence their functionality. We hypothesized that prevailing vitamin D status [25(OH)D] would modulate the bioenergetic profile of PBMCs derived from humans. MATERIALS AND METHODS: 38 participants (16 males, 22 females) ranging in body fat from 14-51% were studied. PBMCs were isolated from whole blood, counted and freshly seeded for bioenergetic analysis using the Seahorse XFe96 flux analyser. Whole body energy metabolism via indirect calorimetry, body composition by dual-energy X-ray absorptiometry, and relevant clinical biochemistry were measured. Data was analysed based on 25(OH)D cut-offs of <50nmol/L (Group 1, n=12), 50-75nmol/L (Group 2, n=15) and ≥75nmol/L (Group 3, n=11). A multivariate general linear model adjusting for age, fat mass, fat-free mass, parathyroid hormone and insulin sensitivity was used. RESULTS: There were significant differences in cellular mitochondrial function between groups. Group 1 had significantly higher basal respiration (p=0.001), non-mitochondrial respiration (p=0.009), ATP production (p=0.001), proton leak (p=0.018), background glycolysis (p=0.023) and glycolytic reserve (p=0.039) relative to either Group 2 or Group 3; the latter two did not differ on any measures. There were no differences in bioenergetic health index (BHI), resting metabolic rates and systemic inflammatory markers between groups. CONCLUSIONS: Inadequate vitamin D status adversely influenced bioenergetic parameters of PBMCs obtained from adults, in a pattern consistent with increased oxidative metabolism and activation of these cells.

The Impact of Vitamin D Levels on Inflammatory Status: A Systematic Review of Immune Cell Studies.

Chronic low-grade inflammation accompanies obesity and its related chronic conditions. Both peripheral blood mononuclear cells (PBMCs) and cell lines have been used to study whether vitamin D has immune modulating effects; however, to date a detailed systematic review describing the published evidence has not been completed. We therefore conducted a systematic review on the effect of vitamin D on the protein expression and secretion of inflammatory markers by human-derived immune cells. The review was registered at the International Prospective Register for Systematic Reviews (PROSPERO, Registration number CRD42015023222). A literature search was conducted using Pubmed, Science Direct, Scopus, Web of Science and Medline. The search strategy used the following search terms: Vitamin D or cholecalciferol or 1,25-dihydroxyvitamin or 25-hydroxy-Vitamin D and Inflam* or cytokine* and supplement* or cell*. These terms were searched in the abstract, title and keywords. Inclusion criteria for study selection consisted of human-derived immune cell lines or cellular studies where PBMCs were obtained from humans, reported in the English language, and within the time period of 2000 to 2015. The selection protocol was mapped according to PRISMA guidelines. Twenty three studies (7 cell line and 16 PBMCs studies) met our criteria. All studies selected except one used the active metabolite 1,25(OH)2, with one study using cholecalciferol and two studies also using 25(OH)D. Four out of seven cell line studies showed an anti-inflammatory effect where suppression of key markers such as macrophage chemotactic protein 1, interleukin 6 and interleukin 8 were observed. Fourteen of sixteen PBMC studies also showed a similar anti-inflammatory effect based on common inflammatory endpoints. Mechanisms for such effects included decreased protein expression of toll-like receptor-2 and toll-like receptor-4; lower levels of phosphorylated p38 and p42/42; reduced expression of phosphorylated signal transducer and activator of transcription 5 and decreased reactive oxygen species. This review demonstrates that an anti-inflammatory effect of vitamin D is a consistent observation in studies of cell lines and human derived PBMCs.

Certain dietary patterns are beneficial for the metabolic syndrome: reviewing the evidence.

The metabolic syndrome (MetS) is a global public health issue of increasing magnitude. The Asia-Pacific region is expected to be hardest hit due to large population numbers, rising obesity, and insulin resistance (IR). This review assessed the protective effects of dietary patterns and their components on MetS. A literature search was conducted using prominent electronic databases and search terms that included in combination: diet, dietary components, dietary patterns, and metabolic syndrome. Articles were restricted to prospective studies and high quality randomized controlled trials that were conducted on humans, reported in the English language, and within the time period of 2000 to 2012. Traditional factors such as age, gender, physical activity, and obesity were associated with risk of MetS; however, these potential confounders were not always accounted for in study outcomes. Three dietary patterns emerged from the review; a Mediterranean dietary pattern, dietary approaches to stop hypertension diet, and the Nordic Diet. Potential contributors to their beneficial effects on prevalence of MetS or reduction in MetS components included increases in fruits, vegetables, whole grains, dairy and dairy components, calcium, vitamin D, and whey protein, as well as monounsaturated fatty acids, and omega-3 fatty acids. Additional prospective and high quality randomized controlled trial studies that investigate Mediterranean dietary pattern, the dietary approaches to stop hypertension diet, and the Nordic Diet would cement the protective benefits of these diets against the MetS.