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

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. 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. 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. 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.

Calton EK ，Keane KN ，Soares MJ ，Rowlands J ，Newsholme P ... -  《Redox Biology》

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

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. 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. 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. 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.

Calton EK ，Keane KN ，Raizel R ，Rowlands J ，Soares MJ ，Newsholme P ... -  《-》

The impact of cryopreservation on human peripheral blood leucocyte bioenergetics.

Keane KN ，Calton EK ，Cruzat VF ，Soares MJ ，Newsholme P ... -  《-》

Serum 25-hydroxyvitamin D concentrations are associated with nuclear factor kappa-B activity but not with inflammatory markers in healthy normoglycemic adults.

Vitamin D has been reported to have anti-inflammatory properties in in vitro and animal studies, which are thought to occur via inhibition of the nuclear factor kappa-B (NFκB) pathway. However, the association between vitamin D and in vivo NFκB activity in humans has not previously been reported. The aim of the present study was to examine the associations between circulating 25-hydroxyvitamin D (25(OH)D) concentrations and NFκB activity in peripheral blood mononuclear cells (PBMCs) as well as plasma inflammatory markers in healthy individuals. We hypothesized that 25(OH)D concentrations would be negatively associated with NFκB activity and pro-inflammatory markers downstream of NFκB, and positively associated with anti-inflammatory markers. We measured circulating 25(OH)D (chemiluminescent immunoassay); anthropometry: body mass index (BMI), waist-to-hip ratio (WHR), and % body fat (dual X-ray absorptiometry); plasma pro- and anti-inflammatory markers: high sensitivity C-reactive protein (hsCRP), tumor necrosis factor (TNF), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and IL-10 (ELISA); and NFκB activity in PBMCs (DNA-binding assay). Forty-nine participants were included in the study (21M/28F; age=31.6±10.2years (mean±SD); BMI=28.4±4.6kg/m2; % body fat=30.2±9.3%). Mean 25(OH)D concentration was 48.2±24.5 nmol/l. There were no differences in 25(OH)D concentrations between genders and no association between 25(OH)D concentrations and age, BMI, or % body fat (all p>0.1). Serum 25(OH)D concentrations were positively associated with NFκB activity in PBMCs (r=0.48, p=0.0008) but not with any of the pro- or anti-inflammatory markers measured (all p>0.1). After adjusting for age, sex, and % body fat, 25(OH)D concentrations remained positively associated with NFκB activity in PBMCs (β=0.55, p<0.0001). Although in-vitro studies suggest that vitamin D inhibits NFκB activity, our novel cross-sectional data from a cohort of healthy individuals suggest that vitamin D may regulate rather than inhibit the NFκB pathway. Large-scale intervention and mechanistic studies are needed to further investigate the effects of vitamin D on NFκB activity in vivo in humans.

Mousa A ，de Courten MPJ ，Forbes J ，de Courten B ... -  《-》

Metabolic Changes in Peripheral Blood Mononuclear Cells Isolated From Patients With End Stage Renal Disease.

As numerous complex pathologies stem from cellular energy dysfunction, we aimed to elucidate mitochondrial function and associated stress pathologies in kidney disease in a cohort of hemodialysis patients with end-stage kidney disease (ESKD). The bioenergetics study was conducted using peripheral blood mononuclear cells (PBMCs) of ESKD patients (n = 29) and healthy controls (no ESKD, n = 10). PBMCs were isolated from whole blood and seeded into assay plates to detect changes in oxidative phosphorylation and glycolysis. The bioenergetics analysis (i.e., mitochondrial stress test) was performed using Seahorse XFe24 flux analyzer. We observed significant reduction in mitochondrial respiration in patient PBMCs in terms of fundamental bioenergetics parameters such as basal respiration, ATP turnover, maximal respiration and spare respiratory capacity. These findings were correlated with the expression levels of proteins coordinating cellular energy status and regulating mitochondrial dynamics. Our data demonstrates an association between mitochondrial oxygen consumption of PBMCs and ESKD. AMPK activity, its downstream effector PGC-1α and mitochondrial fission/fusion proteins are partially responsible for the decrease in oxidative phosphorylation of PBMCs isolated from ESKD patients. We propose a link between mitochondrial dysfunction and ESKD and a role for mitochondria as a potential site for therapeutic interventions.

Altintas MM ，DiBartolo S ，Tadros L ，Samelko B ，Wasse H ... -  《Frontiers in Endocrinology》