Effects of calcitriol on the crosstalk between macrophages and human adipocytes

Macrophage-infiltration of adipocytes in obesity provides an important mechanistic link that may explain the increase in inflammatory mediators produced by adipose tissue. Elucidating the crosstalk mechanisms between macrophages and adipocytes is important as adipose tissue has been shown to be the linchpin linking inflammation and insulin resistance. Growing evidence suggests that vitamin D exerts immunomodulatory effects and adipose tissue could be a target for its action. This study investigated the role of vitamin D3 (calcitriol) in alleviating inflammation mediators expressed and released by human adipocytes under the basal and stimulated conditions. The study also examined the effect of calcitriol on monocyte migration as well as on the NF-κB and MAPK signalling pathways. A simulated model of adipocyte inflammation was created by incubating adipocytes with macrophage-conditioned (MC) medium, and this model was then used to study the effect of vitamin D. High dose calcitriol (10-8M) decreased the basal levels of MCP-1 and IL-6 released by adipocytes. Pretreatment with calcitriol (10-8M) reduced the rise in protein levels of MCP-1 and IL-6 released by adipocytes after stimulation with MC medium. Calcitriol (10-8M) also decreased IL-6 release but it had no effect on MCP-1 production by adipocytes stimulated with TNF-α. Upon MC medium stimulation, calcitriol (10-8M) partially reversed the decrease in gene expression of adiponectin and ZAG although the release of adiponectin or ZAG by adipocytes was unaffected. In addition, calcitriol (10-11M and 10-8M) supplementation decreased THP-1 macrophage migration although it did not reverse the effects of MC medium or TNF-α. Furthermore, calcitriol increased the basal protein abundance of NF-κB IκBα and reversed the inhibitory effect of MC medium on IκBα. Calcitriol supplementation also decreased both basal and MC medium stimulated protein abundance of phosphorylated NF-κB p65 as well as basal and MC medium stimulated levels of phosphorylated p38 MAPK. In conclusion, this study has shown that MC medium potently stimulated inflammatory responses in human adipocytes. Calcitriol supplementation was able to reduce the basal and stimulated production of the proinflammatory mediators by adipocytes. Calcitriol also led to a decrease in chemoattractant ability of adipocytes. In addition, calcitriol exhibited an inhibitory effect on the activation of the NF-κB and MAPK signalling pathways. These results suggest that calcitriol may have a beneficial effect in protecting against adipose tissue inflammation induced by the crosstalk between macrophages and adipocytes

Circulating anti-inflammatory adipokines High Molecular Weight Adiponectin and Zinc-α2-glycoprotein (ZAG) are inhibited in early sepsis, but increase with clinical recovery: a pilot study

BACKGROUND: Adipose tissue has been identified as an endocrine organ secreting adipokines involved in metabolic and inflammatory pathways. Adiponectin, an anti-inflammatory adipokine, is reduced in sepsis. High Molecular Weight (HMW) adiponectin, the biologically most relevant molecule, has been investigated very little in human sepsis. Zinc-alpha2-glycoprotein (ZAG) is a novel adipokine and its expression in adipose tissue is positively correlated with adiponectin expression. It is not yet known whether ZAG has a role in sepsis. In this study we assessed levels of HMW adiponectin and ZAG during different stages of sepsis. METHODS: A prospective observational pilot study was carried out on 21 septic patients. Serum samples were taken on day 1 and 2 post ICU admission and on day of discharge. Samples were analysed for total and HMW adiponectin, HMW/total adiponectin ratio, and ZAG. Results were correlated with clinical and metabolic data. RESULTS: There were no differences in total adiponectin, HMW adiponectin and ZAG plasma concentrations between day 1 (admission) and day 2 of the sepsis episode. Compared to admission, a significant increase in total and HMW adiponectin and ZAG was observed on the day of discharge when clinical improvement had been achieved. There was also an increase in the HMW/total adiponectin ratio at that time. CONCLUSIONS: Our data demonstrate an increase in both HMW adiponectin and total adiponectin in patients who had clinically recovered from sepsis. The increase in HMW/total adiponectin ratio with improvement of the clinical condition suggests that HMW adiponectin may have a greater role in the inflammatory process and insulin resistance seen in sepsis. In this pilot study, we have also demonstrated a significant increase in ZAG in critically ill patients temporally related to recovery from sepsis

Metabolic Effects Associated with ICS in Patients with COPD and Comorbid Type 2 Diabetes: A Historical Matched Cohort Study

Background Management guidelines for chronic obstructive pulmonary disease (COPD) recommend that inhaled corticosteroids (ICS) are prescribed to patients with the most severe symptoms. However, these guidelines have not been widely implemented by physicians, leading to widespread use of ICS in patients with mild-to-moderate COPD. Of particular concern is the potential risk of worsening diabetic control associated with ICS use. Here we investigate whether ICS therapy in patients with COPD and comorbid type 2 diabetes mellitus (T2DM) has a negative impact on diabetic control, and whether these negative effects are dose-dependent. Methods and Findings This was a historical matched cohort study utilising primary care medical record data from two large UK databases. We selected patients aged >= 40 years with COPD and T2DM, prescribed ICS (n = 1360) or non-ICS therapy (n = 2642) between 2008 and 2012. The primary endpoint was change in HbA(1c) between the baseline and outcome periods. After 1:1 matching, each cohort consisted of 682 patients. Over the 12-18-month outcome period, patients prescribed ICS had significantly greater increases in HbA1c values compared with those prescribed non-ICS therapies; adjusted difference 0.16% (95% confidence interval [Cl]: 0.05-0.27%) in all COPD patients, and 0.25% (95% Cl: 0.10-0.40%) in mild-to-moderate COPD patients. Patients in the ICS cohort also had significantly more diabetes-related general practice visits per year and received more frequent glucose strip prescriptions, compared with those prescribed non-ICS therapies. Patients prescribed higher cumulative doses of ICS (> 250 mg) had greater odds of increased HbA(1c) and/or receiving additional antidiabetic medication, and increased odds of being above the Quality and Outcomes Framework (QOF) target for HbA1c levels, compared with those prescribed lower cumulative doses ( Conclusion For patients with COPD and comorbid T2DM, ICS therapy may have a negative impact on diabetes control. Patients prescribed higher cumulative doses of ICS may be at greater risk of diabetes progression

Vitamin D signalling in adipose tissue

Adipose tissue inflammation is characterised by increased infiltration of macrophages and is associated with a marked increase in the synthesis and release of proinflammatory factors such as TNFα, IL-6 and MCP-1. Studies suggest that these chemokines and cytokines contribute to local tissue inflammation and levels of inflammatory mediators in the systemic circulation. The potential role of the vitamin D receptor in modulating inflammation in obesity has received increasing attention, with evidence suggesting that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunoregulatory effects. The data presented in this thesis showed adipose tissue expresses the vitamin D receptor, along with vitamin D metabolising enzymes CYP27B1 and CYP24A1. Treatment of adipocytes with 1,25(OH)2D3 reduced the secretion of key cytokines and chemokines involved in the inflammatory response induced by macrophage-secreted factors. MCP-1, IL-6, IL-8 and RANTES production by human adipocytes was significantly downregulated, and western blotting data supports that this was due to the inhibitory effects of 1,25(OH)2D3 on the NF-κB and MAPK signalling pathways. Treatment with 1,25(OH)2D3 also abolished macrophage-induced activation of NFκB, increasing IκBα expression and reducing NFκB p65 phosphorylation. Mitogen-activated protein kinase (MAPK) signalling activated by MC medium was also inhibited by 1,25(OH)2D3 , reducing phosphorylated p38 MAPK and phosphorylated Erk1/2. Investigations into its downstream effects showed 1,25(OH)2D3 decreased macrophage-induced inflammatory cytokine expression, reducing IL-8, MCP-1, RANTES and IL-6. To confirm the involvement of the vitamin D receptor in modulating the inflammatory response in adipocytes, two 1,25(OH)2D3 analogues, ZK159222 and ZK191784, were investigated for its effects on adipocyte-macrophage crosstalk. The anti-inflammatory effects of ZK159222 and ZK191784 are demonstrated for the first time in human adipocytes. ZK159222 treatment increased IκBα in adipocytes stimulated with macrophage-conditioned medium. In contrast, ZK159222 markedly reduced protein expression of phos-NF-κB p65. In studies of MAPK activation, ZK159222 dose-dependently decreased expression of phosphorylated p38 MAPK. ZK191784 treatment was observed to increase IκBα while reduce phosphorylated NF-κB p65 levels compared to vehicle controls. ZK191784 also showed an inhibitory effect on protein expression of phosphorylated p38 MAPK. In addition, both 1,25(OH)2D3 analogues reduced protein secretion of MCP-1 and IL-6 by human adipocytes. Both compounds also significantly reduced IL-8 expression compared to vehicle controls as well as 1,25(OH)2D3 treatment as well as MC-induced RANTES expression. Overall this research demonstrates that vitamin D3 has anti-inflammatory properties in human adipocytes, probably mediated by inhibition of the NFκB and MAPK signalling pathways. Reviewing the current evidence, the data suggests that targeting the vitamin D signalling system in adipose tissue presents a novel approach for modulating adipose tissue function, and in particular, ameliorating inflammation perpetuated by macrophage-adipocyte crosstalk in obesity

1,25-dihydroxyvitamin D3 protects against macrophage-induced activation of NFκB and MAPK signalling and chemokine release in human adipocytes.

Increased accumulation of macrophages in adipose tissue in obesity is linked to low-grade chronic inflammation, and associated with features of metabolic syndrome. Vitamin D3 may have immunoregulatory effects and reduce adipose tissue inflammation, although the molecular mechanisms remain to be established. This study investigated the effects of vitamin D3 on macrophage-elicited inflammatory responses in cultured human adipocytes, particularly the signalling pathways involved. Macrophage-conditioned (MC) medium (25% with adipocyte maintenance media) markedly inhibited protein expression of the nuclear factor-κB (NFκB) subunit inhibitor κBα (IκBα) (71%, P<0.001) and increased NFκB p65 (1.5-fold, P = 0.026) compared with controls. Treatment with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) abolished macrophage-induced activation of NFκB signalling by increasing IκBα expression (2.7-fold, P = 0.005) and reducing NFκB p65 phosphorylation (68%; P<0.001). The mitogen-activated protein kinase (MAPK) signalling was activated by MC medium, which was also blunted by 1,25(OH)2D3 with a downregulation of phosphorylated p38 MAPK (32%, P = 0.005) and phosphorylated Erk1/2 (49%, P = 0.001). Furthermore, MC medium (12.5% or 25%) dose-dependently upregulated secretion of key proinflammatory chemokines/cytokines (22-368-fold; all P<0.001) and this was significantly decreased by 1,25(OH)2D3: IL-8 (61% and 31%, P<0.001), MCP-1 (37%, P<0.001 and 36%, P = 0.002), RANTES (78% and 62%, P<0.001) and IL-6 (29%, P<0.001 and 34%, P = 0.019). Monocyte migration-elicited by adipocytes treated with 1,25(OH)2D3 was also reduced (up to 25%, P<0.001). In conclusion, vitamin D3 could be anti-inflammatory in adipose tissue, decreasing macrophage-induced release of chemokines and cytokines by adipocytes and the chemotaxis of monocytes. Our data suggests these effects are mediated by inhibition of the NFκB and MAPK signalling pathways

1,25-dihydroxyvitamin D₃ dose dependently decreases MC medium-induced phosphorylation of p38 MAPK in human adipocytes.

<p>Adipocytes were incubated with increasing doses of 1,25(OH)₂D₃ (10⁻¹¹ M, 10⁻¹⁰ M, 10⁻⁹ M, 10⁻⁸ M) or without (control) for 72 h, followed by stimulation with macrophage conditioned (MC) medium (25%) for another 24 h. Protein expression of phosphorylated p38 MAPK in cell lysates was analysed by western blotting, with GAPDH used as loading controls. (A) Representative western blots. (B) Signals were quantified by densitometry; data are means ± SEM, normalised to total Akt levels, n = 3 per group. ^†<i>P</i><0.05 vs 10⁻¹¹ M dose group, ***<i>P</i><0.001 vs MC group.</p

Effects of 1,25-dihydroxyvitamin D₃ on MC medium-induced secretion of the chemokines/cytokine by human adipocytes.

<p>Adipocytes were pretreated with 1,25(OH)₂D₃ (10⁻⁸ M) or without for 48 h, followed by the incubation with RPMI-1640 medium (control) or macrophage conditioned (MC) medium (12.5% or 25%) for another 24 h. A separate group (25% MC medium only without cells) was included to show basal levels of chemokine/cytokines in the MC medium. Protein release of IL-8 (A), MCP-1 (B), RANTES (C) and IL-6 (D) was determined using ELISAs in supernatants. Data are means ± SEM, n = 6 per group. ^†††<i>P</i><0.001 vs controls; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs MC group. The results were confirmed by three independent experiments.</p