Vitamin D effects on sphingosine 1-phosphate signaling and metabolism in monocytes from type 2 diabetes patients and controls.

Elevated sphingosine 1-phopshate (S1P) concentration was observed in type 2 diabetes mellitus (T2D). On the other side, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) can influence the formation of sphingosine 1-phopshate (S1P) and the expression of S1P receptors, which are known to be involved in T2D. In order to evaluate mechanisms for the antiinflammatory potential of 1,25(OH)2D3, we investigated whether 1,25(OH)2D3 alters S1P signaling and metabolism in human CD14+ monocytes. Primary monocytes isolated from healthy controls (HC) and T2D patients were treated for 24 h with 10 nM 1,25(OH)2D3 in the absence or presence of 500 IU/ml interleukin-(IL)-1ß. Thereafter, sphingosine kinase (SPHK)1, SPHK2 and S1P receptor 1-5 (S1P1-5) mRNA expression levels were measured by TaqMan™ analyses. Sphingolipid levels in cell supernatant were determined by high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). 1,25(OH)2D3 treatment downregulated S1P1 and S1P2 mRNA expression compared to untreated monocytes of HC and T2D patients. In contrast, SPHK1, S1P3 and S1P4 mRNA expression levels were upregulated by 1,25(OH)2D3 treatment compared to the respective controls. Furthermore, reduced S1P2 and increased S1P3 and S1P4 mRNA expression levels upon treatment with 1,25(OH)2D3 occurred in the presence of IL-1ß. Additionally, S1P levels in cell supernatants were decreased in monocytes from HC and T2D patients by 1,25(OH)2D3 with or without IL-1ß costimulation. The levels of sphingosine in cell supernatants were not influenced by 1,25(OH)2D3. Overall, our results demonstrate for the first time that 1,25(OH)2D3 treatment can influence S1P receptor and SPHK expression and S1P levels in primary monocytes of both HC and subjects with T2D. These findings justify further investigations into the sphingolipid metabolism and potential benefits of vitamin D treatment in diabetes.

5-Lipoxygenase (ALOX5): Genetic susceptibility to type 2 diabetes and vitamin D effects on monocytes.

The arachidonate 5-lipoxygenase (ALOX5) pathway has been implicated in chronic inflammatory disease which may be influenced by vitamin D due to vitamin D response elements (VDRE). We investigated an ALOX5 polymorphism (rs4987105) in patients with type 2 diabetes (T2D) and the in vitro effects of calcitriol (1,25(OH)2D3) on ALOX5 metabolism in monocytes of T2D patients and healthy controls (HC). 533 T2D and 473 HC were genotyped for the rs4987105 polymorphism. In addition, the 25(OH)D3 and 1,25(OH)2D3 plasma levels were measured in both cohorts. Further C-reactive protein (CRP) was determined in T2D patients. Our results demonstrate, that genotype CC and the allele C of ALOX5 rs4987105 polymorphism were more frequent in T2D compared to HC (OR = 1.44; 95% CI: 1.12-1.84; p < 0.05). Lower levels of both vitamin D metabolites (p < 0.0001 respectively) were found in the CC genotyped T2D patients compared to CC genotyped HC. In addition, CC genotyped T2D patients had higher levels of CRP compared to CT and TT genotyped T2D patients, (p < 0.01). In order to evaluate the impact of calcitriol in primary isolated monocytes, we isolated monocytes of 20 T2D patients and 20 HC. The cells were treated with 1,25(OH)2D3 and interleukin-1beta (IL-1ß) for 24 h. The following genes were analysed for expression changes: ALOX5, leukotriene A4 hydrolase (LTA4H), leukotriene B4 receptor type 1 (LTB4R1) and CD14. Treatment with IL-1ß+1,25(OH)2D3 increased ALOX5, LTA4H and LTB4R1 and CD14 mRNA in both T2D patients and HC (p < 0.0001, respectively). In addition, IL-1ß+1,25(OH)2D3 treatment led to higher ALOX5, LTA4H and CD14 mRNA levels in T2D patients compared to HC (p < 0.001, p < 0.05, p ≤ 0.05, respectively). In conclusion, ALOX5 rs4987105 allele C confers susceptibility to T2D, lower vitamin D metabolites and higher CRP levels complement this association. Additionally, IL-1ß+1,25(OH)2D3 treatment on, ALOX5, LTA4H and CD14 mRNA indicate a diabetes specific modulation. These findings identify a novel pathway in T2D potentially amenable for individualized therapeutic targeting.