Sphingosine-1-phosphate as a key player of insulin secretion induced by high-density lipoprotein treatment

Abstract

Beta cell failure is one of the most important features of type 2 diabetes mellitus (T2DM). High-density lipoprotein (HDL) has been proposed to improve β-cell function. However, the mechanisms involved in this process are still poorly understood. The aim of this study was to investigate the contribution of sphingosine-1-phosphate (S1P) in the impact of HDL treatment on insulin secretion by pancreatic β-cells and to determine its mechanisms. Primary cultures of β-cells isolated from rat were treated with or without HDL in the presence or absence of S1P pathway inhibitors and insulin secretion response was analyzed. The S1P content of HDL (HDL-S1P) isolated from T2DM patients was analyzed and correlated to the HDL-induced insulin secretion. The expression of genes involved in the biosynthesis of the insulin was also evaluated. HDL as well as S1P treatment enhanced glucose-stimulated insulin secretion (GSIS). In HDL isolated from T2DM patients, while HDL-S1P was strongly correlated to its pro-secretory capacity (r = 0.633, p = 0.005), HDL-cholesterol and apolipoprotein AI levels were not. HDL-induced GSIS was blocked by the S1P1/3 antagonist but not by the S1P2 antagonist, and was also accompanied by increased intracellular S1P in β-cells. We also observed that HDL improved GSIS without significant changes in expression levels of insulin biosynthesis genes. Our present study highlights the importance HDL-S1P in GSIS in T2DM patients and demonstrates that HDL induces insulin secretion by a process involving both intra- and extra-cellular sources of S1P independently of an effect on insulin biosynthesis genes.

Keywords: glucose-stimulated insulin secretion; high-density lipoproteins; primary pancreatic beta cells; sphingosine-1-phosphate; type 2 diabetes mellitus.

FIGURE 1

HDL and S1P treatment improves insulin secretion. HDL 400 µg/ml treatment for 24 h improved GSIS (n = 8) (a). S1P 400 nM treatment for 24 h improved GSIS (n = 4) (b). Data are mean ± SEM, statistical p value was calculated using a two‐way ANOVA with Tukey's multiple comparison post‐test. 2.8 mM glucose was compared to respective 16.7 mM glucose and considered significantly different with a p value of *<0.05, **<0.01 and ****<0.0001.

FIGURE 2

HDL S1P content from diabetic patients correlates with pro‐secretory capacity Pancreatic β‐cells were incubated with HDL (400 µg/ml) from diabetic patients (n = 18) for 24 h and insulin secretion was evaluated following the GSIS protocol. The effects were correlated to the HDL S1P content. (a) Correlation between Insulin secretion and HDL‐cholesterol. (b) Correlation between Insulin secretion and apoAI. (c) Analyses used the nonparametric Spearman test.

FIGURE 3

HDL treatment improves insulin secretion via S1P 1/3. RNA expression of S1P receptors subtypes quantified by qPCR, normalized by Rps9 expression and expressed in arbitrary unit (AU). S1P3 expression is increased compared to other S1P receptor subtypes. Data are mean ± SEM, statistical p value was calculated using a one‐way ANOVA with Tukey's multiple comparison post‐test. S1P 1/3 antagonist (VPC23019, 2 µM) (b) and S1P2 antagonist (JTE013, 5 µM) (c) were incubated 1 h before treatment with HDL 400 µg/ml. Graphs represented GSIS and data are mean ± SEM, statistical p value was calculated using a two‐way ANOVA with Tukey's multiple comparison post‐test. 2.8 mM glucose (basal) was compared to respective 16.7 mM glucose (stimulated) and considered significantly different with a p value of ****<0.0001 (n = 5).

FIGURE 4

HDL treatment improves insulin secretion via Sphk2. Nonspecific Sphk inhibitor (DMS, 10 µM) (a) and Sphk1 inhibitor (SK‐II, 10 µM) (b) were incubated 30 min before treatment with or without HDL (400 µg/ml). Graphs represented GSIS and data are mean ± SEM, statistical p value was calculated using a two‐way ANOVA with Tukey's multiple comparison post‐test. 2.8 mM glucose (basal) was compared to respective 16.7 mM glucose (stimulated) and considered significantly different with a p value of * <0.05, ***<0.001 and **** <0.0001 (n = 6)

FIGURE 5

HDL transfert S1P into pancreatic β‐cells.Pancreatic β‐cellswere incubated with HDL 400 μg/ml for 24 h and S1P cellcontent was quantified by LC MS/MS (a). Data are mean ± SEM, statistical p value was calculated using Student‐T test paired (n = 4). β‐cells were treated with control medium (DMEM glucose 5.6 mmol/L, 0.5%BSA) or with control containing HDL‐S1Pd7 (400 µg/ml) (see Figure S2). After 24 h of stimulation, S1P and S1Pd7 concentrations of incubation medium (b) and cells (c) were determined by LC–MS/MS. AUC: area under the curve. Data are mean ± SEM, statistical p value was calculated using Student‐T test paired (n = 4)