To Be or Not to Be: The Divergent Action and Metabolism of Sphingosine-1 Phosphate in Pancreatic Beta-Cells in Response to Cytokines and Fatty Acids

Abstract

Sphingosine-1 phosphate (S1P) is a bioactive sphingolipid with multiple functions conveyed by the activation of cell surface receptors and/or intracellular mediators. A growing body of evidence indicates its important role in pancreatic insulin-secreting beta-cells that are necessary for maintenance of glucose homeostasis. The dysfunction and/or death of beta-cells lead to diabetes development. Diabetes is a serious public health burden with incidence growing rapidly in recent decades. The two major types of diabetes are the autoimmune-mediated type 1 diabetes (T1DM) and the metabolic stress-related type 2 diabetes (T2DM). Despite many differences in the development, both types of diabetes are characterized by chronic hyperglycemia and inflammation. The inflammatory component of diabetes remains under-characterized. Recent years have brought new insights into the possible mechanism involved in the increased inflammatory response, suggesting that environmental factors such as a westernized diet may participate in this process. Dietary lipids, particularly palmitate, are substrates for the biosynthesis of bioactive sphingolipids. Disturbed serum sphingolipid profiles were observed in both T1DM and T2DM patients. Many polymorphisms were identified in genes encoding enzymes of the sphingolipid pathway, including sphingosine kinase 2 (SK2), the S1P generating enzyme which is highly expressed in beta-cells. Proinflammatory cytokines and free fatty acids have been shown to modulate the expression and activity of S1P-generating and S1P-catabolizing enzymes. In this review, the similarities and differences in the action of extracellular and intracellular S1P in beta-cells exposed to cytokines or free fatty acids will be identified and the outlook for future research will be discussed.

Keywords: beta-cells; cytokines; free fatty acids; inflammation; lipotoxicity; sphingolipids; sphingosine-1 phosphate; type 1 diabetes; type 2 diabetes.

Figure 1

Gene and protein expression of sphingosine kinases 1 and 2 in rat INS1E and human EndoC-βH1 beta-cells. Total RNA was extracted from untreated rat INS1E (a kind gift of Prof.C Wollheim, Geneva, Switzerland) and human EndoC-βH1 beta-cells (ENDOCELLS SARL, Paris, France) (RNeasy Kit, Qiagen, Hilden, Germany). Cells were cultured in a humidified atmosphere at 37 °C and 5% CO₂ as described [31], and were free from mycoplasma contamination.The quality of RNA was verified by agarose gel electrophoresis. RNA was quantified spectrophotometrically at 260/280 nm. Thereafter, 2 µg of RNA were reverse transcribed into cDNA using a random hexamer primer (Thermo Fisher Scientific, Braunschweig, Germany) and RevertAid H Minus M-MuLV reverse transcriptase (Thermo Fisher Scientific). QuantiTect SYBR Green^TM technology (Qiagen) was employed. The reactions were performed using rat (rSK1 fw-CTTCTGGAGGAGGCTGAGGT, rev- TCAGACCGTCACCGGACAT; rSK2 fw-CAAGCCCTACACATACAGCG, rev-GCCACGTGGGTAGGTGTAGA, rActin-b fw-GAACACGGCATTGTAACCAACTGG, rev-GGCCACACGCAGCTCATTGTA) and human (huSK1 fw-TGGGACGCTCTGGTGGTCATGT, rev-TACACAGGGGCTTCTGGATGGC, huSK2 fw-TGCTCCATGAGGTGCTGAACGG, rev-AATCCCCCGTGCTGGTTCACTG, huActin-b fw-ATGGATGATGATATCGCCGC, rev-TTCTGACCCATGCCCACCA) specific primers (Microsynth, Balgach, Switzerland) on a ViiA7 real-time PCR system (Thermo Fisher Scientific) with the following protocol: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles comprising a melting step at 95 °C for 15 s, an annealing step at 62 °C for 60 s and extension step at 72 °C for 30 s. The quality of reactions was controlled by analysis of melting curves. Each sample was amplified in triplicate. Data normalization was performed against the housekeeping gene β-actin. Statistical analysis was performed using t-test, *** p < 0.001.Total cell protein was collected in ice-cold PBS containing a cocktail of protease inhibitor (Roche, Mannheim, Germany) and followed by sonication. Protein concentration was determined by BCA assay (Thermo Fisher Scientific). Following denaturation, 50 µg of samples of rat INS1E or human EndoC-βH2 beta-cells were separated onto 12.5% gels, blotted onto nitrocellulose and blocked with 5% dry-fat milk as described [31]. Primary antibodies against SK1 sc-48825 (M209) (Santa Cruz, Heidelberg, Germany), SK2 17096-1-AP (Proteintech, Manchester, UK), beta-actin (ACTB) sc-47778 (C4) (Santa Cruz) were used at the dilution 1:500, secondary peroxidase conjugated Affini Pure IgG (H + L) at the dilution 1:2000. The hybrids were visualized using the enhanced chemiluminescence detection kit and captured by the INTAS chemiluminescence detection system (Intas Science Imaging Instruments, Göttingen, Germany).

Figure 2

Involvement of sphingosine-1 phosphate (S1P) in beta-cell failure in T1DM and T2DM. Data gained from rodent and murine beta-cells suggest that extracellular S1P (at concentrations <5 µM) predominantly protects against cytokine and FFA-induced toxicity (survival and function), though at higher concentrations (>5 µM) it seems to negatively modulate cell viability and GSIS. Short-time exposure to proinflammatory cytokines (15 min–8 h) and PA (<24 h) increase S1P generation in beta-cells. The role of intracellular S1P remains unclear. Overexpression of SK1 leading to increased intracellular S1P generated in cytoplasm and in proximity of cell membrane has been associated with protective effects against PA toxicity. However, SK1 is weakly expressed in beta-cells (rodent, murine and human) and the predominant isoform is SK2. SK2-derived S1P has been shown to be involved in lipotoxicity, but its role in cytokine toxicity has not yet been addressed. Under short exposures (24 h), SPL overexpression seems to protect against cytokine-mediated toxicity, while it accelerates PA toxicity. The high expression of SPL in human beta-cells correlates with OA toxicity.