Ann Pediatr Endocrinol Metab.  2021 Dec;26(4):252-258. 10.6065/apem.2040266.133.

Identification of sphingosine 1-phosphate level and MAPK/ERK signaling in pancreatic β cells

• Park JH
• Park KK
• Choe JY
• Jang KM

• KMID: 2523831
• DOI: http://doi.org/10.6065/apem.2040266.133

Abstract

Purpose
Sphingosine kinase is a lipid kinase that phosphorylates sphingosine to generate sphingosine 1-phosphate (S1P). S1P regulates pancreatic islet β-cell endoplasmic reticulum stress and proliferation. Type 1 and type 2 diabetes share some key pathogenic processes. In this study, we investigated whether secretion of insulin and production of S1P is altered in alloxan and glucose-treated cells from the rat pancreatic β-cell line RIN-5F.
Methods
RIN-5F cells were treated with 2 mM alloxan and 20 mM glucose for 6 hours or 24 hours before being evaluated by enzyme linked immunosorbent assay (ELISA) and Western blotting.
Results
Insulin secretion and expression was higher in RIN-5F cells treated with glucose compared to control cells. In contrast, alloxan treatment did not affect insulin secretion and expression in RIN-5F cells. Interestingly, compared with normal control levels, S1P/EDG-5 was increased in both alloxan and glucose-treated pancreatic β cell than normal control. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) inhibition strongly decreased the expression of insulin and S1P in glucose- or alloxan-treated RIN-5F cells.
Conclusion
We observe that production of S1P is increased in both diabetic cell models. In addition, MAPK/ERK signaling regulates secretion of insulin and S1P expression in pancreatic β-cells. Based on the literature and our findings, S1P may be a promising agent for the treatment of insulin-related disorders.

Keyword

Diabetes; ERK; Insulin; MAPK/ERK signaling; S1P; Sphingolipid metabolism

Figure

• 

  Fig. 1. Effects of different concentrations of glucose and alloxan on viability of RIN-5F cells. (A, B) CCK reduction was calculated to measure cellular proliferation. RIN-5F cells were treated with alloxan ranging from 0.5 to 4 mM and glucose ranging from 5 to 30 mM. (C) Phase-contrast photograph of change in cellular morphology of RIN-5F cells. Magnifications ×40. The data are representative of 3 independent experiments and quantified as mean±standard error of the mean (n=3–5). Kruskal-Wallis test with Dunn post hoc test, ***P<0.001 compared to normal control. CCK, Cell Count Kit; NC, normal control.

• 

  Fig. 2. Effects of insulin and S1P production on glucose and alloxan-treated RIN-5F cells. RIN-5F cells were treated with 2 mM alloxan and 20 mM glucose for 24 hours. (A) The protein extract was determined using by Western blot. β-actin was used to confirm equal sample loading control. (B) Western blotting was quantified by densitometric analysis. The data are representative of 3 independent experiments and quantified as mean±standard error of the mean (n=3–5). Kruskal-Wallis test with Dunn post hoc test, *P<0.05, ***P<0.001 versus normal control; ###P<0.001 versus alloxan treatment group. S1P/EDG-5, sphingosine 1-phosphate/endothelial differentiation, G protein-coupled receptor 5; pSPHK2, phospho-sphingosine kinase 2; tSPHK2, total-sphingosine kinase 2; NS, not significant.

• 

  Fig. 3. Alloxan and glucose-induced insulin signaling in RIN-5F cells. Phosphorylated extracellular signal-regulated kinase (pERK) (A) and phosphorylated AKT (pAKT) (B) were measured in RIN-5F cells treated with alloxan and glucose for 6 hours. β-actin was used to confirm equal sample loading control. Western blotting was quantified by densitometric analysis. The data are representative of 3 independent experiments and quantified as mean±standard error of the mean (n=3–5). Kruskal-Wallis test with Dunn post hoc test, *P<0.05, ***P<0.001 versus normal control cells; ###P<0.001 versus alloxan treatment group. NS, not significant.

• 

  Fig. 4. Inhibition of MAPK/ERK regulates alloxan and glucose-induced insulin, S1P/EDG-5 and pSPHK2 expression. RIN-5F cells were pretreated with ERK inhibitor (PD98059) for 30 min and then stimulated with alloxan and glucose for 24 hours. β-actin was used to confirm equal sample loading control. Western blotting was quantified by densitometric analysis. The data are representative of 3 independent experiments and quantified as mean±standard error of the mean (n=3–5). Kruskal-Wallis test with Dunn post hoc test, ***P<0.001 versus normal control cells; #P<0.05, ###P<0.001 versus alloxan treatment group, $$P<0.01, $$$P<0.001 versus glucose treatment group. MAPK/ERK, mitogenactivated protein kinase/extracellular signal-regulated kinase; S1P/EDG-5, sphingosine 1-phosphate/endothelial differentiation; G protein-coupled receptor 5, pSPHK2: phospho-sphingosine kinase2; NS, not significant.

Reference

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