Endocrinol Metab.  2018 Sep;33(3):403-412. 10.3803/EnM.2018.33.3.403.

Deficiency of Sphingosine-1-Phosphate Reduces the Expression of Prohibitin and Causes β-Cell Impairment via Mitochondrial Dysregulation

Affiliations
  • 1Institute of Medical Research, Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea.
  • 2Division of Endocrinology and Metabolism, Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea. wonyoung2.lee@samsung.com

Abstract

BACKGROUND
Emerging evidence suggests that sphingolipids may be involved in type 2 diabetes. However, the exact signaling defect through which disordered sphingolipid metabolism induces β-cell dysfunction remains unknown. The current study demonstrated that sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SphK), is an essential factor for maintaining β-cell function and survival via regulation of mitochondrial action, as mediated by prohibitin (PHB).
METHODS
We examined β-cell function and viability, as measured by mitochondrial function, in mouse insulinoma 6 (MIN6) cells in response to manipulation of cellular S1P and PHB levels.
RESULTS
Lack of S1P induced by sphingosine kinase inhibitor (SphKi) treatment caused β-cell dysfunction and apoptosis, with repression of mitochondrial function shown by decreases in cellular adenosine triphosphate content, the oxygen consumption rate, the expression of oxidative phosphorylation complexes, the mitochondrial membrane potential, and the expression of key regulators of mitochondrial dynamics (mitochondrial dynamin-like GTPase [OPA1] and mitofusin 1 [MFN1]). Supplementation of S1P led to the recovery of mitochondrial function and greatly improved β-cell function and viability. Knockdown of SphK2 using small interfering RNA induced mitochondrial dysfunction, decreased glucose-stimulated insulin secretion (GSIS), and reduced the expression of PHB, an essential regulator of mitochondrial metabolism. PHB deficiency significantly reduced GSIS and induced mitochondrial dysfunction, and co-treatment with S1P did not reverse these trends.
CONCLUSION
Altogether, these data suggest that S1P is an essential factor in the maintenance of β-cell function and survival through its regulation of mitochondrial action and PHB expression.

Keyword

Sphingosine 1-phosphate; Sphingosine kinase; Insulin-secreting cells; Mitochondria; Prohibitin

MeSH Terms

Adenosine Triphosphate
Animals
Apoptosis
GTP Phosphohydrolases
Insulin
Insulin-Secreting Cells
Insulinoma
Membrane Potential, Mitochondrial
Metabolism
Mice
Mitochondria
Mitochondrial Dynamics
Oxidative Phosphorylation
Oxygen Consumption
Phosphotransferases
Repression, Psychology
RNA, Small Interfering
Sphingolipids
Sphingosine
Adenosine Triphosphate
GTP Phosphohydrolases
Insulin
Phosphotransferases
RNA, Small Interfering
Sphingolipids
Sphingosine

Figure

  • Fig. 1 Lack of sphingosine-1-phosphate (S1P) induces β-cell dysfunction and apoptosis. Mouse insulinoma 6 (MIN6) cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) media (containing 25 mM glucose and 10% fetal bovine serum). At 50% cell confluence, the cells were incubated in serum-free media with a sphingosine kinase inhibitor (15 µM) and/or S1P (D18:1, 10 µM) for 48 hours. (A, B) Cellular levels of S1P and apoptotic proteins, including cleaved caspase-3 and phosphorylated c-Jun N-terminal kinase (p-JNK), were evaluated by an immunoblotting assay. Quantitative levels of p-JNK and cleaved caspase-3 were normalized to total JNK and caspase-3 expression. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is presented as the percent of relative absorbance compared to the vehicle (Veh) group. (D) Transcription of insulin 1 (INS1) was measured by real-time reverse-transcription polymerase chain reaction and normalized to β-actin. (E) Nuclear expression of pancreatic and duodenal homeobox 1 (Pdx1) was analyzed by an immunoblotting assay, and normalized to lamin B1. Each value represents the mean of 3 experiments. (F) The glucose-stimulated (5.5 or 25 mM) insulin secretion of MIN6 cells was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit. The values are representative of four independent experiments. t-JNK, total c-Jun N-terminal kinase; c-casp3, cleaved caspase-3; t-casp3, total caspase-3; SphKi, sphingosine kinase inhibitor. aP<0.05, bP<0.01, cP<0.001 compared with the Veh group; dP<0.05, eP<0.01 compared with the SphKi group.

  • Fig. 2 Lack of sphingosine-1-phosphate (S1P) induces mitochondrial impairment in β-cells. (A) After incubation with a sphingosine kinase inhibitor (SphKi) and/or S1P for 48 hours, mouse insulinoma 6 (MIN6) cells were incubated with tetramethylrhodamine ethyl ester (TMRE) to evaluate the mitochondrial membrane potential. After washing with phosphate-buffered saline, the intensity of red fluorescence in the TMRE-stained cells was detected, and (B) represented by quantitative analysis. Bar=20 µm. (C) Expression of mitochondrial dynamin-like GTPase (OPA1) and mitofusin 1 (Mfn1), key molecules regulating mitochondrial fusion and fission, was analyzed by an immunoblotting assay, and (D) the ratio to β-actin was determined. (E, F) Mitochondrial proteins regulating oxidative phosphorylation (OxPHOS) were detected using an OxPHOS complex antibody cocktail by an immunoblotting assay, and the ratio to β-actin was determined. (G, H) Adenosine triphosphate (ATP) levels and the oxygen consumption rate in MIN6 cells treated with SphKi and/or S1P were compared. The amount of ATP is represented as the percent of control, and the rate of oxygen consumption is described as picomoles per minute. Each value represents the mean of three experiments. Veh, vehicle; M, markers of size. aP<0.05, bP<0.01, cP<0.001 compared with the Veh group; dP<0.05, eP<0.01, fP<0.001 compared with the SphKi group.

  • Fig. 3 Knockdown of the sphingosine kinase 2 (SPHK2) gene decreases prohibitin (PHB) and induces mitochondrial impairment in β-cells. (A) PHB expression was measured by an immunoblotting assay in mouse insulinoma 6 (MIN6) cells that were incubated with a sphingosine kinase inhibitor (SphKi) and/or sphingosine-1-phosphate (S1P) for 48 hours. (B) MIN6 cells were transfected with silencing genes targeting SPHK1 or SPHK2. After incubation for 8 hours, the transcription of PHB was determined by real-time reverse-transcription polymerase chain reaction. (C, D) MIN6 cells were transfected with small interfering RNA (siRNA) for SPHK2 for 8 hours, and a portion of the cells were incubated with S1P for 48 hours. Expression of PHB was analyzed by an immunoblotting assay, and quantitative levels were represented by normalization to β-actin expression. (E) After knockdown of SPHK2 and/or S1P treatment, MIN6 cells were exposed to 100 ng/mL tetramethylrhodamine ethyl ester (TMRE) and 10 ng/mL Hoechst. Mitochondrial membrane potentials were probed by TMRE showing red fluorescence, and the nucleus was visualized by blue fluorescence. Bars=20 µm. (F, G) Adenosine triphosphate (ATP) levels and the oxygen consumption rate were analyzed in MIN6 cells after transfection of SPHK2 siRNA and/or S1P treatment. (H) Glucose-stimulated (5.5 or 25 mM) insulin secretion was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit in MIN6 cells after transfection of SPHK2 siRNA and S1P treatment. Each value represents the mean of three experiments. Veh, vehicle; si, small interfering; Scr, scramble. aP<0.05, bP<0.001 compared with the si-Scr group; cP<0.05, dP<0.01, eP<0.001 compared with the si-SphK2 group.

  • Fig. 4 Restoration of sphingosine-1-phosphate (S1P) levels did not lead to the recovery of function in prohibitin (PHB)-deficient mouse insulinoma 6 (MIN6) cells. At 60% confluence, MIN6 cells were transfected with 10 nM small interfering RNA (siRNA) for PHB, and then incubated in serum-free media with or without 10 µM S1P for 48 hours. (A, B) The messenger RNA and protein levels of PHB were analyzed by real-time reverse-transcription polymerase chain reaction and an immunoblotting assay. (C) After knockdown of PHB and/or S1P treatment, MIN6 cells were exposed to tetramethylrhodamine ethyl ester (TMRE) and Hoechst to observe the mitochondrial membrane potential. Bars=20 µm. (D, E) Adenosine triphosphate (ATP) levels and the oxygen consumption rate were measured in MIN6 cells after transfection of PHB siRNA and S1P treatment. Each value represents the mean of three experiments. (F) Glucose-stimulated (5.5 or 25 mM) insulin secretion was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit in MIN6 cells after transfection of PHB siRNA and S1P treatment. The values are representative of four independent experiments. Veh, vehicle; si, small interfering; Scr, scramble. aP<0.05, bP<0.001 compared with the si-Scr group.


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