Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels

Abstract

Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.

Keywords: Diacylglycerol; Diacylglycerol Kinase; Glucose; Palmitic Acid; Phosphatidic Acid; Phosphatidylcholine-specific Phospholipase C; Phospholipase C; Type 2 Diabetes.

FIGURE 1.

Changes in the total PA and PA molecular species by high glucose stimulation in C2C12 myoblasts and myotubes. A and B, the amounts of the total PAs (A) and major PA molecular species (B) in the glucose-unstimulated or glucose-stimulated C2C12 myoblasts were quantified using the LC/ESI-MS method. The values are presented as the mean ± S.D. (n = 14). *, p < 0.05; **, p < 0.01; ***, p < 0.005 (no stimulation versus glucose stimulation). C and D, the amounts of the total PAs (C) and major PA molecular species (D) that statistically increased in A in the cells stimulated by glucose for 5, 15, or 30 min were detected using the LC/ESI-MS method. The results are presented as the percentage of the value of PA molecular species in glucose-unstimulated cells. The values are presented as the mean (n = 2). Essentially the same results were obtained in two independent experiments. E and F, the amounts of the total PAs (E) and major PA molecular species (F) in the glucose-unstimulated or glucose-stimulated C2C12 myotubes were quantified using the LC/ESI-MS method. The values are presented as the mean ± S.D. (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.005 (no stimulation versus glucose stimulation).

FIGURE 2.

Effects of DGKδ-siRNA-1 and -2 on high glucose-induced increases of PA molecular species in C2C12 myoblasts. A and C, the suppression of DGKδ2 expression by DGKδ-siRNA-1 (A) or DGKδ-siRNA-2 (C) was confirmed by Western blot analysis using the anti-DGKδ antibody. Human DGKδ1 and DGKδ2 (11) expressed in COS-7 cells were electrophoresed as a control (A). B and D, the major PA molecular species in the glucose-unstimulated or glucose-stimulated cells transfected with control siRNA or DGKδ-siRNA-1/2 were detected using the LC/ESI-MS method. The results are presented as the percentage of the value of PA molecular species in glucose-unstimulated cells transfected with control siRNA or DGKδ-siRNA-1/2. DGKδ-siRNA-1/2 did not significantly affect the value of PA molecular species in glucose-unstimulated cells. The values are presented as the mean ± S.D. (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.005 (no stimulation versus glucose stimulation). #, p < 0.05; ##, p < 0.01 (control siRNA versus DGKδ-siRNA-1 or DGKδ-siRNA-2).

FIGURE 3.

Effect of DGKδ-siRNA-1 on high glucose-induced increases of DG molecular species in C2C12 myoblasts. The major DG molecular species in the glucose-unstimulated or glucose-stimulated cells transfected with control siRNA or DGKδ-siRNA-1 were detected using the ESI-MS method. The results are presented as the percentage of the value of DG molecular species in glucose-unstimulated cells transfected with control siRNA or DGKδ-siRNA-1. DGKδ-siRNA-1 did not significantly affect the value of DG molecular species in glucose-unstimulated cells. The values are presented as the mean ± S.D. (n = 7). *, p < 0.05; ***, p < 0.005 (no stimulation versus glucose stimulation).

FIGURE 4.

PA molecular species in C2C12 cells stably expressing DGKδ2. A, the stable expression of AcGFP-DGKδ2 in C2C12 cells was confirmed by Western blot analysis using the anti-DGKδ antibody. B, the major PA molecular species in the glucose-unstimulated or glucose-stimulated cells stably expressing human DGKδ2 were identified and quantified using LC/ESI-MS. The results are presented as the percentage of the value of PA molecular species in glucose-unstimulated cells transfected with AcGFP alone or AcGFP-DGKδ2. Overexpression of DGKδ2 did not significantly affect the value of PA molecular species in glucose-unstimulated cells. The values are presented as the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.005 (no stimulation versus glucose stimulation). #, p < 0.05; ###, p < 0.005 (no overexpression versus DGKδ overexpression).

FIGURE 5.

In vitro DGKδ activity. For measurement of in vitro DGKδ activity, 2 mm (5.4 mol%) 16:0/16:0-, 16:0/18:1-, and 18:0/20:4-DG were used as substrates. The activity of 3×FLAG-tagged DGKδ2 in COS-7 cells was compared with the control. The results are presented as the percentage of the value of activity against 18:0/20:4-DG. The values are presented as the mean ± S.D. (n = 6).

FIGURE 6.

Effects of TOFA and FIPI on the production of PA molecular species in glucose-stimulated C2C12 cells. A, the major PA molecular species in glucose-unstimulated or glucose-stimulated cells treated with DMSO (control) or TOFA were detected using the LC/ESI-MS method. The results are presented as the percentage of the value of PA species in glucose-unstimulated cells treated with DMSO (control) or TOFA. The values are presented as the mean ± S.D. (n = 5). *, p < 0.05; ***, p < 0.005 (no stimulation versus glucose stimulation). #, p < 0.05 (without TOFA versus with TOFA). B, the major PA molecular species in the glucose-unstimulated or glucose-stimulated cells treated with DMSO (control) or FIPI were detected using the LC/ESI-MS method. The results are presented as the percentage of the value of PA species in glucose-unstimulated cells treated with DMSO (control) or FIPI. The values are presented as the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.005 (no stimulation versus glucose stimulation). #, p < 0.05 (without FIPI versus with FIPI).

FIGURE 7.

Effect of D609 on high glucose-induced increases in PA and DG molecular species in C2C12 myoblasts. A, the major PA molecular species in the glucose-unstimulated or glucose-stimulated cells treated with DMSO (control) or D609 were detected using the LC/ESI-MS method. The results are presented as the percentage of the value of PA molecular species in glucose-unstimulated cells treated with DMSO (control) or D609. D609 did not significantly affect the value of PA molecular species in glucose-unstimulated cells. The values are presented as the mean ± S.D. (n = 5). *, p < 0.05; ***, p < 0.005 (no stimulation versus glucose stimulation). ###, p < 0.005 (without D609 versus with D609). B and C, the major DG molecular species in the glucose-unstimulated or glucose-stimulated cells treated with DMSO (control) or D609 were detected using the ESI-MS method. B, comparison of +D609 versus −D609 in the absence of glucose. The results are presented as the percentage of the value of DG species in glucose-unstimulated cells treated with DMSO (control). The values are presented as the mean ± S.D. (n = 3). *, p < 0.05; ***, p < 0.005. C, comparison of +glucose versus −glucose in the absence or presence of D609. The results are presented as the percentage of the value of DG species in glucose-unstimulated cells treated with DMSO (control) or D609. D609 did not significantly affect the value of DG molecular species in glucose-unstimulated cells. The values are presented as the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.005 (no stimulation versus glucose stimulation). #, p < 0.05; ##, p < 0.01; ###, p < 0.005 (without D609 versus with D609).

FIGURE 8.

Examination of the functional linkage between PC-PLC and DGKδ. Effects of D609 and DGKδ-siRNA-1 on high glucose-induced increases of PA molecular species in C2C12 myoblasts were compared. A, the suppression of DGKδ2 expression by DGKδ-siRNA-1 was confirmed by Western blot analysis using the anti-DGKδ antibody. B, 30:0-, 32:0-, and 34:0-PA in the glucose-unstimulated or glucose-stimulated cells treated with DMSO (control), D609, or D609 and DGKδ-siRNA-1 were detected using the LC/ESI-MS method. The results are presented as the percentage of the value of PA molecular species in glucose-unstimulated cells. The values are presented as the mean ± S.D. (n = 5). ***, p < 0.005 (no stimulation versus glucose stimulation). #, p < 0.05; ##, p < 0.01; ###, p < 0.005 (without D609 versus with D609). †, p < 0.05; †††, p < 0.005 (control siRNA versus DGKδ-siRNA-1). C and D, co-immunoprecipitation of PC-PLC activity with DGKδ2. C, immunoprecipitation (IP) of DGKδ2 using the anti-DGKδ antibody was confirmed by Western blot analysis using the anti-DGKδ antibody. D, PC-PLC activity in the precipitates was measured using the Amplex Red® PC-PLC assay kit. The values are presented as the mean ± S.D. (n = 4). ***, p < 0.005. When the assay was performed in the absence of alkaline phosphatase, the activity was not detectable.

FIGURE 9.

Model for the metabolism pathway utilized by DGKδ. IR, insulin receptor; IRS, insulin receptor substrate.