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  • We elucidated that the ten DGK


    We elucidated that the ten DGK isozymes have different relative 1-MGK and 2-MGK activities vs DGK activity under the same conditions. However, because the octylglucoside-mixed micellar assay was developed for DGK, it is not known whether the DGKs exhibit maximum 1-MGK and 2-MGK activities in this assay. Although the deoxycholate assay was reported to be more suitable for the measurement of the 1-MGK and 2-MGK activities of AGK than the octylglucoside-mixed micellar assay [38], we did not detect a marked enhancement of these activities of AGK and DGK using the deoxycholate assay (data not shown). If an improved MGK assay method is developed, higher intrinsic 1-MGK and 2-MGK activities of DGK isozymes may be found. The 1-MGK activity of DGKθ and the 2-MGK activity of DGKα were stronger than those of AGK (Fig. 7, Fig. 8), which has DGK, 1-MGK and 2-MGK activities [38], under several assay conditions. Therefore, it is possible that the type I–III DGKs and type V DGK act as primary 2-MGK and 1-MGK in cells, respectively. AGK lacks a corresponding region homologous to the C-terminal half of the catalytic domain of DGK [38]. Rittiner et al. [40] and we (Murakami, E. and Sakane, F. unpublished work) revealed that DGKη lacking the C-terminal half of the catalytic domain did not show DGK activity. Therefore, this deletion may cause the low DGK, 1-MGK and 2-MGK activities of AGK. DG content represented 0.7–4mol% of total lipids in isolated cell membranes [41]. The concentrations of MG are 2–10-fold higher than those of DG in macrophages [42] and 3T3-L1 adipocytes [43]. Therefore, the contribution of the 1-MGK and 2-MGK activities of DGK may be considerably great in these MG-rich cells, macrophages and adipocytes. Moreover, it is possible that the concentrations of 1-MG and 2-MG are high in a focal manner. In the 1-MG- and 2-MG-rich regions, DGK isozymes (type I–III and V) would generate substantial amounts of 1-LPA and 2-LPA. 2-Arachidonoyl-glycerol acts as endogenous cannabinoid [44], [45], so its metabolism must be strictly regulated. Because DGKε prefers DG species containing arachidonic aa utp synthesis at the sn-2 position [46], [47], this isozyme may play an important role in 2-arachidonoyl-glycerol kinase. In addition to DGKε [23], DGKα [48], [49], β [50], γ [51], [52], δ [53] and η [34], [53], [54] are also highly expressed in the brain. Moreover, DGKα, β, γ, δ and η have broad selectivities to fatty acids at the sn-2 position of DG [3]. For example, DGKγ indeed showed comparable phosphorylation activities against 2-oleoyl(18:1)-glycerol and 2-arachidonoyl-glycerol (Suppl. Fig. 7). Therefore, these type I and II isozymes may also act as 2-arachidonoyl-glycerol kinase in the brain. Our preliminary experiments showed that DGKδ-specific siRNA attenuated a high glucose-dependent increase of LPA (Sakai, H. and Sakane, F., unpublished work). However, a phospholipase A inhibitor (methyl arachidonyl fluorophosphonate) [55], [56] did not inhibit the high glucose-dependent increase of LPA (Sato, Y., Mizuno, S. and Sakane, F. unpublished work). These results suggest that DGKδ produces LPA through the direct phosphorylation of MG, but not the PA hydrolysis by phospholipase A. Thus, although further works are needed to confirm, it is possible that DGKδ serves as MGK in cells. The ten DGK isozymes are divided into five groups (type I: α, β and γ; type II: δ, η and κ; type III: ε; type IV: ζ and ι; type V: θ) according to their structural features [1], [2], [3], [4], [5], [6]. Arachidonic acid-containing DG-selectivity can also be used to categorize the isozymes into two groups: an arachidonoyl DG-selective isozyme (ε) versus non-selective isozymes (α, β, γ, δ, η, κ, ζ, ι and θ) [1], [2], [3], [4], [5], [6]. DGK isozymes can also be grouped into high DG affinity type (η) versus low DG affinity type (α, ε and ζ) (the affinities of the other isozymes for DG are presently unknown) [57]. In addition to these categorizations, the results of this study provide a new classification based on the different activities for 1-MG and 2-MG: DGK isozyme(s) having (1) 2-MGK activity relatively stronger than their 1-MGK activity (type I–III DGKs), (2) only negligible 1-MGK and 2-MGK activities (type IV DGK), and (3) 1-MGK activity stronger than its 2-MGK (type V DGK). These differences in regard to the relative 1-MGK and 2-MGK activities are probably essential for the DGK isozymes in serving their individual functions.