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  • Furthermore we explored the effect of PKC


    Furthermore, we explored the effect of PKCζ on SIRT6 phosphorylation. Accumulating data showed that aPKC isoforms are involved in regulating lipid metabolism [41], [42], [43], [44], [45], [46], [47]. The aPKC activity was reported to play a dominant role in normal insulin signaling by activating PI3K activity and contributing to insulin-stimulated lipogenesis in the liver [42], [43]. The activity of PKC ι/ζ stimulated by insulin was reduced 57% in obese and 65% in diabetic subjects. Importantly, weight loss in obese subjects normalized PKC ι/ζ activity and simultaneously increased PI3K activity, indicating the effect of PKC ι/ζ on obese and type 2 diabetic subjects [44]. In obesity, the critical role of aPKC in activating hepatic SREBP-1c and NF-κB, which are the major regulators of hepatic lipid synthesis and systemic insulin resistance, was shown in the fed state. Conserved hepatic aPKC-dependent activation of SREBP-1c and NF-κB contributed to hepatic lipogenesis, as well as the development of hyperlipidemia, and systemic insulin resistance [45]. Also, other studies reported a similar mechanism that aPKC mediated SREBP-1c to promote lipogenesis [43], [46]. In addition, PKCζ was suggested to involve in mitogenic factor-stimulated preadipocytes proliferation and insulin-stimulated preadipocytes differentiation through the rapid increase of PKCζ in the cytosolic compartment and the translocation change into the nucleus [47]. In our study, we found that enrichment of SIRT6 on chromatin was blocked after PA stimulation in PKCζ siRNA-treated ldk378 (Figure 4E). Also, the enrichment of SIRT6 on chromatin was significantly reduced in the Flag-SIRT6 T294A mutant transfected cells compared with Flag-SIRT6 WT transfected cells after PA stimulation, showing that PKCζ is a response for the SIRT6 enrichment on chromatin after PA treatment through SIRT6 phosphorylation at Thr 294 residues (Figure 4F). Phosphorylation can have diverse consequences on a protein, such as regulating its enzymatic activity or subcellular localization[48]. Our data showed that SIRT6 phosphorylation at Thr 294 residues is required for its enrichment on chromatin. To further functional study, we found that the mRNA levels of fatty acid β-oxidation–related genes such as ACSL1, CPT1, CACT, and HADHB were increased after PA treatment and regulated by PKCζ (Figure 5). The SIRT6 RNAi and T294A mutant SIRT6 transfected study also showed that phosphorylation SIRT6 at Thr294 residue is a key point on the regulation of PA-induced gene expression of fatty acid β-oxidation–related genes (Figure 6A, B). In further study of the mechanism, we found that the binding of SIRT6 to the ACSL1, CPT1, CACT, and HADHB promoters was increased after PA treatment and regulated by PKCζ through SIRT6 phosphorylation (Figure 6C-J). Recently, Khan et al. showed that SIRT6 transcriptionally regulated the expression of pyruvate dehydrogenase kinase 4 by binding to its promoter to further mediate glucose metabolism in heart [49]. SIRT6 transcriptional activation is less reported, but one study has shown that SIRT6 can interact with and recruit RNAP II to coactivate nuclear factor erythroid 2-related factor 2 in human mesenchymal stem cells [50]. It needs to be further explored whether SIRT6 can recruit certain activator to the promoters of fatty acid β-oxidation–related genes to regulate fatty acid β-oxidation. Taken together, we have identified a novel function of PKCζ on fatty acid β-oxidation. We found that PKCζ physically interacts with SIRT6 in vitro and in vivo, and phosphorylates SIRT6 at Thr294 residue after PA treatment. PKCζ mediated SIRT6 phosphorylation could recruit SIRT6 to the promoters of fatty acid β-oxidation–related genes and further regulated the expression of these genes. Understanding the new role of PKCζ on fatty acid β-oxidation will be useful for the future design of effective therapeutic targets to help regulate lipid homeostasis or treat metabolic diseases.