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. 2018 Jan 2;9:30. doi: 10.1038/s41467-017-02537-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Deacetylation of K126 on GKRP by Sirt2 facilitates GK−GKRP dissociation and hepatocyte glucose uptake. a, b Comparison of the acetylation levels of wild-type (WT) GKRP, eight GKRP Lys-to-Arg mutants (a), and eight GKRP Lys-to-Gln mutants (b) in HEK293 cells expressing Flag-tagged Sirt2 or not. c The acetylation levels of GKRP in primary hepatocytes derived from db/db mice transfected with adenovirus encoding Flag-tagged GKRP-wild-type (Flag-GKRP-WT) or Flag-tagged GKRP-K126R (Flag-GKRP-K126R). Endo. endogenous GKRP, Exo. exogenous GKRP. d Effect of Sirt2 knockdown on glucokinase (GK)−GKRP binding in primary hepatocytes transfected with adenovirus encoding HA-tagged GK (HA-GK) and Flag-GKRP-WT or Flag-GKRP-K126R in low- and high-glucose medium. e Effect of nicotinamide (NAM) on GK−GKRP binding in mouse primary hepatocytes transfected with adenovirus encoding GK and Flag-GKRP-WT or Flag-GKRP-K126R in high-glucose medium. f, g Effect of Sirt2 knockdown (f) or NAM (g) on 2-deoxyglucose (2-DG) uptake in mouse primary hepatocytes transfected with adenovirus vector encoding Flag-GKRP-WT or Flag-GKRP-K126R (n = 4). h Immunoprecipitation and immunoblot analysis of GK−GKRP binding in db/db mouse primary hepatocytes transfected with adenovirus encoding HA-GK and Flag-GKRP-WT or Flag-GKRP-K126R in low- and high-glucose medium. i, j NAD+ (i) and 2-DG uptake (j) levels in db/db mouse primary hepatocytes transfected with adenovirus vector encoding Flag-GKRP-WT and Flag-GKRP-K126R (n = 5). k, l Effect of Sirt2 knockdown (k) and NAM (l) on GK−GKRP binding in primary hepatocytes transfected with adenovirus vectors encoding HA-GK or GK and Flag-GKRP-WT or Flag-tagged GKRP-K126Q (Flag-GKRP-K126Q) in high-glucose medium. m, n Effect of Sirt2 knockdown (m) or NAM (n) on 2-DG uptake in primary hepatocytes transfected with adenovirus vectors encoding Flag-GKRP-WT or Flag-GKRP-K126Q (n = 4). *P < 0.05; one-way ANOVA with the Fisher’s PLSD post-hoc test (f, g, m, n) and Student’s t-test (i, j). Data in a–e, h, k, l are representative of at least three independent experiments. Error bars show s.e.m