Acetylation of glucokinase regulatory protein decreases glucose metabolism by suppressing glucokinase activity

Abstract

Glucokinase (GK), mainly expressed in the liver and pancreatic β-cells, is critical for maintaining glucose homeostasis. GK expression and kinase activity, respectively, are both modulated at the transcriptional and post-translational levels. Post-translationally, GK is regulated by binding the glucokinase regulatory protein (GKRP), resulting in GK retention in the nucleus and its inability to participate in cytosolic glycolysis. Although hepatic GKRP is known to be regulated by allosteric mechanisms, the precise details of modulation of GKRP activity, by post-translational modification, are not well known. Here, we demonstrate that GKRP is acetylated at Lys5 by the acetyltransferase p300. Acetylated GKRP is resistant to degradation by the ubiquitin-dependent proteasome pathway, suggesting that acetylation increases GKRP stability and binding to GK, further inhibiting GK nuclear export. Deacetylation of GKRP is effected by the NAD(+)-dependent, class III histone deacetylase SIRT2, which is inhibited by nicotinamide. Moreover, the livers of db/db obese, diabetic mice also show elevated GKRP acetylation, suggesting a broader, critical role in regulating blood glucose. Given that acetylated GKRP may affiliate with type-2 diabetes mellitus (T2DM), understanding the mechanism of GKRP acetylation in the liver could reveal novel targets within the GK-GKRP pathway, for treating T2DM and other metabolic pathologies.

Figure 1. GKRP is acetylated by p300.

(A) Effects of histone deacetylase inhibitors on GKRP. HeLa cells transfected with Myc-tagged GKRP were treated with 5 mM NAM and 1 μM TSA 6 hr before harvest. (B) Band intensities of acetylated Myc-GKRP were quantified by Image J software. The values from samples not treated with NAM and TSA were set to 1.0. Data are shown as the means ± SEM of four independent experiments. (C) Identification of the acetyltransferase responsible for GKRP acetylation. Expression vectors of various acetyltransferases (ATs) were co-transfected with pSG-Myc GKRP into HeLa cells. The immunoprecipitates (from antibodies against the various ATs) were then subjected to immunoblot with antibodies against Ac-Lys or Myc. (D) LC-MS/MS spectrum of GKRP peptides showing that acetylation occurs at K5. (E) Effects of site-specific mutation on the potential acetylation site, GKRP K5. Substitutions of Lys (K) with Arg (R) or Glu (Q) at the indicated sites are shown in parenthesis. HeLa cells transfected with the indicated mutant or wild-type plasmids were lysed and immunoprecipitated by an anti-Myc antibody. Acetylated GKRP was detected by an anti-Ac-Lys antibody. (F) Sequence alignment of the GKRP region containing K5 from various species. NAM, nicotinamide; TSA, Trichostatin A. Data are expressed as means ± SEMs, n = 4, *p ≤ 0.05; **p ≤ 0.01.

Figure 2. Ubiquitin-dependent GKRP degradation is decreased by acetylation.

(A) Effects of histone deacetylase inhibitors on GKRP levels in. HeLa cells transfected with Myc-tagged GKRP. (B) Effect of p300 on GKRP protein levels in HeLa cells transfected with Myc-tagged GKRP and full-length p300. (C) Effect of anti-p300 siRNA on GKRP protein levels. HeLa cells transfected with Myc-tagged GKRP and negative control or p300 siRNA (10 nM) were incubated for 48 hr, and GKRP protein levels assessed by immunoblot using an anti-Myc antibody. (D) Effect of ubiquitin on GKRP stability. HeLa cells cotransfected with Myc-tagged GKRP and HA-tagged ubiquitin were maintained in the absence or presence of MG132 (10 μM) for 2 hr. Cell lysates were precipitated with an anti-Myc antibody and immunoblotting used to detect ubquitinated GKRP by anti-HA antibody. (E) Effect of various K5 mutants on GKRP ubiquitination. Myc-tagged wild type, deacetyl-mimic (K5R), or acetyl-mimic (K5Q) GKRP was cotransfected with HA-ubiquitin in the presence of the proteasome inhibitor MG132 (10 μM) for 2 hr. Cell lysates were precipitated by an anti-Myc antibody, and immunoblot with an anti-HA antibody used to detect ubiquitinated GKRP. (F) Effects of HDACIs on GKRP stability. HeLa cells were transfected with Myc-tagged GKRP were treated with NAM (5 mM) and TSA (1 μM) 4 hr before treating CHX. 24 hr after transfection, cells were treated with 50 μg/ml of the protein synthesis inhibitor CHX for the indicated time periods. GKRP protein levels from cells collected at time zero were defined as 100%. GAPDH and β-actin were used as an internal control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. HDACIs, histone deacetylase inhibitor, NAM, nicotinamide, TSA, Trichostatin A, and CHX, cycloheximide. Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.

Figure 3. GKRP acetylation is critical for binding and retaining GK in the nucleus.

(A,B) Interaction between GKRP and human glucokinase (hGK). HeLa cells transfected with expression vectors for GKRP and hGK were lysed, protein immunoprecipitated with anti-Myc or anti-V5 antibodies, and immunoblotted by anti-His or anti-Myc antibodies. (C) Effect of K5 mutations on GKRP interaction with hGK. HeLa cells cotransfected with hGK and wild type or the indicated GKRP mutants were lysed, protein precipitated with an anti-Myc antibody, and blotted with an anti-His antibody. β-actin expression was used as internal control. (D) Immunofluorescence micrographs showing the subcellular locations of GKRP and hGK. HeLa cells transfected with Myc-tagged GKRP and V5/His-tagged GK were maintained in 5.5 mM or 25 mM glucose for 4 hr in the absence (−) or presence (+) of a mixture of the HDACIs NAM (5 mM) and TSA (1 μM). Data are expressed as means ± SEMs, n = 4, ***p ≤ 0.001.

Figure 4. Acetylation of GKRP suppresses glycolytic flux.

(A–D) HeLa cells were seeded in V7 cell plates at a density of 10,000 cells/well. Glycolysis assays were performed using a glycolytic stress test kit, according to the manufacturer’s protocol, on a XF24 instrument (Seahorse Biosciences). (A) A representative XF24 graph showing the ECAR response to glucose, oligomycin, and 2-deoxyglucose in Seahorse glucose-free medium. (B) Basal glycolysis calculated relative to the control after subtraction of non-glycolytic acidification. (C) Glycolytic capacity was calculated relative to the control following the addition of oligomycin. (D) Glycolytic reserve (the difference between the basal glycolysis and glycolytic capacity rates). A minimum number of n = 5 with 3–4 replicate wells per group was employed for all experiments. ECAR, extracellular acidification rate. 2-DG, 2-deoxyglucose. Con, Control. Values are expressed as means ± SEMs, *p ≤ 0.05; ***p ≤ 0.001. NS, not significant.

Figure 5. Increased acetylation of GKRP in db/db mice.

(A) Western blot of acetylated GKRP in db/db mouse livers. Cell lysates were immunoprecipitated by an anti-GKRP antibody and immunoblot performed to detect endogenous acetylated GKRP using an anti-Ac-Lys antibody. (B) Band intensities of acetylated GKRP, as quantified by Image J software. The values from db/m + mice were set to 1.0 (n = 8). (C) Interactions between GK and GKRP in control and db/db mice. Endogenous GKRP was immunoprecipitated from liver homogenates of db/m + and db/db mice, using GKRP antibody, and then immunoblotted by an anti-GK antibody. (D) Band intensities of the GK-GKRP complexes were quantified using Image J software. The values from db/m + mice were set to 1.0 (n = 4). Values are expressed as means ± SEM, *p ≤ 0.05.

Figure 6. SIRT2 deacetylates GKRP.

(A) Effect of the HDAC inhibitors NAM and TSA on GKRP acetylation. HeLa cells transfected with Myc-tagged GKRP expression vector were treated with NAM (5 mM) or TSA (1 μM) for 6 h before harvest. (B) Identification of interaction between SIRT2 and GKRP. HeLa cells transfected with expression plasmids of Myc-tagged GKRP and Flag-tagged SIRT2 were lysed, immunoprecipitated, and subjected to immunoblot with antibodies to Flag or Myc. (C) SIRT2 deacetylates GKRP in HeLa cells transfected with the indicated plasmids. Protein was precipitated with anti-Myc antibody and immunoblotted using anti-Ac-Lys or anti-Flag or anti-Myc antibodies, respectively. (D) GKRP deacetylation by SIRT2 is reversed by NAM. HeLa cells were transfected with the indicated plasmids, treated with NAM (5 mM) and TSA (1 μM) for 6 h, and precipitates subjected to immunoblot with antibodies to Ac, Myc or Flag. β-actin, protein level were used as an internal control. Deacetylation of GKRP regulates glycolytic flux. (E-H) HeLa cells were seeded in V7 cell plates at a density of 10,000 cells/well. Glycolysis assays were performed using a glycolytic stress test kit, according to manufacturer’s protocol, on a XF24 instrument (Seahorse Biosciences). (E) A representative XF24 graph showing the ECAR response to glucose, oligomycin, and 2-deoxyglucose in Seahorse glucose-free medium. (F) Basal glycolysis calculated relative to the control after subtraction of non-glycolytic acidification. (G) After the addition of oligomycin, glycolytic capacity was calculated relative to control. (H) Glycolytic reserve (the difference between basal glycolysis and glycolytic capacity rate). A minimum number of n = 5 with 3–4 replicate wells per group was employed for all experiments. ECAR, extracellular acidification rate. 2-DG, 2-deoxyglucose. Con, Control. SIRT2 H187Y, catalytic mutant of SIRT2. NAM, nicotinamide; TSA, Trichostatin A. Data are expressed as means ± SEMs, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. NS, not significant.