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

Park JM, Kim TH, Jo SH, Kim MY, Ahn YH - Sci Rep (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f6: 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.

Mentions: As shown in Figs 1A and 6A, GKRP acetylation increased when HeLa cells were treated with nicotinamide (NAM), an inhibitor of the NAD+-dependent class III (“sirtuin”) family of histone deacetylases (HDACs)25 (Fig. 1A). Co-immunoprecipitation assays and immunoblots further showed sirtuin 2 (SIRT2) interaction with GKRP (Fig. 6B), although other sirtuins (1 and 3–7) interacted with GKRP (see Supplementary Fig. S5A). To determine if any other sirtuins deacetylate GKRP, HeLa cells were co-tranfected with expression vectors for Myc-GKRP, various sirtuins, and p300. As shown in Supplementary Fig. S5B, GKRP was deacetylated only by SIRT2, but no other sirtuins. Further, co-overexpression of SIRT2, but not its catalytically inactive H187Y mutant, with p300, resulted in decreased deacetylation of GKRP (Fig. 6C). In addition, GKRP deacetylation by SIRT2 was reversed by NAM, but not by TSA (Fig. 6D), which does not inhibit class III HDACs. These results suggest that GKRP deacetylation is catalyzed by SIRT2.


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

Park JM, Kim TH, Jo SH, Kim MY, Ahn YH - Sci Rep (2015)

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.
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f6: 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.
Mentions: As shown in Figs 1A and 6A, GKRP acetylation increased when HeLa cells were treated with nicotinamide (NAM), an inhibitor of the NAD+-dependent class III (“sirtuin”) family of histone deacetylases (HDACs)25 (Fig. 1A). Co-immunoprecipitation assays and immunoblots further showed sirtuin 2 (SIRT2) interaction with GKRP (Fig. 6B), although other sirtuins (1 and 3–7) interacted with GKRP (see Supplementary Fig. S5A). To determine if any other sirtuins deacetylate GKRP, HeLa cells were co-tranfected with expression vectors for Myc-GKRP, various sirtuins, and p300. As shown in Supplementary Fig. S5B, GKRP was deacetylated only by SIRT2, but no other sirtuins. Further, co-overexpression of SIRT2, but not its catalytically inactive H187Y mutant, with p300, resulted in decreased deacetylation of GKRP (Fig. 6C). In addition, GKRP deacetylation by SIRT2 was reversed by NAM, but not by TSA (Fig. 6D), which does not inhibit class III HDACs. These results suggest that GKRP deacetylation is catalyzed by SIRT2.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus