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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

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.
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f4: 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.

Mentions: We next hypothesized that if GKRP acetylation increases its interaction with GK in the nucleus, cytosolic glycolysis should be reduced. Consequently, we overexpressed GK in HeLa cells and measured glycolytic flux (i.e., basal glycolysis, glycolytic capacity, and glycolytic reserve) by assessing the extracellular acidification rate (ECAR), after sequential treatment with glucose, oligomycin (an inhibitor of mitochondrial respiration), and 2-deoxyglucose (an inhibitor of glycolysis), in glucose-free Seahorse assay media (Fig. 4A and Supplementary Fig. S4A). Under those conditions, glycolytic flux significantly increased in HeLa cells overexpressing GK (Fig. 4B–D, p ≤ 0.001); that effect was negated by expression of the WT or K5Q mutant GKRP (Fig. 4B–D, p ≤ 0.05 (*) or p ≤ 0.001 (***)). In contrast, there was no significant difference in glycolytic flux in GK- or GKRP-K5R mutant-overexpressing cells (Fig. 4B–D), nor was it changed in the WT or K5Q or K5R GKRP mutants, in the absence of GK (see Supplementary Fig. S4B–D). Together, these data strongly suggest decreased glucose utilization when GKRP is acetylated.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664969&req=5

f4: 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.
Mentions: We next hypothesized that if GKRP acetylation increases its interaction with GK in the nucleus, cytosolic glycolysis should be reduced. Consequently, we overexpressed GK in HeLa cells and measured glycolytic flux (i.e., basal glycolysis, glycolytic capacity, and glycolytic reserve) by assessing the extracellular acidification rate (ECAR), after sequential treatment with glucose, oligomycin (an inhibitor of mitochondrial respiration), and 2-deoxyglucose (an inhibitor of glycolysis), in glucose-free Seahorse assay media (Fig. 4A and Supplementary Fig. S4A). Under those conditions, glycolytic flux significantly increased in HeLa cells overexpressing GK (Fig. 4B–D, p ≤ 0.001); that effect was negated by expression of the WT or K5Q mutant GKRP (Fig. 4B–D, p ≤ 0.05 (*) or p ≤ 0.001 (***)). In contrast, there was no significant difference in glycolytic flux in GK- or GKRP-K5R mutant-overexpressing cells (Fig. 4B–D), nor was it changed in the WT or K5Q or K5R GKRP mutants, in the absence of GK (see Supplementary Fig. S4B–D). Together, these data strongly suggest decreased glucose utilization when GKRP is acetylated.

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