Increased Potency and Efficacy of Combined Phosphorylase Inactivation and Glucokinase Activation in Control of Hepatocyte Glycogen Metabolism
Laura J. Hampson, and
Loranne Agius
From the School of Clinical Medical SciencesDiabetes, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K
 |
ABSTRACT
|
---|
Glucokinase and phosphorylase both have a high control strength over hepatocyte glycogen metabolism and are potential therapeutic targets for type 2 diabetes. We tested whether combined phosphorylase inactivation and glucokinase activation is a more effective strategy for controlling hepatic glycogen metabolism than single-site targeting. Activation of glucokinase by enzyme overexpression combined with selective dephosphorylation of phosphorylase-a by an indole carboxamide that favors the T conformation of phosphorylase caused a greater stimulation of glycogen synthesis than the sum of either treatment alone. This result is explained by the complementary roles of elevated glucose-6-phosphate (G6P; a positive modulator) and depleted phosphorylase-a (a negative modulator) in activating glycogen synthase and also by synergistic inactivation of phosphorylase-a by glucokinase activation and the indole carboxamide. Inactivation of phosphorylase-a by the indole carboxamide was counteracted by 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside, which is metabolized to an AMP analog; this effect was reversed by G6P. Our findings provide further evidence for the inverse roles of G6P and AMP in regulating the activation state of hepatic phosphorylase. It is proposed that dual targeting of glucokinase and phosphorylase-a enables improved potency and efficacy in controlling hepatic glucose metabolism.
In type 2 diabetes, the livers failure to maintain blood glucose homeostasis is a significant contributing factor to hyperglycemia during both the absorptive and the fasting state (1,2). Accordingly, targeting hepatic glucose utilization or production is a potential therapeutic strategy for type 2 diabetes. The optimal targets for drug intervention are proteins with a high control strength over glucose metabolism (3), such as glucokinase (4) and phosphorylase (5).
Activators of glucokinase lower blood glucose in animal models of type 2 diabetes by potentiating glucose-induced insulin secretion (6) and stimulating hepatic glucose metabolism (7). Their efficacy in lowering blood glucose concentrations is consistent with the effect of activating mutations of the glucokinase gene that cause hyperinsulinemia and hypoglycemia in humans (8). The high control strength of glucokinase on liver metabolism is explained by the reciprocal control of glucokinase activity by its regulatory protein (9) and the role of glucose-6-phosphate (G6P), the product of the glucokinase-catalyzed reaction, as an activator of glycogen synthase (10,11) and inactivator of phosphorylase (12,13).
Inhibitors of liver phosphorylase also lower blood glucose in animal models of type 2 diabetes (1416). Phosphorylase catalyzes the flux-generating step in glycogen degradation (17) and is a key regulatory component of the mechanism by which insulin activates glycogen synthase in liver (18,19). Various classes of phosphorylase inhibitors have been identified that inhibit hepatic glycogenolysis by interacting with diverse binding sites (20). They include glucose analogs (21), dihydropyridine derivatives that bind to the adenine nucleotide activation site (22,23), flavopiridol derivatives that bind to the purine nucleoside inhibitor site (24), and indole carboxamides that bind to a novel site (14,25). Many, but not all, of these compounds promote dephosphorylation of phosphorylase-a (conversion of phosphorylase-b) by favoring the T conformation, which is a better substrate for phosphorylase phosphatase (26). Because phosphorylase-a is a potent allosteric inhibitor of glycogen synthase phosphatase associated with the glycogen-targeting protein GL, phosphorylase inhibitors that promote dephosphorylation of phosphorylase-a also cause stimulation of glycogen synthesis (5,17,26).
Targeting of a single site in a metabolic pathway is predicted to have a limited effect on metabolic flux (27). In this study, we tested the hypothesis that simultaneous targeting of two enzymes with a high control strength over glycogen metabolism is more effective at increasing pathway flux than targeting a single site. We determined the combined effects of glucokinase overexpression and inactivation of phosphorylase with an indole carboxamide (14) on hepatocyte glycogen synthesis. Our results demonstrate the increased potency of the phosphorylase inhibitor as well as the increased efficacy achieved by combined targeting of the two enzymes.
 |
RESEARCH DESIGN AND METHODS
|
---|
CP-91149 was a kind gift from Pfizer Global Research and Development (Groton/New London Laboratories, Groton, CT); the adenovirus for glucokinase expression (28) was a kind gift from Dr. C. Newgard (Duke University, Durham, NC).
Hepatocytes were isolated by collagenase perfusion of the liver from male Wistar rats (180250 g body wt; B & K, Hull, U.K.) fed ad libitum. The hepatocytes were cultured in monolayer in minimum essential medium (MEM) containing 6% vol/vol newborn calf serum for 2 h to allow cell attachment (5). This was followed by a 2-h incubation in serum-free medium with varying titers of adenovirus encoding rat liver glucokinase (28). The medium was then replaced by serum-free MEM containing 10 nmol/l dexamethasone, and the hepatocytes were cultured for 16 h.
Glycogen synthesis was determined during a 3-h incubation in MEM containing 10 mmol/l glucose and 2 µCi/ml [U-14C]glucose (5). Incubations without radiolabel were performed to determine enzyme activity and G6P levels (12). Glucokinase activity was measured spectrometrically after hepatocytes were permeabilized with digitonin (4). To determine phosphorylase-a and glycogen synthase, hepatocyte monolayers were snap frozen in liquid N2. Phosphorylase-a levels were measured spectrometrically (29). The concentration of CP-91149 necessary to cause the half-maximal effect (EC50) was determined using the Fig.P program (Biosoft, Cambridge, U.K.). Glycogen synthase activity was determined in homogenate extracts from the incorporation of [3H]glucose from uridine diphosphate [3H]glucose into glycogen in the absence or presence of G6P, representing active and total enzyme, respectively (30). Translocation of glycogen synthase was determined from the distribution of total glycogen synthase (assayed with G6P) in the 13,000-g supernatant and pellet fractions of homogenates and is expressed as total glycogen synthase in the pellet percent (supernatant + pellet) fractions (13). Enzyme activities are expressed as milliunits per milligram of cell protein, where one milliunit is the amount converting 1 nmol of substrate/min.
Data are expressed as means ± SE for the number of hepatocyte preparations indicated. Statistical analysis was by the paired t test.
 |
RESULTS
|
---|
Combined effects of glucokinase overexpression and CP-91149 on glycogen synthesis.
Previous studies have shown that graded overexpression of glucokinase (4) or inactivation of phosphorylase with CP-91149, an indole carboxamide (5), stimulates glycogen synthesis. We determined the combined effects of graded glucokinase overexpression and CP-91149 on glycogen synthesis (Fig. 1). Overexpression of glucokinase by up to fivefold relative to endogenous activity caused a progressive increase in glycogen synthesis, and CP-91149 (2.5 µmol/l) further increased glycogen synthesis (Fig. 1A and B). The increment in glycogen synthesis caused by CP-91149 was significantly greater in cells overexpressing glucokinase (Fig. 1C); similarly, the increment caused by glucokinase overexpression was greater in incubations with CP-91149 than in controls without the inhibitor (Fig. 1D), indicating that the combined effects of glucokinase overexpression and CP-91149 were more than additive.

View larger version (27K):
[in this window]
[in a new window]
|
FIG. 1. Combined effects of glucokinase overexpression and CP-91149 on glycogen synthesis. Hepatocytes were either untreated (1.0-fold) or treated with four titers of adenovirus for expression of glucokinase by 1.3-, 1.8-, 2.9-, and 5.2-fold relative to endogenous activity. After a 16-h culture, they were incubated for 3 h in MEM containing 10 mmol/l glucose and [U-14C]glucose without or with 2.5 µmol/l CP-91149 for determination of glycogen synthesis. A: Rates of glycogen synthesis. B: Double log plot of glycogen synthesis versus glucokinase activity. C: Increment in glycogen synthesis caused by CP-91149. D: Increment in glycogen synthesis caused by glucokinase overexpression. Data are means ± SE, n = 4. *P < 0.05 vs. untreated hepatocytes (1.0-fold); **P < 0.05 vs. no inhibitor.
|
|
Role of phosphorylase inactivation and glycogen synthase activation.
Because both CP-91149 and glucokinase overexpression cause inactivation of phosphorylase (5,12,13) and activation of glycogen synthase (5,31,32), we tested whether glycogen synthesis correlates with the activation state of phosphorylase or glycogen synthase. The combined effects of glucokinase overexpression and CP-91149 on inactivation of phosphorylase (Fig. 2A) and activation of glycogen synthase (Fig. 3A) were greater than the effects of either treatment alone.

View larger version (21K):
[in this window]
[in a new window]
|
FIG. 2. Combined effects of glucokinase overexpression and CP-91149 on phosphorylase-a. Experimental conditions were as described for Fig. 1. Hepatocytes overexpressing glucokinase by 1.3- to 5.2-fold were incubated without or with 2.5 µmol/l CP-91149. A: Activity of phosphorylase-a. B: Rates of glycogen synthesis from Fig. 1, relative to phosphorylase-a activity. Data are means ± SE, n = 5. *P < 0.05; **P < 0.005 vs. untreated cells (1.0).
|
|

View larger version (35K):
[in this window]
[in a new window]
|
FIG. 3. Combined effects of glucokinase overexpression and CP-91149 on activation and translocation of glycogen synthase. Hepatocytes were either untreated (1.0-fold) or treated for overexpression of glucokinase by 2.9- and 5.2-fold. They were then incubated for 1 h in medium containing 10 mmol/l glucose without or with 2.5 or 10 µmol/l CP-91149. A: Active glycogen synthase. B: Glycogen synthesis versus active glycogen synthase. C: Glycogen synthase distribution: pellet percent (supernatant + pellet). D: Glycogen synthesis vs. synthase distribution. Data are means ± SE, n = 5. *P < 0.05; **P < 0.005 effects of CP-91149; aP < 0.05; aaP < 0.01 effects of glucokinase overexpression.
|
|
A plot of glycogen synthesis against phosphorylase-a activity (Fig. 2B) shows that CP-91149 caused greater inactivation of phosphorylase than glucokinase expression for corresponding rates of glycogen synthesis. This finding is consistent with the selective action of CP-91149 on phosphorylase inactivation (14,26) and with the activation of glycogen synthase by G6P (10,11). In contrast, when glycogen synthesis was plotted against glycogen synthase activity, the data for incubations without and with CP-91149 were superimposed (Fig. 3B). Similar to activation of glycogen synthase, both glucokinase overexpression and CP-91149 caused movement of glycogen synthase from a soluble to a pellet fraction (Fig. 3C), and this translocation correlated with glycogen synthesis (Fig. 3D). This observation is consistent with the role of activation and translocation of glycogen synthase in the mechanism(s) by which phosphorylase inactivation and glucokinase activity regulate glycogen synthesis (13).
The cellular G6P content was increased by glucokinase overexpression by 4- to 12-fold in the absence of CP-91149, a result in agreement with previous findings (31,32). Although CP-91149 alone did not affect the G6P content in control conditions, it did suppress the increase by glucokinase overexpression to two- to sixfold (Fig. 4).
Glucokinase expression increases the affinity for CP-91149 on glycogen synthesis.
To determine the basis for the synergistic stimulation of glycogen synthesis by increased glucokinase activity and phosphorylase inactivation, we tested whether glucokinase overexpression increases the affinity for CP-91149 on glycogen synthesis. The concentration of CP-91149 that caused the EC50 was decreased (2.9 ± 0.3 to 1.8 ± 0.3 µmol/l; P < 0.002) (Fig. 5A) in cells with twofold glucokinase overexpression, and the increase in glycogen synthesis in cells overexpressing glucokinase and incubated with CP-91149 (Fig. 5B,
) was greater than the arithmetic sum of the stimulation by CP-91149 and glucokinase expression alone (Fig. 5B,

).
Converse effects of glucokinase overexpression and 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside on phosphorylase inactivation by CP-91149.
Because glucokinase overexpression is associated with an increase in G6P (32) and the effects of G6P on phosphorylase are counteracted by 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR), which is metabolized to an AMP analog (13), we tested the separate and combined effects of glucokinase overexpression and AICAR on the inactivation of phosphorylase-a by CP-91149. AICAR activated phosphorylase (Fig. 6A) and increased the EC50 for CP-91149 (Fig. 6B), whereas glucokinase overexpression partially reversed the effect of AICAR.

View larger version (18K):
[in this window]
[in a new window]
|
FIG. 6. Converse effects of AICAR and glucokinase overexpression on the dephosphorylation of phosphorylase-a by CP-91149. Hepatocytes were untreated or treated ( and ) for overexpression of glucokinase by 1.8-fold. Phosphorylase-a activity was determined after a 60-min incubation with varying amounts of CP-91149 in the absence or presence of 100 µmol/l AICAR. A: Phosphorylase-a activity. B: EC50 for CP-91149. Data are means ± SE, n = 4. *P < 0.05 and **P < 0.005 for effects of glucokinase overexpression; #P < 0.05 for effect of AICAR.
|
|
Because inactivation of phosphorylase by a high glucose concentration is in part due to the increase in G6P (12,13), we tested whether the affinity for CP-91149 is affected by a high glucose concentration and whether this involves a role for G6P. 5-Thioglucose (5TG), a glucokinase inhibitor (13), suppressed the increase in G6P caused by 25 mmol/l glucose (5 mmol/l glucose, 0.27 ± 0.07; 25 mmol/l glucose, 1.54 ± 0.18; 25 mmol/l glucose + 3 mmol/l 5TG, 0.40 ± 0.08 nmol/mg; n = 6) and partially counteracted the decrease in the EC50 for CP-91149 caused by 25 mmol/l glucose (Fig. 7), consistent with the roles for both glucose and G6P in synergizing with CP-91149.

View larger version (16K):
[in this window]
[in a new window]
|
FIG. 7. 5TG partially counteracts the effects of glucose on the inactivation of phosphorylase-a by CP-91149. Hepatocytes were incubated for 1 h with varying concentrations of CP-91149 (0.510 µmol/l) at 5 or 10 mmol/glucose or 25 mmol/l glucose without or with 3 mmol/l 5TG. The EC50 for CP-91149 was determined as in Fig. 6. Data are means ± SE, n = 4. *P < 0.05 and ** P < 0.005 vs. 25 mmol/l glucose.
|
|
We tested other substrate conditions that are associated with an increase in G6P (12,13). Incubation with dihydroxyacetone (1 mmol/l), which caused a greater than twofold increase in G6P (0.22 ± 0.03 to 0.60 ± 0.07 nmol/mg protein), decreased the EC50 for CP-91149 (2.5 ± 0.5 to 1.5 ± 0.1 µmol/l; n = 6; P < 0.03), and incubation with 0.2 mmol/l octanoate, which increased G6P by 60% (0.22 ± 0.03 to 0.35 ± 0.03 nmol/mg protein), also decreased the EC50 for CP-91149 (2.0 ± 0.4 to 1.7 ± 0.4 µmol/l; n = 6; P < 0.02), consistent with the synergism between G6P and the indole carboxamide.
 |
DISCUSSION
|
---|
The concept of combination therapy for type 2 diabetes has been widely explored with drugs that target different organs such as insulin secretagogues and insulin sensitizers or metformin (3335). In this study, we tested the hypothesis that combination therapy for targets within the same organ and metabolic pathway has the potential advantage of increased efficacy and potency, thereby enabling the usage of lower drug concentrations. We tested the combined effects of upregulation of glucokinase and downregulation of phosphorylase-a on hepatocyte glycogen metabolism because both enzymes have a high control strength on glycogen metabolism (4,5) and both glucokinase activators (6) and phosphorylase inhibitors (1416,36) lower blood glucose in animal diabetes when used separately.
Glucokinase is regulated by changes in enzyme expression (37,38) and subcellular compartmentation (39). Pharmacological activators of glucokinase increase the enzyme affinity for glucose and cause enzyme translocation from the nucleus (6,7), thus mimicking the effects of physiological activators. In this study, we expressed glucokinase by 30500% above endogenous to mimic the changes induced by nutritional state or pharmacological activators. These activities of glucokinase increase the G6P content, which causes both activation of glycogen synthase (10,11,32) and inactivation of phosphorylase (12,13). The combined effects of glucokinase activation and phosphorylase inactivation might be expected to be less than additive because phosphorylase inactivation is downstream of glucokinase activation (Fig. 8). However, glucokinase expression caused increased efficacy and potency of the phosphorylase inhibitor on glycogen synthesis. This result is partially explained by the synergy between G6P and the indole carboxamide in promoting dephosphorylation (inactivation) of phosphorylase-a, but it may also involve the synergy between the elevated G6P levels and depletion of phosphorylase-a in promoting activation of glycogen synthase (Fig. 8).

View larger version (24K):
[in this window]
[in a new window]
|
FIG. 8. Dual role of G6P in regulating the activation state of phosphorylase and glycogen synthase. Glucokinase activity is a major determinant of the G6P content of hepatocytes (32). G6P promotes dephosphorylation (activation) of glycogen synthase from a less active phosphorylated state (GSB) to a more active dephosphorylated (GSA) state (GSB to GSA) by synthase phosphatase (SP) and dephosphorylation (inactivation) of phosphorylase-a (phos-a) by phosphorylase phosphatase (PP). AMP antagonizes the effect of G6P by competing for the same site. Glucose and CP-91149 promote dephosphorylation synergistically with G6P by binding to different sites. Phosphorylase-a is a potent inhibitor of dephosphorylation of GSB by synthase phosphatase and also a determinant of glycogen synthase translocation. An increase in G6P and depletion of phosphorylase-a promote translocation of glycogen synthase to the pellet fraction.
|
|
The partial counteraction by the phosphorylase inhibitor of the G6P increase caused by glucokinase overexpression indicates partitioning of G6P toward glycogen synthesis in conditions of elevated glucokinase flux and activation of glycogen synthase. This result is consistent with the hypothesis of Schafer et al. (40) that the phosphorylation state of glycogen synthase determines the G6P concentration and suggests that this mechanism operates in hepatocytes in conditions of elevated glucokinase activity. Synergy between the indole carboxamide and G6P on dephosphorylation of phosphorylase-a was observed despite the partial lowering of G6P by the inhibitor. Evidence for the synergy between G6P and the phosphorylase inhibitor is supported by the decrease in the EC50 for CP-91149 in cells overexpressing glucokinase or incubated with octanoate or dihydroxyacetone, both of which raise the cell content of G6P, and by the partial reversal of the effects of 25 mmol/l glucose with the glucokinase inhibitor 5TG, which counteracts the rise in G6P. This indicates that synergy between CP-91149 and high glucose is due in part to the elevated G6P and in part to a direct effect of glucose. The converse effect of AICAR, which is metabolized to an AMP analog, in decreasing the potency for CP-91149 and counteracting the effect of glucokinase expression is further support for the role for G6P, as AMP and G6P compete for the same site, but promote the R and T conformations, respectively (20) (Fig. 8).
The synergy between G6P and CP-91149 in causing dephosphorylation of phosphorylase-a is unlikely to be a unique property of these ligands. Studies on the purified enzyme have shown synergy between glucose analogs and purine-site (41) or AMP-site (42,43) ligands and between indole carboxamides and glucose or purine-site ligands (14) in causing allosteric inhibition. Studies on hepatocytes have shown synergy between nonmetabolizable glucose analogs and indole carboxamides on the inhibition of glycogenolysis (44) and between glucose and glucose analogs on the inactivation of phosphorylase-a (45). The latter can be explained by synergy between the G6P derived from glucose and the glucose analog (13). Synergy with glucokinase activators is therefore predicted for phosphorylase inhibitors that favor the T conformation.
The lack of correlation between rates of glycogen synthesis and the activity of phosphorylase-a in cells overexpressing glucokinase supports a fundamental role for G6P in promoting stimulation of glycogen synthesis by mechanisms other than inactivation of phosphorylase, such as activation and translocation of glycogen synthase (10,11,46). It is noteworthy that conditions that cause activation of glycogen synthase in the absence of inactivation of phosphorylase, such as inhibitors of glycogen synthase kinase-3, have a negligible effect on glycogen synthesis (19). This observation highlights a role for mechanisms downstream of phosphorylase-a depletion that are essential for the stimulation of glycogen synthesis (19).
The synergistic effects of glucokinase activation and phosphorylase inactivation are relevant for understanding insulin action. Insulin signaling involves multiple pathways with convergent effects on metabolism (47). In hepatocytes, insulin-induced inactivation of phosphorylase is part of the mechanism leading to activation of glycogen synthase (19). Nonetheless, the fractional inactivation of phosphorylase is modest compared with the increase in glycogen synthesis. Stimulation of glucokinase translocation by insulin (48), with a consequent increase in G6P, may cause both inactivation of phosphorylase and activation of synthase, and synergy between convergent pathways may explain the large effect of insulin on glycogenic flux, despite modest effects on individual enzyme activities.
 |
ACKNOWLEDGMENTS
|
---|
We thank Diabetes U.K. for project grant and equipment grant support and Dr. Judith Treadway for the gift of CP-91149 and useful discussions.
 |
FOOTNOTES
|
---|
L.A. has received research grant support from AstraZeneca.
Address correspondence and reprint requests to Loranne Agius, School of Clinical Medical SciencesDiabetes, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, U.K. E-mail: loranne.agius{at}ncl.ac.uk
Received for publication July 22, 2004
and accepted in revised form November 30, 2004
AICAR, 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside; G6P, glucose-6-phosphate; MEM, minimum essential medium; 5TG, 5-thioglucose
 |
REFERENCES
|
---|
- Bogardus C, Lillioja S, Howard BV, Reaven G, Mott D: Relationships between insulin secretion, insulin action, and fasting plasma glucose concentration in nondiabetic and noninsulin-dependent diabetic subjects.
J Clin Invest74
:1238
1246,1984[Medline]
- DeFronzo RA, Simonson D, Ferrannini E: Hepatic and peripheral insulin resistance: a common feature of type 2 (non-insulin-dependent) and type 1 (insulin-dependent) diabetes mellitus.
Diabetologia23
:313
319,1982[Medline]
- Kacser H, Burns JA: Molecular democracy. Who shares the controls?
Biochem Soc Trans7
:1149
1160,1979[Medline]
- Agius L, Peak M, Newgard CB, Gomez-Foix AM, Guinovart JJ: Evidence for a role of glucose-induced translocation of glucokinase in the control of hepatic glycogen synthesis.
J Biol Chem271
:30479
30486,1996[Abstract/Free Full Text]
- Aiston S, Hampson L, Gomez-Foix AM, Guinovart JJ, Agius L: Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis.
J Biol Chem276
:23858
23866,2001[Abstract/Free Full Text]
- Grimsby J, Sarabu R, Corbett WL, Haynes NE, Bizzarro FT, Coffey JW, Guertin KR, Hilliard DW, Kester RF, Mahaney PE, Marcus L, Qi L, Spence CL, Tengi J, Magnuson MA, Chu CA, Dvorozniak MT, Matschinsky FM, Grippo JF: Allosteric activators of glucokinase: potential role in diabetes therapy.
Science301
:370
373,2003[Abstract/Free Full Text]
- Brocklehurst KJ, Payne VA, Davies RA, Carroll D, Vertigan HL, Wightman HJ, Aiston S, Waddell ID, Leighton B, Coghlan MP, Agius L: Stimulation of hepatocyte glucose metabolism by novel small molecule glucokinase activators.
Diabetes53
:535
541,2004[Abstract/Free Full Text]
- Matschinsky FM: Regulation of pancreatic ß-cell glucokinase: from basics to therapeutics.
Diabetes51 (Suppl. 3)
:S394
S404,2002[Medline]
- Van Schaftingen E, Detheux M, Veiga da Cunha M: Short-term control of glucokinase activity: role of a regulatory protein.
FASEB J8
:414
419,1994[Abstract/Free Full Text]
- Ferrer JC, Favre C, Gomis RR, Fernandez-Novell JM, Garcia-Rocha M, de la Iglesia N, Cid E, Guinovart JJ: Control of glycogen deposition.
FEBS Lett546
:127
132,2003[Medline]
- Cadefau J, Bollen M, Stalmans W: Glucose-induced glycogenesis in the liver involves glucose 6-phosphate dependent dephosphorylation of glycogen synthase.
Biochem J322
:745
750,1997[Medline]
- Aiston S, Andersen B, Agius L: Glucose 6-phosphate regulates hepatic glycogenolysis through inactivation of phosphorylase.
Diabetes52
:1333
1339,2003[Abstract/Free Full Text]
- Aiston S, Green A, Mukhtar M, Agius L: Glucose 6-phosphate causes translocation of phosphorylase in hepatocytes and inactivates the enzyme synergistically with glucose.
Biochem J377
:195
204,2004[Medline]
- Martin WH, Hoover DJ, Armento SJ, Stock IA, McPherson RK, Danley DE, Stevenson RW, Barrett EJ, Treadway JL: Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo.
Proc Natl Acad Sci U S A95
:1776
1781,1998[Abstract/Free Full Text]
- Fosgerau K, Westergaard N, Quistorff B, Grunnet N, Kristiansen M, Lundgren K: Kinetic and functional characterization of 1,4-dideoxy-1, 4-imino-d-arabinitol: a potent inhibitor of glycogen phosphorylase with anti-hyperglycaemic effect in ob/ob mice.
Arch Biochem Biophys380
:274
284,2000[Medline]
- Ogawa AK, Willoughby CA, Bergeron R, Ellsworth KP, Geissler WM, Myers RW, Yao J, Harris G, Chapman KT: Glucose-lowering in a db/db mouse model by dihydropyridine diacid glycogen phosphorylase inhibitors.
Bioorg Med Chem Lett13
:3405
3408,2003[Medline]
- Bollen M, Keppens S, Stalmans W: Specific features of glycogen metabolism in the liver.
Biochem J336
:19
31,1998[Medline]
- Tavridou A, Agius L: Phosphorylase regulates the association of glycogen synthase with a proteoglycogen substrate in hepatocytes.
FEBS Lett551
:87
91,2003[Medline]
- Aiston S, Coghlan MP, Agius L: Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis.
Eur J Biochem270
:2773
2781,2003[Abstract/Free Full Text]
- Newgard CB, Hwang PK, Fletterick RJ: The family of glycogen phosphorylases: structure and function.
Crit Rev Biochem Mol Biol24
:69
99,1989[Abstract]
- Massillon D, Bollen M, De Wulf H, Overloop K, Vanstapel F, Van Hecke P, Stalmans W: Demonstration of a glycogen/glucose 1-phosphate cycle in hepatocytes from fasted rats: selective inactivation of phosphorylase by 2-deoxy-2-fluoro-alpha-D-glucopyranosyl fluoride.
J Biol Chem270
:19351
29356,1995[Abstract/Free Full Text]
- Bergans N, Stalmans W, Goldmann S, Vanstapel F: Molecular mode of inhibition of glycogenolysis in rat liver by the dihydropyridine derivative, BAY R3401: inhibition and inactivation of glycogen phosphorylase by an activated metabolite.
Diabetes49
:1419
1426,2000[Abstract]
- Zographos SE, Oikonomakos NG, Tsitsanou KE, Leonidas DD, Chrysina ED, Skamnaki VT, Bischoff H, Goldmann S, Watson KA, Johnson LN: The structure of glycogen phosphorylase b with an alkyldihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor.
Structure5
:1413
1425,1997[Medline]
- Oikonomakos NG, Schnier JB, Zographos SE, Skamnaki VT, Tsitsanou KE, Johnson LN: Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site.
J Biol Chem275
:34566
34573,2000[Abstract/Free Full Text]
- Rath VL, Ammirati M, Danley DE, Ekstrom JL, Gibbs EM, Hynes TR, Mathiowetz AM, McPherson RK, Olson TV, Treadway JL, Hoover DJ: Human liver glycogen phosphorylase inhibitors bind at a new allosteric site.
Chem Biol7
:677
682,2000[Medline]
- Latsis T, Andersen B, Agius L: Diverse effects of two allosteric inhibitors on the phosphorylation state of glycogen phosphorylase in hepatocytes.
Biochem J368
:309
316,2002[Medline]
- Kacser H, Acerenza L: A universal method for achieving increases in metabolite production.
Eur J Biochem216
:361
367,1993[Abstract]
- Becker TC, Noel RJ, Johnson JH, Lynch RM, Hirose H, Tokuyama Y, Bell GI, Newgard CB: Differential effects of overexpressed glucokinase and hexokinase I in isolated islets: evidence for functional segregation of the high and low Km enzymes.
J Biol Chem271
:390
394,1996[Abstract/Free Full Text]
- Aiston S, Agius L: Leptin enhances glycogen storage in hepatocytes by inhibition of phosphorylase and exerts an additive effect with insulin.
Diabetes48
:15
20,1999[Abstract]
- Thomas JA, Schlender KK, Larner J: A rapid filter paper assay for UDP-glucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP 14C-glucose.
Anal Biochem25
:486
499,1968[Medline]
- ODoherty RM, Lehman DL, Seoane J, Gomez-Foix AM, Guinovart JJ, Newgard CB: Differential metabolic effects of adenovirus-mediated glucokinase and hexokinase I overexpression in rat primary hepatocytes.
J Biol Chem271
:20524
20530,1996[Abstract/Free Full Text]
- Seoane J, Gomez-Foix AM, ODoherty RM, Gomez-Ara C, Newgard CB, Guinovart JJ: Glucose 6-phosphate produced by glucokinase, but not hexokinase I, promotes the activation of hepatic glycogen synthase.
J Biol Chem271
:23756
23760,1996[Abstract/Free Full Text]
- Horton ES, Foley JE, Shen SG, Baron MA: Efficacy and tolerability of initial combination therapy with nateglinide and metformin in treatment-naive patients with type 2 diabetes.
Curr Med Res Opin20
:883
889,2004[Medline]
- Del Prato S, Volpe L: Rosiglitazone plus metformin: combination therapy for type 2 diabetes.
Expert Opin Pharmacother5
:1411
1422,2004[Medline]
- Tan MH, Glazer NB, Johns D, Widel M, Gilmore KJ: Pioglitazone as monotherapy or in combination with sulfonylurea or metformin enhances insulin sensitivity (HOMA-S or QUICKI) in patients with type 2 diabetes.
Curr Med Res Opin20
:723
728,2004[Medline]
- Treadway JL, Mendys P, Hoover DJ: Glycogen phosphorylase inhibitors for treatment of type 2 diabetes mellitus.
Exp Opin Invest Drugs10
:439
454,2001
- Iynedjian PB: Mammalian glucokinase and its gene.
Biochem J293
:1
13,1993[Medline]
- Vandercammen A, Van Schaftingen E: Species and tissue distribution of the regulatory protein of glucokinase.
Biochem J294
:551
556,1993[Medline]
- Agius L: The physiological role of glucokinase binding and translocation in hepatocytes.
Adv Enzyme Regul38
:303
331,1998[Medline]
- Schafer JR, Fell DA, Rothman D, Shulman RG: Protein phosphorylation can regulate metabolite concentrations rather than control flux: the example of glycogen synthase.
Proc Natl Acad Sci U S A101
:1485
1490,2004[Abstract/Free Full Text]
- Kasvinski PJ, Shechosky S, Fletterick RJ: Synergistic regulation of phosphorylase a by glucose and caffeine.
J Biol Chem253
:9102
9106,1978[Abstract]
- Kosmopoulou MN, Leonidas DD, Chrysina ED, Bischler N, Eisenbrand G, Sakarellos CE, Pauptit R, Oikonomakos NG: Binding of the potential antitumour agent indirubin-5-sulphonate at the inhibitor site of rabbit muscle glycogen phosphorylase b: comparison with ligand binding to pCDK2-cyclin Acomplex.
Eur J Biochem271
:2280
2290,2004[Abstract/Free Full Text]
- Kristiansen M, Andersen B, Iversen LF, Westergaard N: Identification, synthesis, and characterization of new glycogen phosphorylase inhibitors binding to the allosteric AMP site.
J Med Chem47
:3537
3545,2004[Medline]
- Andersen B, Westergaard N: The effect of glucose on the potency of two distinct glycogen phosphorylase inhibitors.
Biochem J367
:443
450,2002[Medline]
- Board M, Bollen M, Stalmans W, Kim Y, Fleet GW, Johnson LN: Effects of C-1-substituted glucose analogue on the activation states of glycogen synthase and glycogen phosphorylase in rat hepatocytes.
Biochem J311
:845
852,1995[Medline]
- Fernández-Novell JM, Ariño J, Vilaró S, Bellido D, Guinovart JJ: Role of glucose 6-phosphate in the translocation of glycogen synthase in rat hepatocytes.
Biochem J288
:497
501,1992[Medline]
- Saltiel AR, Kahn CR: Insulin signalling and the regulation of glucose and lipid metabolism.
Nature414
:799
806,2001[Medline]
- Agius L, Peak M: Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin.
Biochem J296
:785
796,1993[Medline]