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Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo - PubMed

  • ️Thu Jan 01 1998

Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo

W H Martin et al. Proc Natl Acad Sci U S A. 1998.

Abstract

An inhibitor of human liver glycogen phosphorylase a (HLGPa) has been identified and characterized in vitro and in vivo. This substance, [R-(R*, S*)]-5-chloro-N-[3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)pr opyl]-1H-indole-2-carboxamide (CP-91149), inhibited HLGPa with an IC50 of 0.13 microM in the presence of 7.5 mM glucose. CP-91149 resembles caffeine, a known allosteric phosphorylase inhibitor, in that it is 5- to 10-fold less potent in the absence of glucose. Further analysis, however, suggests that CP-91149 and caffeine are kinetically distinct. Functionally, CP-91149 inhibited glucagon-stimulated glycogenolysis in isolated rat hepatocytes (P < 0.05 at 10-100 microM) and in primary human hepatocytes (2.1 microM IC50). In vivo, oral administration of CP-91149 to diabetic ob/ob mice at 25-50 mg/kg resulted in rapid (3 h) glucose lowering by 100-120 mg/dl (P < 0.001) without producing hypoglycemia. Further, CP-91149 treatment did not lower glucose levels in normoglycemic, nondiabetic mice. In ob/ob mice pretreated with 14C-glucose to label liver glycogen, CP-91149 administration reduced 14C-glycogen breakdown, confirming that glucose lowering resulted from inhibition of glycogenolysis in vivo. These findings support the use of CP-91149 in investigating glycogenolytic versus gluconeogenic flux in hepatic glucose production, and they demonstrate that glycogenolysis inhibitors may be useful in the treatment of type 2 diabetes.

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Figures

Figure 1
Figure 1

Synthetic route to CP-91149. a, HCl-MeOH, 60 h, 23°C, then NaHCO3/H2O, 77%; b, 1.0 equiv. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl, 1.5 equiv. 1-hydroxybenzotriazole, dichloromethane, 23°C, 18 h, 100% crude; c, 2.0 equiv. NaOH, MeOH, H2O, 25°C, 2 h, 80%; d, as for b, but 1.1 equiv. dimethylamine HCl and 1.1 equiv. triethylamine in dimethylformamide, 18 h, 25°C, 62%.

Figure 2
Figure 2

Inhibition of HLGPa by CP-91149 and caffeine. HLGPa activity was measured at varied concentrations of CP-91149 (▪) or caffeine (•). The data are plotted as percent inhibition vs. concentration of compound. The data (±SEM) are from a representative experiment performed in triplicate.

Figure 3
Figure 3

The effect of glucose on the potency of CP-91149 (▪) and caffeine (•). IC50 values for HLGPa inhibition were determined at varied glucose concentrations, and then normalized by dividing the values by the IC50 value obtained in the absence of glucose. The normalized results are plotted as function of glucose concentration.

Figure 4
Figure 4

Synergistic inhibition of HLGPa by CP-91149, caffeine and theophylline. (A) HLGPa activity was measured in the presence of varied concentrations of CP-91149 under control conditions (▪), or with 240 μM caffeine (•) or 480 μM theophylline (▴) in the assay buffer. (B) HLGPa activity was measured in the presence of varied caffeine concentration under control conditions (▪), or with 1.0 μM CP-91149 (•) or 490 μM theophylline (▴) in the assay buffer.

Figure 5
Figure 5

Inhibition of glycogenolysis by CP-91149 in isolated rat hepatocytes. Isolated rat hepatocytes were preincubated with (A) 100 μM CP-91149 or DMSO or (B) 1–100 μM CP-91149 or DMSO for 5 min, then in the absence (filled bars) or presence (open bars) of 0.3 nM GGN for 60 min in buffer containing 7.5 mM glucose, followed by glycogen determination. Results (μmol/g) are the mean ± SD of triplicate determinations. ∗∗, P < 0.01; ∗∗∗, P < 0.001 between treatment (GGN, CP-91149) vs. control (no GGN); †, P < 0.05; ‡, P < 0.01; §, P < 0.001 between CP-91149 + GGN vs. control + GGN.

Figure 6
Figure 6

Inhibition of glycogenolysis by CP-91149 in primary human hepatocytes. Primary human hepatocytes were incubated with [14C-U]glucose plus 1 μM insulin, then with 10 mM glucose and (A) 50 μM forskolin (F) or 25 nM GGN (G), plus 30 μM CP-91149 as indicated, or (B) in the absence (filled bars) or presence (open bars) of 25 nM GGN, plus CP-91149 as indicated for 2 h. (A) Results (dpm 14C-glycogen) are the mean ± SEM of triplicate determinations from a representative experiment. ∗, P < 0.001 between GGN, forskolin, or CP-91149 vs. untreated control; ‡, P < 0.01 between control + GGN or forskolin vs. CP-91149 + GGN or forskolin. (B) Results are expressed relative (%) to untreated control in the absence of GGN (100%) and are the mean ± SEM from 3 to 10 independent experiments, each performed in triplicate. ∗, P < 0.05 between CP-91149 vs. untreated control; ‡, P < 0.001 between CP-91149 + GGN vs. GGN-treated control.

Figure 7
Figure 7

Dose-response of glucose lowering activity by CP-91149 in (A) obese, diabetic ob/ob mice, and (B) lean, nondiabetic control mice. Mice were treated with vehicle or CP-91149 as indicated, and plasma glucose concentration was determined 3 h later. Values (mg/dl) represent the mean ± SEM for 10–60 mice per group. ∗, Significant (P < 0.01) decrease by CP-91149 compared with the vehicle-treated group.

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