D1AtIE'J']Zl ]llZllEMiCIEI AffD
Diabetes Research and Clinical Practice 28 Suppl. (1995) S105-S108
Extrapancreatic effects of sulfonylurea drugs Kohei Kaku", Yasushi Inoue, Toshio Kaneko Third Department of Internal medicine, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, Yamaguchi 755, Japan
Abstract Extrapancreatic action of sulfonylurea (SU) drugs were extensively summarized. Hypoglycemic SU drugs stimulate glycolytic pathway and inhibit gluconeogenic pathway in the liver through regulating key enzymes such as the bifunctional enzyme PFK2jF-2,6-P2ase and PEPCK. It is possible that SUs improve the primary defects in NIDDM through both pancreatic and extrapancreatic actions. Keywords: Sulfonylurea; Extrapancreatic; NIDDM; Glycolysis; Gluconeogenesis; Fructose-2,6-bisphosphate (F-2,6-P2 )
From tolbutamide in late 1950s, many kinds of sulfonylurea (SU) drugs have appeared on the stage of non-insulin dependent diabetes mellitus (NIDDM) treatment. Although an acute administration of sulfonylureas stimulates the system for insulin-secretion from pancreas , a long-term treatment of NIDDM patients with sulfonylureas improves glucose tolerance without significant increase in insulin secretion [2,3]. This evidence suggests that SU drugs exert their action as an insulin-sensitizer, and extrapancreatic actions are involved in the anti-diabetic effect of these agents. Several observations have demonstrated that SU drugs enhance insulin action, e.g., stimulation of glucose uptake, glycolysis and glycogen synthesis, or inhibition of glucose output, primarily through affecting a postreceptor mechanism of insulin action [4-7]. Biochemical or molecular modes of
* Corresponding author.
SU actions have entirely unknown for several decades. 2. Extrapancreatic actions of SU drugs 2.1. SU receptors in hepatocytes
SU binding sites on the membrane of the rat hepatocytes were characterized using 3[H]_ glibenclamide. The binding affinities of glibenclamide, acetohexamide and tolbutamide were paralleled with their clinical potency. The inactive tolbutamide metabolite, carboxytolbutamide, and biguanide, buformin only partially displaced the tracer from the receptor. Thus, the existence of sulfonylurea binding sites in hepatocytes suggests that the liver is one of extra pancreatic action sites for sulfonylureas. 2.2. Effects of SU drugs on hepatic F-2 6-P2 production
The liver fructose-2,6-bisphosphate (F-2,6-P2) is the potent allosteric factor of phosphofructokinase-I, one of the key enzymes in hepatic glycoly-
0168-8227/95 /$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved. S S D I 0 I 6 8 - 8 2 2 7 ( 95 ) 0 I 0 7 8- R
K Kaku et al. / Diabetes Research and Clinical Practice 28 Suppl. (1995) S105-S108
sis, and is also an inhibitor of fructose-Lo-bisphophatase, a gluconeogenic enzyme [8-10]. Therefore, elevation of F-2,6-Pz level stimulates glycolysis and inhibits gluconeogenesis. The F-2,6Pz level is determined by a unique bifunctional enzyme that catalyzes both the synthesis and degradation of F-2,6-Pz. This bifunctional enzyme is a homodimer whose activities are regulated by cAMP-dependent protein kinase-catalyzed phosphorylation at a single N-terminal seryl residue . This phosphorylation results in activation of fructose-2,6-bisphosphatase (F-2,6-Pzase), and inhibition of 6-phosphofructo-2-kinase (PFK2). Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of F-2,6-Pz level. Thus, this bifunctional enzyme provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver. Tolbutamide stimulated the liver F-2,6-Pz formation in a dose-dependent manner . Glibenclamide also stimulated F-2,6-Pz formation, and its activity was 103 times more potent than tolbutamide. We also found a significant effect of other SU drugs as well as tolbutamide and glibenclamide . As a rule, the effects of sulfonylure as on their respective stimulation parallel with their hypoglycemic potency, and there is a good relationship between the stimulatory effects of SU drugs and their binding affinities to the receptor. 2.3. Effects of tolbutamide on the liver PFK2 / F2,6-P2ase Tolbutamide stimulated PFK2 activity as a function of fructose-o-phosphate concentration in a dose-dependent manner by lowering the K rn value. In contrary, tolbutamide inhibited F-2,6Fzase activity by increasing the K rn value. On the other hand, the maximum activities of the bifunctional enzyme, Vrnax , were not altered by tolbutamide . Glucagon inhibited PFK2 activity and stimulated F-2,6-Pzase activity, resulting in a decrease in the F-2,6-Pz level. Tolbutamide restored the enzyme activity suppressed by glucagon . Thus, these data clearly show that tolbutamide stimulates hepatic F-2,6-Pz production through activat-
ing the synthesizing enzyme, PFK2, and inactivating the degrading enzyme, F-2,6-P2ase. It is also demonstrated that tolbutamide acts counter-regulatory to glucagon on the bifunctional enzyme activity. 2.4. Effects of tolbutamide on the bifunctional enzyme phosphorylation The glucagon-induced phosphorylation of the hepatic bifunctional enzyme detected as 55 kDa protein. In the presence of glucagon, the bifunctional enzyme was significantly phosphorylated, and tolbutamide inhibited the bifunctional enzyme phosphorylation in the presence of glucagon in a dose-dependent manner . In the same condition as phosphorylarion study, glucagon decreased the kinase activity and reciprocally increased the phosphatase activity . Tolbutamide suppressed the effect of glucagon. In the reconstruction system using the purified enzyme, the enzyme phosphorylation was inhibited by addition of tolbutamide in a dose-dependent manner . These observations strongly suggest that hypoglycemic sulfonylureas inhibit cAMP-dependent protein kinase-catalyzing phosphorylation of the liver bifunctional enzyme PFK2/F2,6-Pzase associated with the significant regulation of these enzyme activities (Fig. 1). The inhibitory effect of the drug on the enzyme phosphorylation induces the activation of PFK2, the synthesizing enzyme Glucose
t~K2f-2~~1 F~1,6-p,as~j r ~' PFI
~F-16'P~ fl a
Fig. 1. Regulatory mechanism of sulfonylurea drugs on hepatic fructose-2,6-bisphosphate level. SU drugs inhibit cAMPdependent protein kinase-catalyzing phosphorylation of the liver bifunctional enzyme PFK2/F-2,6-P2ase.
K Kaku et al. / Diabetes Research and Clinical Practice 28 Suppl. (1995) S105-5108
- ... ........_-
Fig. 2. Sulfonylurea actions on liver glucose metabolism. SU drugs stimulate glycolytic and glycogenic pathways, and inhibit gluconeogenic pathway through regulating key enzyme activities.
of F-2,6-P2 , and the inactivation of F-2,6-P2ase, the degrading enzyme of F-2,6-P2 • The elevated F-2,6-P2 level by sulfonylureas may stimulate the glycolytic pathway and inhibit the gluconeogenic pathway in the liver. 2.5. Effects of SU drugs on hepatic glucose pathway In Fig. 2, the effects of hypoglycemic sulfonylurea on hepatic glucose pathway were summarized. So far studied, sulfonylurea stimulates glycolytic pathway and inhibited gluconeogenic pathway through regulating some key enzymes such as the bifunctional enzyme PFK2/F-2,6-P2ase [14-16] and PEPCK . Our recent study also demonstrated that sulfonylurea stimulates acetyl CoA carboxylase, a rate limiting enzyme of lipid synthesis . As a result of this effect, the increased level of malonyl CoA inhibits carnitine acyltransferase activity, a key enzyme of ketogenesis. In addition, sulfonylureas directly inhibit carnitine palmitoyltransferase activity , inhibiting ketogenesis . On the other hand, we also confirmed that SU drug-stimulated F-2,6-P2 production is observed in the muscle . Thus, it is possible that SU drugs play a role to enhance insulin action not only in the liver but also peripheral tissues such as muscle and adipose tissue.
The current concept of the pathophysiology for the deranged glucose metabolism in NIDDM can be indicated by impaired insulin secretion due to abnormal pancreatic islet J3-cell function and insulin resistance which are the primary defects in NIDDM pathogenesis. In addition, by a prolonged hyperglycemia pancreatic J3-cell function and insulin resistance further deteriorate. Sulfonylureas probably improve the primary defects in NIDDM pathogenesis through both pancreatic and extrapancreatic actions. Thus, hypoglycemic sulfonylureas are considered to be reasonable drugs for NIDDM treatment. References Grodsky, G.M., Epstein, G.R, Fanska, R. et al. (1972) Pancre~tic action of the sulfonylureas. Fed. Proc. 31, 2714-2719.  Duckworth, W.e., Solomon, 5.5. and Kitabchi, AE. (1972) Effect of chronic sulfonylurea therapy on plasma insulin and proinsulin levels. J. Clin. Endocrinol. Metab. 35, 585-591. [3) Lebovitz, RE., Feinglos, M.N., Bocholtz, H.K. et al. (1977) Potentiation of insulin action: a probable mechanism for the anti-diabetic action of sulfonylurea drugs. J. Clin. Endocrinol. Metab. 45, 601-604. [I]
K. Knku et al. / Diabetes Research and Clinical Practice 28 Suppl. (1995) Sl05-S108
 Lockwood, D.H., Maloff, B.L., Nowak, S.L. and McCaleb, M.L. (1983) Extrapancreatic effects of sulfonylureas: potentiation of insulin action through post binding mechanisms. Am. 1. Med. 74, suppl. lA, 102-108.  Simonson, D.C., Ferrannini, E, Bevilacqua, S. et al. (1984) Mechanism of improvement in glucose metabolism after chronic glyburide therapy. Diabetes 33, 838-845.  Salhanick, AI., Konowitz, P. and Amatruda, 1.M. (1983) Potentiation of insulin action by sulfonylurea in primary cultures of hepatocytes, from normal and diabetic rats. Diabetes 32, 206-212.  Fleig, W.E., Noether-Fleig, G., Fussgaenger, R. et al. (1984) Modulation by a sulfonylurea of insulin-dependent glycogenesis but not of insulin binding in cultured rat hepatoeytes. Diabetes 33, 285-290.  Van Schaftingen, E and Hers, H.G. (1980) Synthesis of a stimulator of phosphofructokinase. Biochem. Biopys. Res. Commun. 96, 1524-1531.  Furuya, E and Uyeda, K (1980) An activator of liver phosphofructokinase. Proc. Natl. Acad. Sci. USA 77, 5861-5864.  Pilkis, S.J., El-Maghrabi, M.R., Pilkis, 1. et al. (198]) Inhibition of fructose 1,6-bisphosphate by fructose 2,6bisphosphate. 1. BioI. Chern. 256, 3619-3622.  Pilkis, S.J., Regen, D.M., Stewart, H.B. et al. (1983) Rat liver 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase: A unique bifunctional enzyme regulated by cyclic AMP-dependent phophorylation. Mol. Aspect Cell. Regul. 3, 95-122.  Matsutani, A, Kaku, K and Kaneko, T. (1984) Tolbutamide stimulates fructose-2,6-bisphosphate formation in perfused rat liver. Diabetes 33, 495-498.  Hatao, K, Kaku, K, Matsuda, M. et al. (1985) Sulfony-
lure a stimulates liver fructose-2,6-bisphosphate formation in proportion to its hypoglycemic action. Diab. Res. Clin. Prac. 1, 49-53. Kaku, K, Matsuda, M., Matsutani, A et al. (1986) Effect of tolbutamide on fructose-2,6-bisphosphate, 2kinase and fructose-2,6-bisphosphatase in rat liver. Biochem, Biophys, Res, Commun. 139, 687-692. Ayame, H., Matsutani, A, Inoue, H. et al. (1995) Tolbutamide inhibits glucagon-induced phophorylation of 6phosphofructo-2-kinase / fructose-2,6-bisphosphatase in rat hepatocytes. Am. 1. Physiol. 268, E391-E396. Aoki, M., Kaku, K, Inoue, H. et al. (1992) Tolbutamide inhibits cAMP-dependent phosphorylation of liver 6phosphofructo-2-kinase.fructose-2,6-bisphosphatase. Diabetes 41, 334-338. Emoto, M., Inoue, Y, Kaku, K et al. (1993) The inhibitory effect of tolbutamide on phosphoenolpyruvate carboxykinase activity in rat hepatoma H4IIE cells. Biochem. Biophys. Res. Commun. 191,465-471. Matsuda, M., Kaku, K, Aoki, M. et al. (1991) Sulfonylurea enhances insulin-induced acetyl coenzyme, A carboxylase activity in rat adipocytes. Horm, Metab. Res. 23,209-212. Cook, G.A. (1987) The hypoglycemic sulfonylureas glyburide and tolbutamide inhibit fatty acid oxidation by inhibiting carnitine palmitoyltransferase. 1. BioI. Chern. 262, 4968-4972. Mori, K, Kaku, K, Inoue, H. et al. (1992) Effects of tolbutamide on fructose-2,6-bisphosphate formation and ketogenesis in hepatocytes from diabetic rats. Metabolism 41, 706-710. Matsuda, M., Kaku, K and Kaneko, T. (1986) Regulation of muscle fructose 2,6-bisphosphate levels by sulfonylureas. Endocrinol, lap. 33, 913-917.