Mechanism of Action of Brazilin on Gluconeogenesis in Isolated Rat Hepatocytes

Abstract

The present study was undertaken to investigate the mechanism of action of brazilin on gluconeogenesis and ketogenesis in isolated rat hepatocytes and to elucidate the hypoglycemic mechanism of brazilin. Brazilin decreased gluconeogenesis at 100 μM in hepatocytes isolated from diabetic rats. Brazilin also decreased basal and glucagon-induced gluconeogenesis in hepatocytes from normal rats. Fatty acids (octanoate or oleate)-induced gluconeogenesis was significantly reduced by brazilin, but ketogenesis was not influenced. The depletion of extracellular or intracellular calcium decreased gluconeogenesis in hepatocytes and brazilin inhibited gluconeogenesis in calcium-depleted media. Brazilin lowered dibutyryl cAMP (Bt2cAMP)-induced gluconeogenesis and the intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) level in glucagon-treated hepatocytes. It was also found that brazilin does not require calcium for inhibition of gluconeogenesis, but may inhibit the down-stream of cAMP signaling pathways. These data suggest that a decreased gluconeogenic flux in hepatocytes might at least partly contribute to the hypoglycemic effects of brazilin.

Abbreviations

STZ:streptozotocin

BSA:bovine serum albumin

SD rat:Sprague-Dawley rat

i. p.:intraperitoneal

EDTA:ethylenediaminetetraacetic acid

cAMP:adenosine 3′,5′-cyclic monophosphate

Bt2cAMP:dibutyryl cAMP

PBS:phosphate buffered saline

Key words

Brazilin - gluconeogenesis - ketogenesis - hepatocytes - cAMP - fatty acid

References

• 1 Hikino H, Taguchi T, Fujimura H, Hiramatsu Y. The validity of oriental medicine.  Planta Medica. 1977;  31 214-20
• 2 Moon C K, Lee S H, Chung J H, Won H S, Kim J Y, Khil L Y. et al . Effects of brazilin on glucose metabolism in isolated soleus muscles from streptozotocin induced diabetic rats.  Arch Pharm Res. 1990;  13 359-64
• 3 Moon C K, Lee S H, Lee M O, Kim S G. Effects of brazilin on glucose oxidation, lipogenesis and therein involved enzymes in adipose tissues from diabetic KK-mice.  Life Sci. 1993;  53 1291-7
• 4 Khil L Y, Han S S, Kim S G, Chang T S, Jeon S D, So D S. et al . Effects of brazilin on GLUT4 recruitment in isolated rat epididymal adipocytes.  Biochem Pharmacol. 1999;  58 1705-12
• 5 Pilkis S J, El-Maghrabi M R, Claus T H. Hormonal regulation of hepatic gluconeogenesis and glycolysis.  Annu Rev Biochem. 1988;  57 755-83
• 6 Boden G. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM.  Diabetes. 1997;  46 3-10
• 7 Randle P J. Regulatory interactions between lipids and carbohydrates.  Diabetes Metab Rev. 1998;  14 263-83
• 8 Unger R H, Muller W A, Faloona G R. Insulin glucagon ratio.  Trans Assoc Am Physicians. 1971;  84 122-9
• 9 Menahan L A, Wieland O. Interaction of glucagon and insulin on the metabolism of perfused livers from fasted rats.  Eur J Biochem. 1969;  9 55-62
• 10 Exton J H, Mallette L E, Jefferson L S, Wong E H, Friedman N, Miller TB J r. et al . The hormonal control of hepatic gluconeogenesis.  Recent Prog Horm Res. 1970;  26 411-61
• 11 Bonner-Weir S, Trent D F, Honey R N, Weir G C. Responses of neonatal rat islets to streptozotocin-limited β-cell regeneration and hypoglycemia.  Diabetes. 1981;  30 64-9
• 12 Berry M N, Friend D S. High yield preparation of isolated rat liver parenchymal cells.  J Cell Biol. 1969;  43 506-20
• 13 Hue L, Bartrons R. Role of fructose 2, 6-bisphosphate in the control by glucagon of gluconeogenesis from various precursors in isolated rat hepatocytes.  Biochem J. 1984;  218 165-70
• 14 Williamson J R, Corkey B E. Assay of citric acid cycle intermediates and related compounds-update with tissue metabolite levels and intracellular distribution.  Methods Enzymol. 1979;  55 200-22
• 15 Feliu J E, Mojena M, Silvestre R A, Monge L, Marco J. Stimulatory effect of vasoactive intestinal peptide on glycogenolysis and gluconeogenesis in isolated rat hepatocytes: antagonism by insulin.  Endocrinology. 1983;  112 2120-7
• 16 Peterson G L. A simplification of the protein assay method of Lowry et al. which is more generally applicable.  Anal Biochem. 1977;  83 346-56
• 17 Otto M, Breinholt J, Westergaard N. Metformin inhibits glycogen synthesis and gluconeogenesis in cultured rat hepatocytes.  Diabetes Obes Metab. 2003;  5 189-94
• 18 Rosler M, Schoner W. Antagonizing effects of phorbol 12-myristate 13-acetate on hormonally stimulated gluconeogenesis in isolated rat hepatocytes involve activity changes of pyruvate kinase.  Arch Biochem Biophys. 1990;  281 185-90
• 19 Pilkis S J, Granner D K. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis.  Annu Rev Physiol. 1992;  54 885-909
• 20 Boobis A R, Seddon C E, Nasseri-Sina P, Davies D S. Evidence for a direct role of intracellular calcium in paracetamol toxicity.  Biochem Pharmacol. 1990;  39 1277-81