Life Sciences, Vol. 50, pp. PL-203 - PL-208 Printed in the U S A

Pergamon P r e s s

PHARMACOLOGY LETTERS Accelerated Communication

EFFECT OF TOLBUTAMIDE AND GLYBURIDE ON PYRUVATE KINASE FLUX IN ISOLATED RAT HEPATOCYTES Robert L. Judd °, Vidya V. Kunjathoor, Usha A. Pillai, Paul W. Ferguson, and Philip J. Medon Division of Pharmacology and Toxicology, School of Pharmacy, Northeast Louisiana University, Monroe, Louisiana (Submitted February 19, 1992; accepted March 2, 1992; received in final form March 26, 1992)

Abstract. The effects of two representative sulfonylureas, tolbutamide and glyburide, on pyruvate kinase (PK) flux were examined in fasted rat hepatocytes. PK flux was estimated by trapping 14Cfrom NaH~4CO3 in a 2 mM lactate pool, accounting for any incomplete trapping by parallel incubations with L-[I~4C]alanine. Glyburide (20 pM) and tolbutamide (1 mM) decreased glucose formation by 34.9% and 54.8%, respectively, from 2 mM lactate. This decrease in glucose formation was associated with a proportional decrease in pyruvate carboxylase (PCOX) flux (32.7% and 50.5%, respectively). Under these conditions, no net change in PK flux was observed. When hepatocytes were preincubated with lactate and/or sulfonylurea addition for 30 min prior to radiolabeling with NaHI'CO3, the metabolic state of the cells changed markedly. Glyburide produced a 34.6% decrease in glucose formation and a 31.3% decrease in PCOX flux, but no change in PK flux. In contrast, tolbutamide decreased glucose formation by 12.5% and increased PK flux by 53.2%, but no change in PCOX flux was observed. Such an increase in PK flux may be linked to tolbutamide-mediated increases in fructose-l,6-bisphosphate (F16P) via fructose-2,6-bisphosphate (F26P). These findings demonstrate that tolbutamide and glyburide decrease hepatic glucose production through various alterations in carbohydrate metabolism, depending upon the metabolic state of the cell. In addition, F26P may play a larger role in the hypoglycemic mechanism of action of toibutamide than glyburide, since pyruvate carboxylase accounted for most of the decrease in glucose formation observed with glyburide and because preincubation with tolbutamide resulted in an activation of PK.

Introduction The sulfonylureas are a group of oral hypoglycemic agents commonly used in the treatment of non-insulin dependent diabetes mellitus (NIDDM). The sulfonylureas reduce blood glucose, in part, by acutely stimulating pancreatic insulin secretion (1-3). This stimulation of insulin secretion was formerly thought to be the sole mechanism by which these agents were effective in the treatment of NIDDM. However, other studies indicate that the sulfonylureas are also involved in maintaining blood glucose homeostasis in the diabetic through their extrapancreatic effects (4-6). Extrapancreatic effects of the sulfonylureas have been observed in muscle, myocardial and fat tissue (4,5-7,8). However, the most profound extrapancreatic effects have been observed in the liver, primarily because of its major role in carbohydrate metabolism. Numerous studies have demonstrated that the sulfonylureas decrease hepatic glucose output (913). Further studies have linked this decrease in glucose output to an inhibition of *Corresponding Author: Dr. Robert Judd, Mayo Clinic, Endo. Res. Unit, Rochester, MN 55905 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.

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Sulfonylureas and Pyruvate Kinase Flux

Vol. 50, No. 23, 1992

gluconeogenesis from various substrates (11) and a stimulation of glycolysis and glycogenesis (12,14). Elevations in fructose-2,6-bisphosphate (F26P) (15,16), activation of glycogen phosphorylase (17), inhibition of pyruvate carboxylase (PCOX)(18) and proteolytic mechanisms (19) have all been identified as possible sites of action responsible for the gluconeogenic/glycolytic alterations observed upon administration of the sulfonylureas. The majority of these studies have focused on sites of action responsible for gluconeogenic regulation. However, relatively few studies have investigated possible sites of glycolytic regulation. To this end, pyruvate kinase (PK; EC# 2.7.1.40), a major glycolytic regulatory enzyme, may be a site of action of the sulfonylureas in the liver. Such a possibility was first proposed by Patel (12) to explain the stimulation of lactate and pyruvate production from dihydroxyacetone, glycerol and fructose observed when tolbutamide was infused into the perfused rat liver. An increase in PK activity could explain the inhibition of gluconeogenesis from lactate and pyruvate observed under these same experimental conditions. Sulfonylurea administration could increase PK activity, which would increase glycolysis and explain the decrease in hepatic glucose output observed. Additionally, PK is allosterically activated by fructose-l,6-bisphosphate (F16P). Therefore, it seems plausible that the sulfonylureas may stimulate F26P, thereby increasing F16P concentrations and subsequently increasing PK activity, resulting in an increased glycolytic rate. In this paper, the effects of two representative sulfonylureas, glyburide and tolbutamide, on PK flux were evaluated in isolated rat hepatocytes during varying periods of exposure to the gluconeogenic substrate, lactate. PK flux was determined by measuring the rate of passage of a radiolabeled tracer 14C from NaH14CO3 through the PK pathway. This method of measuring PK flux using radiolabeled tracers was chosen over the enzymatic PK activity measurements due to the better sensitivity of the flux measurements.

Materials and Methods Isolated Hepatocyte Preparation. Isolated rat liver parenchymal cells were isolated from fasted, male Sprague-Dawley rats (175-225 g) by a recirculation in situ perfusion technique as previously described (20). Viability of cells was assessed by exclusion of 0.4% trypan blue and ranged between 88 and 95 %. Data from all hepatocyte experiments were expressed as concentrations per unit wet weight of hepatocytes. Flux Studies. PK flux studies were conducted by the method of Rognstad (21). Hepatocytes (2030 mg dry weight) were incubated in 25 ml Erlenmeyer flasks stoppered with rubber serum caps from which a 500 tzl plastic well (Kontes) was suspended. Hepatocytes were: 1) incubated with 2 mM lactate as the gluconeogenic precursor (lactate group) or 2) incubated with 2 mM lactate plus the specified sulfonylurea (20 t~M glyburide or 1 mM tolbutamide - Sigma). Either NaH~4CO3 (New England Nuclear) or L-[1-~nc]alanine (ICN Radiochemicals) was added to the flasks at concentrations of 50 ~tCi and 1 tzCi, respectively. Hepatocyte treatment groups were radiolabeled immediately or preincubated for 30 rain with substrate and drug prior to addition of the radiolabeled substrates. Bicarbonate groups were then additionally incubated for 10 min and the alanine groups for 5 min. Incubations were conducted at 37°C in KHBC • 20 mM HEPES and terminated by adding 0.5 ml of 21% perchloric acid. In studies where radiolabeled alanine was utilized, 300 ~1 of 4N NaOH was injected into the center well to absorb radiolabeled carbon dioxide liberated by further shaking for 3 hr. Wells were placed in 20 ml of liquid scintillation fluid (BCS-Amersham) and radioactivity counted.

Vol. 50, No. 23, 1992

Sulfonylureas

and Pyruvate Kinase Flux

PL-205

Mctabolite Separation. Cells plus medium were diluted to 10 ml and centrifuged for 5 rain at 3000 rpm to sediment the deproteinized hepatocytes. A 9 ml aliquot of the supernatant was subjected to ion exchange chromatography. The supernatant was placed on a 0.8 X 4 cm Dowex 50 (H ÷) column (Bio-Rad) on top of a 1 X 8 cm Dowex 1 (acetate) column (Bio-Rad). After separation of the initial 9 ml aliquot was complete, both columns were washed with 21 ml of water to ensure that all of the glucose was removed from the columns. The 14C yield in lactate and pyruvate was subsequently determined by respective elutions of the original 1 X 8 acetate columns with 40 ml of 2N acetic acid and 40 ml of 3N formic acid. Eluant aliquots were added to scintillation cocktail for subsequent scintillation counting. Calculations. Glucose formation, PK flux and PCOX flux were calculated as described by Rognstad (22). Glucose formation was estimated from the isotopic yield in glucose from NaH14CO3 and the NaI-I14CO3 specific activity. PK flux was calculated as the product of 2 X rate of glucose formation X the quotient of the radioactivity in the lactate fraction and glucose fraction, multiplied by a partition factor obtained from parallel incubation with L-[1-14C]alanine (21). The alanine partition factor was calculated as the ratio of products of metabolism of L-[114C]alanine:[lactate + pyruvate + glucose + CO2]/[lactate + pyruvate] (22). PCOX flux is calculated from two times glucose formation plus PK flux. Statistics. Data from all experiments were expressed as the mean plus or minus the standard error of the mean (SEM). Sulfonylurea treatment groups were compared to the lactate control group by using an analysis of variance (ANOVA) and a Duncan's Multiple Range test were performed. The minimum significance level was set at p

Effect of tolbutamide and glyburide on pyruvate kinase flux in isolated rat hepatocytes.

The effects of two representative sulfonylureas, tolbutamide and glyburide, on pyruvate kinase (PK) flux were examined in fasted rat hepatocytes. PK f...
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