of Tolbutamide




on Fructose-2,fGBisphosphate in Hepatocytes From Diabetic Kaku, Hiroshi





Formation Rats Matsutani,

and Ketogenesis

and Toshio


To assess the extrapancreatic action of sulfonylurea directly in the diabetic, effects of tolbutamide on hepatocyte fructose-2,6-bisphosphate (F-2,6-P2) formation and ketone production were investigated using isolated hepatocytes from streptozotocin (STZ)-induced diabetic rats. The basal level of hepatocyte F-2,6-P* was significantly higher in diabetic rats within 2 weeks after STZ (40 mg/kg body weight) injection compared with that in the nondiabetic control group. Ultimately, a marked decrease in the F-2,6-Pz level was observed at 4 weeks after STZ administration (10% of the control). Although the addition of tolbutamide further increased the hepatocyte F-2,6-P* level during the first week after STZ injection, no significant effect was observed after the second week and on from the initial STZ. Treatment of diabetes with insulin restored the stimulatory effect of tolbutamide on the hepatocyte F-2,6-P* formation. Tolbutamide, independently of insulin treatment, lowered the ketone production of hepatocytes from diabetic rats. The present results indicate that insulin is necessary, in advance, for sulfonylurea to stimulate the liver F-2,6-P* formation, while tolbutamide inhibition of hepatocyte ketone production is independent of insulin. These results provide further support for the role of sulfonylurea in regulating hepatic energy metabolism in the diabetic. Copyright

0 7992 by W.B. Saunders Company


YPOGLYCEMIC sulfonylureas are used in the management of non-insulin-dependent diabetes mellitus (NIDDM). The stimulatory effect of sulfonylurea drugs on pancreatic insulin secretion’” suggested that these drugs were effective in the treatment of NIDDM primarily by elevating plasma insulin levels. On the other hand, recent reports have indicated that sulfonylurea drugs, by their extrapancreatic effects, may also directly contribute to the maintenance of blood glucose homeostasis in the diabetic. Tolbutamide increases the rate of glucose uptake in the perfused rat hindlimb. Tolbutamide also stimulated the glycolytic flux in the rat myocardium5Jj and in isolated perfused rat liver.7 Glyburide has been demonstrated to potentiate insulin-stimulated glycogenesis in cultured rat hepatocytes,8 and chlorpropamide and carbutamide inhibited the liver glycogenolysis induced by catecholamines.9 Chlorpropamide has been reported to inhibit hepatic glucose production in NIDDM.‘O Similarly, sulfonylureas inhibited gluconeogenesis in the rat liver7J1J? and also lowered the endogenous rate of hepatic ketogenesis.7J3-16 Although these studies have accumulated evidences for extrapancreatic actions of sulfonylurea drugs, effects of the drugs in the diabetic state, especially in the lack of insulin, have not been well known. The novel sugar diphosphate, fructose-2,6-bisphosphate (F-2,6-P& is an allosteric activator of 6-phosphofructo-lkinase (PFK-1) and an inhibitor of fructose-1,6-bisphosphatase. 17-19Regulation of the F-2,6-PZ level by hormones and substrates is one of the important factors in the control of glycolysis and gluconeogenesis in the liver.20 The addition of glucose to isolated hepatocytes from fed rats further increased the level of F-2,6-P2,21.22 and the addition of

lactate or pyruvate lowered the F-2,6-P2 leve1.20,23While glucagon addition to isolated hepatocytes resulted in a decrease in the F-2,6-P2 level, insulin opposed the action of glucagon.22,24 Glucagon acts through a mechanism that involves cyclic adenosine monophoshate (cAMP)-dependent phosphorylation, resulting in an inactivation of the synthesizing enzyme, 6-phosphofructo-2-kinase (PFK-2), and in an activation of the degrading enzyme, fructose-2,6bisphosphatase (F-2,6-Pase). 25,26Our previous studies demonstrated that sulfonylureas also stimulated the synthesis of the liver F-2,6-P* through activating PFK-2 and inactivating F-2,6-Pase.27-29 The hepatic F-2,6-P* level was lowered in the streptozototin (STZ)- or alloxan-induced diabetic rats.30-32However, long-term effects of diabetes on the hepatic concentration of F-2,6-Pz, have not been demonstrated. In the present study, we examined the F-2,6-P2 level and ketone production of hepatocytes from rats in the chronic phase of diabetes induced by administration of intermediate-dose STZ (40 mg/kg body weight). Effects of tolbutamide on hepatocyte F-2,6-PZ production and ketone production in diabetic rats were also investigated. To avoid the drug’s pancreatic action of stimulating endogenous insulin secretion, we used the isolated hepatocytes from diabetic rats. Furthermore, the aims of our experiment are to make clear how the sulfonylurea deranged metabolism

drug contributes

MATERIALS From the Third Department of Medicine, Yamaguchi University School of Medicine, Ube, Japan. Address reprint requests to Kohei Kaku, Third Depattment of Medicine, Yarnaguchi University School of Medicine, 1144 Kogushi, Ube 755, Japan. Copyright 0 1992 by W.B. Saunders Company 0026-049Sl92/4107-0006$03.00/O


to ameliorating


in the mild diabetes associated with a relative lack of insulin, such as in NIDDM. To answer the question, the intermediate dose of STZ was used to induce the diabetes, and a relatively low dose of insulin was used to maintain the moderate hyperglycemia. AND METHODS

Collagenase type I, trypsin inhibitor type II-S, pyrophosphate-

dependent phosphofructokinase (PPi-PFK), F-2,6-P*, oleic acid palmityl ester (palmityl oleate), tolbutamide, and STZ were purchased from Sigma (St Louis, MO). NADH, NAD, fructose-6phosphate (F-6-P), glycerin-3-phosphate dehydrogenase, triosephosphate isomerase, aldolase, and 3-hydroxybutyrate (3-HB) dehydrogenase were products of Boehringer (Mannheim, Ger-

Metabolism, Vol41,No 7(July),1992: pp 706-710




many). Hanks balanced salt solution (310-4060 10x Liquid) was obtained from Gibco Laboratories (Grand Island, NY). All other reagents were of analytical grade. Animals Seven-week-old male Wistar rats were obtained and were injected either with vehicle (0.1 kmol/L sodium citrate, pH 4.5) or vehicle containing STZ (40 mg/kg body weight) after overnight fastmg. The animals were allowed unlimited access to tap water and standard chow. After STZ injection, the degree of diabetes was monitored by measuring blood glucose levels in the evening. Two weeks after STZ injection, rats with blood glucose values greater than 300 mg/dL were identified as diabetic. Then, a group of diabetic rats were given Monotard MC insulin (6 U/d) subcutaneousiy.

Isoia tion of Hepa tocytes Isolated hepatocytes were prepared according to a modification of the method of Seglen.33 The viability of hepatocytes used in the experiments was greater than 85% as judged by the trypan blue excbision test. Incubation Study Isolated hepatocytes were preincubated in Hanks 10 mmol/L HEPES buffer, pH 7.5, with 0.5% bovine serum albumin (BSA), 5.5 mmol/L glucose, and 1 mmol/L palmityl oleate at 37°C for 15 minutes, and then centrifuged (15 seconds at 40 x g) at room temperature. The supernatant was discarded. Hepatocytes were resuspended in the same buffer with 0.5% BSA, 5.5 mmol/L glucose, and 1 mmol/L palmityl oleate, and were incubated in various concentrations of tolbutamide at 37°C. After 15 minutes of incubation, the samples were put on ice to stop the reaction. We made preliminary measurements of F-2,6-P2 and 3-HB levels in hepatocytes at various time points in a l-hour incubation, and confirmed that these parameters were linearly increased for the first 30 minutes. Therefore, all measurements to evaluate the effects of tolbutamide were performed at 15 minutes of incubation. One half of the sample in each tube was homogenized at 4°C and kept at -40°C until needed for 3-HB determination. The remainders were centrifuged (60 seconds at 170 x g) at 4°C. The pellet was homogenized in the mixed solution with 800 FL of 1 mmol/L EGTA, pH 7.4, containing 10 mmol/L MgClz plus 200 FL of 0.5N NaOH by sonication with pulsed 50% duty cycle for 1 minute (Sonicator Model W2OOP, Heat Systems-Ultrasonics, Plainview, NY). The homogenate was heated at 80°C for 20 minutes, and then the same volume of 400 mmol/L TRIS-HCI buffer, pH 7.5, was added. After centrifugation at 17.500 x g for 5 minutes at room temperature, the supernatant was kept at -40°C until needed for F-1!.6-P2determination. All experiments were performed in triplicate. Assays for F-2,6-P,

3-HB was assayed by a modification of the method of Williamson et al.35 3-HB dehydrogenase was added to the deproteinized sample of homogenized hepatocytes, which were previously incubated with or without tolbutamide for 15 minutes as described in the incubation study, in 0.1 mmol/L TRIS-HCI buffer, pH 8.5, containing 2.7 mmol/L EDTA and 3.5 mmol/L NAD. To determine a total level of 3-HB produced in hepatocytes incubated for 15 minutes, the reaction should be completed. We first confirmed that the reaction reached a plateau within 10 minutes after addition of 3-HB dehydrogenase to the sample. This indicates that 3-HB, a substrate of the enzyme produced in hepatocytes for 15 minutes incubation, was completely exhausted in the reaction within 10 minutes. Therefore, the reaction was performed for 10 minutes, and production of NADH was estimated spectrophotometrically. Assays for Insulin and Glucagon Rat plasma insulin and glucagon were determined by radioimmunoassay kits (Dai-ichi Radioisotope, Tokyo, Japan). RESULTS

Body Weight, Blood Glucose, and Plasma Insulin and Glucagon of STZ-Induced Diabetic Rats Changes in body weight, blood glucose, and plasma insulin and glucagon of rats after STZ injection are shown in Fig 1. While normal rats increased in body weight with age, STZ-induced diabetic rats did not gain weight. On the other hand, an increase in body weight was observed in




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and 3-HB

F-2,6-P2 was assayed by the modified method of Furuya and Uyeda.34 The reaction mixture in a final volume of 1 mL consisted of 54 mmol/L TRIS-HCl, pH 9.0, 1 mmol/L F-6-P, 0.2 mmol/L NADH, 7.5 mmol/L dithiothreitol, 0.5 mmol/L EDTA, 0.01 U PPi-PFK, 0.4 U aldolase, 1 U glycerin-3-phosphate dehydrogenase, and 3 U triosephosphate isomerase. The sample (20 ~.LL)and 0.5 mmol/L PPi (50 ~.LL)were added to 930 l.r,Lof the reaction mixture in the assay cuvette. The reaction velocity was determined spectrophotometrically at 37°C and compared with that determined by using a known amount of standard F-2,6-P*.





b t


age (vieek)


9 a9e (week)


Fig 1. Body weight, blood glucose, and plasma insulin and glucagon of nondiabetic IO), untreated ST&diabetic (0). and insulintreated diabetic rats (A). Each value indicates the mean 2 SD (n = 6). lp c .06, lV c .Ol versus 7-week. tP < .06, ttP c .Ol vetsus nondiabetic.



diabetic rats treated with insulin. Blood glucose levels were 320 + 15 mg/dL (mean & SD, n = 6) at 2 weeks and 430 -r23 mg/dL at 4 weeks after STZ injection. Insulin treatment (6 U/d) significantly lowered the blood glucose concentration of diabetic rats, but it was still higher than that of normal rats (P < .Ol). The plasma insulin level at 2 weeks after the STZ injection was significantly lowered, but still detectable, from 81 ? 21.2 to 5.1 f 5.2 pU/mL (mean t SD, n = 6, P < .OOl). However, plasma insulin at 5 weeks after STZ was undetectable by our assay system. The plasma glucagon level of diabetic rats at 2 weeks after STZ injection was significantly increased up to 2.2 times the level before STZ, and at 5 weeks after STZ injection, the glucagon level was still higher than that of the normal rats. Hepatoqte F-2,6-PJ in STZ-Induced Diabetic Rats

A significant increase in the basal level of hepatocyte F-2,6-Pz was observed in the diabetic rats within 2 weeks after STZ injection (Fig 2). The diabetic rats had approximately 40% higher F-2,6-P2 levels at 1 week and 30% higher levels at 2 weeks after STZ injection compared with those of the nondiabetic animals. However, hepatocyte F-2,6-P2 levels were decreased at 3 weeks or more after STZ. The basal level of hepatocyte F-2,6-P* at 4 weeks after STZ injection was extremely lower than that in the nondiabetic rats (61 + 8.6 pmol/mg protein in nondiabetic controls, and 5.9 ? 2.2 in diabetic rats; mean +- SD, n = 6, P < ,001). On the other hand, the basal level of hepatocyte F-2,6-P2 was restored to the control level or higher in the insulintreated rats. Effects of Tolbutamide on Hepatocyte F-2,6-P2 Formation

Tolbutamide stimulated hepatocyte F-2,6-P2 formation in nondiabetic rats in a dose-dependent manner. The significant effect of tolbutamide was observed at the concentration of 0.5 to 2 mmol/L of the drug (data not shown). This observation reconfirmed our previous result.*’ As

shown in Fig 2, the addition of 2 mmol/L tolbutamide stimulated F-2,6-P2 formation in hepatocytes from diabetic rats at 1 week after STZ injection. However, this stimulatory effect was not observed in hepatocytes from diabetic rats at 2 weeks or more after STZ injection. On the other hand, tolbutamide further increased F-2,6-PZ levels in hepatocytes from diabetic rats treated with insulin for 2 weeks. Effects of Tolbutamide on Hepatocyte 3-HB Production

Plasma 3-HB concentration in STZ-induced diabetic rats was extremely higher than that in nondiabetic animals (2.4 ? 0.9 mmol/L and 0.2 5 0.07 mmol/L; mean 4 SD, n = 6, P < .OOl). Fig 3 shows the dose-dependent manner of tolbutamide effects on the 3-HB production in isolated hepatocytes from nondiabetic and diabetic rats. The 3-HB production was determined as the total level of 3-HB in hepatocytes incubated for 15 minutes. We also determined 3-HB levels in the incubation medium, and confirmed that the levels in the incubation medium were in parallel with those in hepatocytes (data not shown). The basal levels of hepatocyte 3-HB were significantly higher in diabetic rats than in nondiabetic rats. A significant decrease in hepatocyte 3-HB levels was observed in both untreated and insulin-treated diabetic rats by the addition of tolbutamide. DISCUSSION

The present study observed the changes in hepatocyte F-2,6-P* level in the development of diabetes. Direct effects of tolbutamide on hepatocyte F-2,6-P2 formation and 3-HB production in the diabetic state were also evaluated, using isolated hepatocytes from STZ-induced diabetic rats. The previous reports demonstrated that the liver F-2,6-Pz level was greatly reduced in severely diabetic rats, resulting in an increased rate of gluconeogenesis.30-32 On the other hand, our data indicated that the basal level of hepatocyte F-2,6-Pr varied in the development of diabetes after STZ


Effects of tolbutamide on fructose-2,6-bisphosphate formation and ketogenesis in hepatocytes from diabetic rats.

To assess the extrapancreatic action of sulfonylurea directly in the diabetic, effects of tolbutamide on hepatocyte fructose-2,6-bisphosphate (F-2,6-P...
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