BIOCHEMISTRY ondmolecular BIOLOGY INTERNATIONAL Poges

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1 Vol. 40, No. 3, October 1996 BIOCHEMISTRY ondmolecular BIOLOGY INTERNATIONAL Poges ATTENUATION BY NICOTINAMIDE OF THE STREPTOZOCIN INDUCED EARLY HYPERGLYCAEMIA IN FASTING RATS Kai K. Wong Department of Pharmacology, National Yang Ming University Taipei, Taiwan, Republic of China ReceivedJulyll, 1996 Received after~vision August28,1996 SUMMARY: The effect of nicotinamide on the streptozocin induced early hyperglycaemia in 48-hour fasted rats was investigated. It was shown that 240 or 400 mg/kg nicotinamide significantly attenuated the streptozocin induced early hyperglycaemia. Nicotinamide should be administered no later than I hour after the streptozocin administration. Lower level doses of nicotinamide such as 80 mg/kg were unable to attenuate the streptozocin induced early hyperglycaemia. The possibility of nicotinamide offsetting the streptozocin effect was discussed. INTRODUCTION Streptozocin (STZ) induces an irreversible increase in blood glucose concentration in mice, rats, dogs, and monkeys (1,2). After an intravenous administration of STZ in rats, an early hyperglycaemic phase appears, followed by a hypoglycaemic phase and then a permanent diabetic phase at approximately 4, 7 and 24 hours, respectively (3,4). The action of STZ in the induction of an early hyperglycaemic phase may be mechanistically different to that of the permanent diabetic phase which is speculated to be the result of a cytotoxic action by STZ on pancreatic beta cells (2,5). The early hyperglycaemic phase which appears 4 hours after STZ administration is not attributed to an increase in insulin release (4) nor to an increase of glucagon secretion from the pancreas (6). Moreover, the levels of serum and pancreatic immunoreactive insulin detected 4 hours after STZ administration are not significantly different from those found before STZ administration, even though the pancreatic immunoreactive insulin is reduced 24 hours after STZ admission (5,7). 515 i /96/ /0 Copyright 1996 by Academic Press Australia. All rights of reproduction in any fi~rm reserved.

2 Vol. 40, No. 3, 1996 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Anderson et al. postulate that the diabetogenic effect of STZ may be related to its interference in pyridine nucleotide metabolism (8). Loftus et al. (9) have suggested that STZ may induce a decrease in pancreatic NAD concentration. If the administration of STZ induces a decrease in pancreatic NAD +, an increase of pancreatic NAD + induced by nicotinamide may be able to offset the STZ induced NAD + degration and result in an attenuation of the STZ effect. It is reported that if nicotinamide dissolved as a 5% aqueous solution is injected subcutaneously in a dose of 0.5 mg/g body weight of mice daily, nicotinamide prevents the develop- ment of diabetes in nonobese diabetic mice (10). Nicotinamide is reported to protect C57BL/KsJ mice from developing the acute phase of STZ induced hyperglycaemia, although it was unsuccessful in preventing 13 out of 18 test mice from eventually developing hyperglycaemic and pancreatic insuli- tis (11). Kaplan et al. (12,13) showed that the livers of rats and mice had increased NAD + levels after intraperitoneal administration of nicoti- namide. The purpose of this study was an attempt to demonstrate whether nicotinamide treatment would attenuate the magnitude of STZ induced early hyperglycaemia. MATERIALS AND METHODS Ten week old male Sprague Dawley rats were fasted for 48 hours. The STZ solution was prepared by dissolving STZ in saline solution (0.9% NaCl) containing 50 mm citrate with ph adjusted to 4.5. The STZ solution was administered intravenously (60 mg/kg) into the tail vein of fasting rats. Glucose in the blood collected from the tail vein was determined by means of a Reflolux II glucose analyser (Boehringer, Germany). Nicotinamide was dissolved in saline solution before being administered intraperitoneally. Blood glucose at 4 hours after the STZ administration was expressed as a ratio of the control before the STZ administration. STZ and nicotinamide were purchased from Sigma Chemical Co. (St. Louis, Mo. USA). ANOVA was used for statistical analysis. RESULTS In order to evaluate whether nicotinamide affected the magnitude of the STZ induced early hyperglycaemia under fasting conditions used, 48- hour fasted rats were treated with various doses of nicotinamide before 516

3 Vol. 40, No. 3, 1996 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL STZ administration. It was shown that 80 mg/kg nicotinamide was unable to change the magnitude of the STZ induced early hyperglycaemia, but 240 mg/kg or 400 mg/kg nicotinamide were effective to attenuate the STZ induced early hyperglycaemia (Fig. I). It was also found that pretreatment with nicotinamide (400 mg/kg) I hour before or immediately before the STZ administration, attenuated the STZ induced early hyperglycaemia 4 hours after the STZ administration. However, nicotinamide (400 mg/kg) adminis- tration I hour after STZ administration was ineffective (Fig. 2). These data suggested that the nicotinamide effect is dose dependent, and the effective nicotinamide concentration may be above 80 mg/kg. Moreover, the nicotinamide effect was also time dependent, because it was insignificant when the nicotinamide was applied I hour after the streptozocin administration. DISCUSSION Since nicotinamide in a dose of 80 mg/kg was ineffective and a further increase of nicotinamide admission is required in order to attenuate the STZ induced early hyperglycaemia, the nicotinamide effect is judged to be concentration dependent. Kaplan et al. showed that nicotinamide administration (500 mg/kg) induces a 10 fold increase in the hepatic NAD level (12,13). Therefore, it is presumed that nicotinamide treatment in this investigation will induce an increase of hepatic NAD, even though the hepatic NAD content was not determined. It is reported that the hepatic NAD is decreased I hour after STZ administration (14,15). If nicotinamide is administered I hour after STZ administration, the STZ induced hepatic NAD degradation has already begun, and the protective effect of externally admitted nicotinamide is likely to be diminished and/or ineffective. It is of interest to note that adminis- tration of 400 mg/kg nicotinamide I hour after STZ administration is unable 517

4 Vol. 40, NO. 3, 1996 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL o "~ 80 _ ~ 6o,o 0 o 4 Time (hours) Fig. I. Attenuation by nicotinamide of the streptozocin induced early hyperglycaemia in rats fasted for 48 hours. Ordinate: blood glucose concentration (mg/dl). Abscissa: time after streptozocin administration (60 mg/kg, iv). Nicotinamide was administered intraperitoneally immediately before streptozocin administration. Open column, without nicotinamide pretreatment; shaded, crossed, and solid columns, with nicotinamide treatment of 80 mg/kg, 240 mg/kg, and 400 mg/kg, respectively. Bar is mean±sem (n = 5). Asterisk represents a significant difference, p < to reduce the STZ induced early hyperglycaemia, confirming the claim that the process is time dependent. The protective effect by nicotinamide against the STZ action in inducing early hyperglycaemia may be due to the preservation of cellular NAD levels, since nicotinamide prevents NAD destruction (16). Changes in the NADH/NAD ratio may affect the maintenance of the overall cellular redox potential. A decrease in the relative NAD level may facilitate cellular anabolism and enhance glucose biosynthesis; while an increase in the relative NAD content may facilitate glucose dissimila- tion through glycolysis and tricarboxylic acid cycle reactions. If STZ decreases the hepatic NAD content, then the resulting increase in the NADH/NAD ratio may favour glucose biosynthesis. Moreover, the administration of STZ is expected to lead to enhanced hepatic gluconeogenesis and hepatic glucose availability and secretion. This speculation may account 518

5 VOI. 40, NO. 3, 1996 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL 140 m 120 ~ ' Time (hours) Fig. 2. Lack of attenuation, by delayed intraperitoneal nicotinamide administration, of the streptozocin induced early hyperglycaemia in rats fasted for 48 hours. Ordinate: blood glucose concentration (mg/dl). Abscissa: time after streptozocin administration (60 mg/kg,iv). Open column, without nicotinamide treatment; shaded column, with nicotinamide pretreatment immediately before streptozocin; crossed column, with nicotinamide pretreated at I hour before streptozocin administration; solid column, with nicotinamide postinjected I hour after streptozocin administration. Bar is mean±sem (n = 5). Asterisk represents a significant difference, p < for the reason that blood glucose in fasting rats is still increased during the STZ induced early hyperglycaemic phase, even though hepatic glycogen is almost depleted (17). Moreover, an attenuation by nicotinamide of the STZ induced early hyperglycaemia may be due to an increase in hepato- cyte NAD following the nicotinamide administration to offset the loss of cellular NAD + induced by STZ. These latter speculations are the subject of further investigation. REFERENCES I. Rakieten, N., Rakieten, M.L. and Nadkarni, M.V. (1963) Cancer Chemother. Rep. 29, Arison, R.N., Ciaccio, E.I. and Clitzer, M.S. (1967) Diabetes 16, Ounod, A., Lambert, A.E., Orci, L., Pictet, R., Gonet, A.E. and Renold, A.E. (1967) Proc. Soc. Exp. Biol. Med. 126,

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