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0022-3565/02/3013-930 –937$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics JPET 301:930–937, 2002 Vol. 301, No. 3 4648/985115 Printed in U.S.A. Nicotine Increases Hepatic Oxygen Uptake in the Isolated Perfused Rat Liver by Inhibiting Glycolysis BRIAN J. DEWAR, BLAIR U. BRADFORD, and RONALD G. THURMAN Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina Received October 15, 2001; accepted February 13, 2002 This article is available online at http://jpet.aspetjournals.org Currently, it is estimated that more than 1.1 billion people smoke tobacco worldwide (Sellers, 1998). Of the 2000 components of tobacco, -pyridyl-␣-N-methyl-pyrrolidine, nicotine, is one of the principal ingredients (Fukumoto et al., 1997). A commonly reported effect of smoking is that upon commencement, individuals tend to lose weight, whereas cessation of smoking leads to weight gain (Troisi et al., 1991). This phenomenon also occurs in experimental animals (Ashakumary and Vijayammal, 1997) and indicates that nicotine affects energy metabolism, but mechanisms responsible for this remain unclear. Many studies have documented the effects of nicotine on metabolic events in the body. Nicotine administration in rats as well as in humans increases serum cholesterol, triglycerides, phospholipids, and free fatty acids (Latha et al., 1988; Ashakumary and Vijayammal, 1997). Moreover, overall fat oxidation correlates positively with excretion of the primary metabolite of nicotine, cotinine, indicating that smokers burn more lipid (Jensen et al., 1995). In addition, acute nicotine exposure causes a rapid increase in circulating catecholamines (Grunberg et al., 1988; Anderson et al., 1993), and administration of nicotine decreases insulin levels in This work was supported, in part, by a grant from R. J. Reynolds Tobacco Company. nicotine. Infusion of atractyloside, potassium cyanide, or glucagon blocked the nicotine-induced increase in respiration. Intracellular calcium was increased in isolated hepatocytes by nicotine, a phenomenon prevented by incubation of cells with d-tubocurarine, a nicotinic acetylcholine receptor antagonist. Respiration was also increased ⬃30% in hepatocytes isolated from fed rats by nicotine, whereas hepatocytes isolated from fasted rats showed little response. In the presence of N-[2-(pbromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), an inhibitor of cyclic AMP-dependent protein kinase A, nicotine failed to stimulate respiration. These data support the hypothesis that inhibition of glycolysis by nicotine increases oxygen uptake due to an ADP-dependent increase in mitochondrial respiration. rats (Grunberg et al., 1988). The direct effects of nicotine on hepatic metabolic functions are not well documented; therefore, the purpose of this study was to assess changes in hepatic oxygen and carbohydrate metabolism due to nicotine in the isolated perfused rat liver. The perfused liver was used to exclude possible effects of hormones and metabolites from other organs on hepatic intermediary metabolism. Materials and Methods Animals. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 176 to 200 g were used for all experiments and were allowed free access to laboratory chow and tap water. Animals were fasted in suspended wire cages 24 h before perfusion to prevent coprophagia. All animals were given humane care in compliance with institutional guidelines. Liver Perfusion. Details of the liver perfusion technique have been described elsewhere (Thurman et al., 1979). Briefly, livers were perfused with Krebs-Henseleit bicarbonate buffer (pH 7.4, 37°C) saturated with an oxygen/carbon dioxide mixture (95:5) in a nonrecirculating system. Perfusate was delivered at flow rates of approximately 4 ml/g liver weight/min via a cannula inserted in the portal vein. Perfusate exited the liver via a cannula placed in the inferior vena cava and was channeled past a Teflon-shielded, Clark-type oxygen electrode. Oxygen uptake was calculated from influent minus effluent oxygen concentration differences, the flow rate, and wet ABBREVIATIONS: ANOVA, analysis of variance; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide. 930 Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017 ABSTRACT Nicotine influences energy metabolism, yet mechanisms remain unclear. Since the liver is one of the largest organs and performs many metabolic functions, the goal of this study was to determine whether nicotine would affect respiration and other metabolic functions in the isolated perfused liver. Infusion of 85 M nicotine caused a rapid 10% increase in oxygen uptake over basal values of 105 ⫾ 5 mol/g/h in perfused livers from fed rats, and an increase of 27% was observed with 850 M nicotine. Concomitantly, rates of glycolysis of 105 ⫾ 8 mol/g/h were decreased to 52 ⫾ 9 mol/g/h with nicotine, whereas ketone body production was unaffected. Nicotine had no effect on oxygen uptake in glycogen-depleted livers from 24-h fasted rats. Furthermore, addition of glucose to perfused livers from fasted rats partially restored the stimulatory effect of Nicotine and Oxygen Uptake Results Effects of Nicotine on Oxygen Uptake in Perfused Livers from Fed and Fasted Rats. Results from a representative experiment demonstrating the effects of nicotine on oxygen uptake in perfused liver are shown in Fig. 1A. Infusion of 85 M nicotine caused an immediate 10% increase in respiration from a basal level of 100 mol/g/h to a value of 110 mol/g/h in a perfused liver from a fed rat. Infusion of higher concentrations of nicotine (850 M) increased oxygen consumption to a maximal value around 124 mol/g/h. Average basal rates of respiration in perfused livers from fed rats were 105 ⫾ 5 mol/g/h. When 85 and 850 M nicotine were infused, average rates were increased to 116 ⫾ 6 and 134 ⫾ 8 mol/g/h, respectively (Table 1). In contrast, in livers from 24-h fasted rats, basal rates of oxygen consumption (113 ⫾ 6 Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017 tissue weight. In experiments using livers from fed animals, samples of effluent perfusate were collected and analyzed for glucose, pyruvate, lactate, -hydroxybutyrate, and acetoacetate by standard enzymatic techniques (Bergmeyer, 1988). In experiments with livers from fasted animals, only -hydroxybutyrate and acetoacetate were measured. (⫺)-Nicotine (Sigma-Aldrich, St. Louis, MO) was dissolved in Krebs-Henseleit buffer and infused with a precision infusion pump. Six concentrations ranging from 85 to 850 M nicotine were infused in a step-wise fashion for 6 min each. Hepatocyte Isolation and Respiration. Hepatocytes from fed and fasted rats were isolated by standard techniques described elsewhere (Qu et al., 1999). Briefly, livers were perfused with a KrebsRinger-HEPES buffer containing collagenase (Sigma-Aldrich). Livers were isolated and cells were dispersed by gentle shaking and filtered through sterile nylon gauze. The cells were washed two times with sterile phosphate-buffered saline and then purified by centrifugation in 50% isotonic Percoll (Sigma-Aldrich). Cells were resuspended with Krebs-Ringer-HEPES ⫹ Ca2⫹ buffer to a total volume of 10 ml. Viability was validated via trypan blue exclusion and routinely exceeded 90%. Cells were incubated with or without 10 M N-[2-(pbromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) (BioMol Co., La Jolla, CA) in the presence or absence of 1 mM nicotine. Respiration was measured polarographically at 37°C in a closed well with a Clark-type oxygen electrode and calculated in micromoles of O2 per minute per gram of wet weight cells. Mitochondrial Isolation and Respiration. Mitochondria were isolated from rat livers by standard techniques of differential centrifugation. Briefly, livers from fed rats were rapidly removed after decapitation and homogenized using a Teflon-glass homogenizer. Nuclear and cellular debris were removed by centrifugation at 2,000g for 10 min and the supernatant was centrifuged at 10,000g for 10 min. The resulting mitochondrial pellet was washed three times. After the last centrifugation, the pellet was resuspended to a final concentration of approximately 1.4 mg of protein/ml. Respiration was measured at 25°C with a Teflon-shielded, Clark-type oxygen electrode in 2 ml of reaction buffer, pH 7.2. After addition of 1 mM rotenone, state 4 respiration was initiated by addition of succinate to a final concentration of 2.5 mM. After a steady rate of oxygen consumption was observed, state 3 respiration was initiated by adding ADP (final concentration 0.25 mM). Rates of oxygen uptake were calculated as nanomoles of O2 per minute per milligram of protein. Calcium Measurement. Isolated hepatocytes were plated on collagen-coated cover slips at a concentration of 5 ⫻ 105 cells per plate for 4 h in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, and sometimes 500 M d-tubocurarine (Sigma-Aldrich). Fura-2/acetoxymethyl ester (Molecular Probes, Eugene, OR) was added to plates at a final concentration of 5 M and incubated for 30 min at room temperature. The cells were washed and placed in a chamber with Krebs-Ringer-HEPES buffer ⫹ Ca2⫹. Calcium uptake was monitored in at least four viable cells per isolation, and there were seven rats in each treatment group. Changes in fluorescence intensity of Fura-2 at excitation wavelengths 340 and 380 nm were monitored using a dual-wavelength fluorescence imaging system (Intracellular Imaging Inc., Cincinnati, OH). Statistics. Statistical comparisons were made using Student’s t test or analysis of variance (ANOVA), and post hoc comparisons were made in pairwise fashion using Tukey’s method as appropriate. A value of p ⬍ 0.05 was selected before the study as the level of significance. 931 Fig. 1. The effect of nicotine infusion on oxygen uptake and lactate ⫹ pyruvate production in perfused livers. Livers were perfused with KrebsHenseleit bicarbonate buffer (pH 7.4) at 37°C in a nonrecirculating system as described under Materials and Methods. A, after steady-state oxygen uptake was established in perfused livers from fed and fasted rats, nicotine was infused in a step-wise fashion from 85 M to 850 M using a precision infusion pump. Oxygen uptake was calculated from the difference in influent minus effluent O2 concentration, flow rate, and the liver weight. Typical experiment. B, samples of effluent perfusate were collected every 2 min and assayed enzymatically for lactate and pyruvate as described under Materials and Methods. F, rates of lactate ⫹ pyruvate production. Each point is a mean from four experiments. 932 Dewar et al. TABLE 1 Effect of nicotine on oxygen uptake, ketogenesis, glucose production, and lactate ⫹ pyruvate production in perfused livers from fed and fasted rats Nicotine at concentrations indicated was infused into livers perfused with Krebs-Henseleit bicarbonate buffer saturated with oxygen/carbon dioxide mixtures (95:5) in a nonrecirculating system. Oxygen uptake was calculated from influent minus effluent oxygen concentration differences, the flow rate and tissue wet weight. Samples of effluent perfusate were collected and analyzed for metabolites as described under Materials and Methods. The cytosolic redox ratio (L/P) was calculated from lactate and pyruvate concentrations in the perfusate. Results are expressed as mean ⫾ S.E.M., with n in parentheses. Treatment Oxygen Uptake Ketone Bodies Glucose L⫹P L/P 102 ⫾ 10 (5) 90 ⫾ 10 (5) 88 ⫾ 8 (5) 105 ⫾ 8 (5) 93 ⫾ 8 (5)b 52 ⫾ 9 (5)a 6 ⫾ 1 (4) 13 ⫾ 3 (4) 11 ⫾ 2 (4) mol/g/h Fed Basal 85 M Nicotine 850 M Nicotine Fasted Basal 85 M Nicotine 850 M Nicotine 105 ⫾ 5 (5) 116 ⫾ 6 (5) 134 ⫾ 8 (5)a 13 ⫾ 3 (5) 10 ⫾ 2 (5) 9 ⫾ 2 (5) 113 ⫾ 6 (5) 114 ⫾ 7 (5) 110 ⫾ 8 (5) 26 ⫾ 3 (6)c 23 ⫾ 4 (3) 18 ⫾ 5 (3) p ⬍ 0.05 for comparison with basal. p ⬍ 0.05 for comparison with higher concentration of nicotine (850 M) in the same column by ANOVA with post hoc comparisons in pair-wise fashion using Tukey’s method. c p ⬍ 0.05 for comparison of rates between nutritional status by Student’s t test. a b Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017 mol/g/h) were not altered by any concentration of nicotine studied (Fig. 1A and Table 1). Changes in Carbohydrate Metabolism due to Nicotine in Perfused Livers from Fed Rats. In the fed state, rat livers release glucose, lactate, and pyruvate at high rates from endogenous glycogen (Ross et al., 1967). Basal rates of glucose output as well as lactate ⫹ pyruvate production were both around 100 mol/g/h (Table 1). Infusion of nicotine decreased rates of glucose output slightly, whereas rates of glycolysis were decreased dramatically (Fig. 1B). Average basal values of 105 ⫾ 8 mol/g/h lactate ⫹ pyruvate production were lowered significantly to 52 ⫾ 9 mol/g/h by nicotine (850 M) (Table 1). When the increase in oxygen uptake was plotted against the decreases in glycolysis, a positive relationship was observed (r ⫽ 0.933) (Fig. 2). The cytosolic redox ratio, calculated by lactate/pyruvate production, was not affected by any concentration of nicotine (Table 1). Effect of Nicotine on Ketogenesis. Ketone bodies (i.e., acetoacetate and -hydroxybutyrate) are released from liver when lipids are metabolized (McGarry and Foster, 1971). To determine the effects of nicotine on fatty acid metabolism in the liver, ketone body production was monitored. Basal rates of ketone body release were significantly greater in perfused livers from fasted compared with fed rats as expected (Table 1). Infusion of nicotine in a step-wise fashion up to 850 M did not significantly alter rates of ketone body release in perfused livers from either fed or fasted rats (Table 1). The Effect of Glucose on Nicotine-Induced Changes in Oxygen Uptake. In contrast to perfused livers from fed rats, oxygen uptake was not affected by infusion of nicotine in perfused livers from fasted rats (Fig. 1A). Since glycogen is absent, livers from fasted rats produce glucose, lactate, and pyruvate at minimal rates. Therefore, livers from fasted rats were perfused with an exogenous source of carbohydrate, glucose, providing enough substrate to stimulate glycolysis (Thurman and Scholz, 1977). When glucose (50 mM) was infused, basal rates of O2 uptake significantly increased 30% to a rate of 132 ⫾ 5 mol/g/h (p ⬍ 0.05 by ANOVA). Under these conditions, average rates of 140 ⫾ 5 and 148 ⫾ 4 mol/g/h were achieved with the infusion of 85 and 850 M nicotine, respectively. Representative data of the effects of nicotine (85 and 850 M) in perfused livers from fasted rats in the presence of glucose (50 mM) are shown in Fig. 3A. Fig. 2. Relationship between increase in oxygen uptake and decrease in lactate ⫹ pyruvate after infusion of nicotine. Increases in oxygen uptake were plotted against decreases in glycolysis due to nicotine. Points (F) show mean data (n ⫽ 5) for each concentration of nicotine used in perfusion experiments with livers from fed rats (r ⫽ 0.933). As expected, initial rates of lactate ⫹ pyruvate production were low. When glucose was added, the average lactate ⫹ pyruvate output was increased approximately 10-fold to a rate of 57 ⫾ 1 mol/g/h. Infusion of 850 M nicotine de- Nicotine and Oxygen Uptake 933 creased lactate ⫹ pyruvate production to 28 ⫾ 2 mol/g/h (Fig. 3B) (p ⬍ 0.05 by ANOVA). Inhibition of the Electron Transport Chain Blocks Nicotine-Induced Increases in Oxygen Uptake. To determine whether increases in oxygen uptake were of mitochondrial origin, perfused livers from fed animals were infused with potassium cyanide (2 mM), a potent inhibitor of the mitochondrial respiratory chain. Basal rates of 122 ⫾ 9 mol O2/g/h were decreased to 38 ⫾ 3 mol/g/h by KCN (2 mM), similar to results reported elsewhere (Thurman and Scholz, 1969) (Fig. 4A). Under these conditions, infusion of nicotine had little effect on O2 uptake. Oxygen uptake returned to normal levels of 101 ⫾ 6 mol/g/h after termination of KCN infusion. Isolated mitochondria were incubated with concentrations of nicotine ranging from 0.05 to 1.25 mM. Control values for state 4 and state 3 respiration were 33 ⫾ 2 and 170 ⫾ 17 nmol O2/min/mg protein, respectively. No significant change in mitochondrial respiration was observed with any concentration of nicotine used (data not shown). Fig. 4. Effect of potassium cyanide and atractyloside on nicotine-induced increases in oxygen uptake. Livers from fed rats were perfused with Krebs-Henseleit bicarbonate buffer (pH 7.4) at 37°C in a nonrecirculating system. KCN (2 mM, A) or atractyloside (50 M, B) dissolved in buffer (pH 7.4) was infused as indicated by arrows. Nicotine was infused at concentrations indicated. Oxygen uptake was calculated from the difference in influent minus effluent O2 concentration, flow rate, and the liver weight. Figure depicts representative data (n ⫽ 4). Inhibition of the Adenine Nucleotide Translocase Blocks the Increase in Oxygen Uptake due to Nicotine. Rates of mitochondrial respiration are controlled, in part, by ADP availability through the adenine nucleotide translocase in the mitochondrial membrane. Atractyloside is an effective inhibitor of this translocase (Klingenberg, 1976); therefore, perfused livers were infused with atractyloside to determine whether a link between the effect of nicotine upon glycolysis and the observed increase in mitochondrial respiration exists. Infusion of atractyloside (50 M) significantly decreased basal rates of O2 uptake by 13% to a rate of 112 ⫾ 3 mol/g/h (n ⫽ 4, p ⬍ 0.05 by ANOVA). When 85 and 850 M nicotine were infused under these conditions, average rates of oxygen Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017 Fig. 3. Effect of glucose on rates of oxygen uptake and lactate ⫹ pyruvate production from perfused livers from fasted rats. A, livers from 24-h fasted rats were perfused with Krebs-Henseleit bicarbonate buffer containing 50 mM glucose (pH 7.4) at 37°C and oxygen uptake was measured. B, rates of lactate ⫹ pyruvate production were monitored as described under Materials and Methods. Nicotine, 85 and 850 M, was infused for 6 and 8 min, respectively, as indicated by the arrow. Figure depicts representative data (n ⫽ 4). 934 Dewar et al. TABLE 2 Effect of nicotine on respiration in hepatocytes isolated from fed and fasted rats Nicotine (1 mM) was added to hepatocytes isolated from fed or fasted rats. In some experiments hepatocytes from fed rats were incubated with H-89 (10 M) for 1 min prior to addition of nicotine. Respiration was measured in a 2-ml closed well and monitored via a Teflon-shielded, Clark-type oxygen electrode as described under Materials and Methods. Rates of respiration are presented as mol O2/min/g wet wt cells. Results are expressed as mean ⫾ S.E.M. with number of trials in parentheses. Oxygen Uptake Treatment Untreated H-89 Treated mol/min/g wet wt cells Fed Basal Nicotine (1 mM) Fasted Basal Nicotine (1 mM) 1.28 ⫾ 0.05 (15) 1.67 ⫾ 0.06 (15)a 1.39 ⫾ 0.06 (12) 1.45 ⫾ 0.06 (12)b 1.44 ⫾ 0.05 (11) 1.39 ⫾ 0.06 (11)b p ⬍ 0.05 for comparison with basal. p ⬍ 0.05 for comparison with fed, untreated 1 mM nicotine group by ANOVA with post hoc comparisons made in pair-wise fashion using Tukey’s method. a b Fig. 5. d-Tubocurarine blunts the increase in intracellular [Ca2⫹]i due to nicotine in isolated hepatocytes. [Ca2⫹]i was measured with the fluorescent indicator Fura-2 as described under Materials and Methods. A, trace from isolated hepatocytes treated with 1 mM nicotine. B, hepatocytes were incubated in medium with 500 M d-tubocurarine and then treated with 1 mM nicotine. Figure depicts representative data. infusion of nicotine failed to increase oxygen uptake in perfused livers from fed rats (Fig. 6). Cotinine Influences Hepatic Respiration. The liver converts nearly 70% of nicotine to its major metabolite cotinine (Jacob et al., 1988). Therefore, the effects of cotinine infusion upon hepatic respiration, carbohydrate, and lipid metabolism were measured in perfused livers from fed rats. The concentrations of cotinine used were greater than nicotine concentrations since concentrations in vivo are reported to be 10 times greater (Kyerematen and Vesell, 1991). Oxygen uptake was significantly increased from basal levels of 118 ⫾ 5 to 137 ⫾ 2 mol/g/h after infusion of 1.25 mM cotinine (Table 3). No significant changes in lactate ⫹ pyruvate production, glucose, or ketone body output were observed at any concentration of cotinine (Table 3). Discussion Changes in Hepatic Oxygen Uptake due to Nicotine in the Perfused Liver. After cigarette smoking, blood plasma levels of nicotine are estimated to be around 6.0 M (Henningfield et al., 1993). Rapid removal of nicotine from Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017 uptake (114 ⫾ 3 and 112 ⫾ 2 mol/g/h, respectively) were not significantly increased (Fig. 4B). Oxygen Uptake in Isolated Rat Hepatocytes. The effect of nicotine on respiration was also studied in isolated hepatocytes. Various concentrations ranging from 0.25 to 1.0 mM nicotine were added to hepatocytes (data not shown). Respiration was stimulated 30% by addition of 1.0 mM nicotine to hepatocytes and was therefore used in subsequent experiments. Similar to the perfused liver, nicotine failed to increase rates of respiration in hepatocytes isolated from rats fasted 24 h (Table 2). H-89 is a potent inhibitor of cyclic AMP-dependent protein kinase A (Chijiwa et al., 1990). To determine whether protein kinase A is involved in the mechanism of increased respiration due to nicotine, isolated hepatocytes from fed rats were incubated in the presence or absence of H-89 and oxygen uptake was measured. The increase in oxygen uptake due to nicotine was blocked significantly by H-89 (Table 2). Effect of d-Tubocurarine on Increases in Intracellular Calcium due to Nicotine. Nicotinic acetylcholine receptors are ligand-gated ion channels (Stroud et al., 1990). Isolated hepatocytes were treated with 1 mM nicotine to test the hypothesis that nicotine affects intracellular calcium levels via nicotinic acetylcholine receptors. As depicted in Fig. 5, the average increase of calcium in hepatocytes treated with 1 mM nicotine was 70 ⫾ 6.0 nM [Ca2⫹]i (n ⫽ 7). However, 1 mM nicotine only increased calcium 13 ⫾ 4.0 nM [Ca2⫹]i (n ⫽ 7), in hepatocytes preincubated in medium containing 500 M d-tubocurarine. Representative data show that when nicotine was added to hepatocytes, intracellular calcium increased from basal levels of 90 to a peak value of 145 nM [Ca2⫹]i (Fig. 5A). When nicotine was added in the presence of d-tubocurarine, a nicotinic acetylcholine receptor antagonist, no stimulation of intracellular calcium was observed (Fig. 5B). Glucagon Blocks the Effect of Nicotine on Hepatic Oxygen Uptake. Glucagon stimulates hepatic respiration through the activation of the cAMP-dependent protein kinase A pathway (Williamson et al., 1969). This pathway is similarly activated in liver when adrenergic hormones such as epinephrine are present in circulation. After hepatic respiration was stimulated by the infusion of glucagon, further Nicotine and Oxygen Uptake TABLE 3 Effect of cotinine infusion on oxygen uptake, ketogenesis, glucose production, and glycolysis in perfused livers from fed rats Cotinine at concentrations indicated was infused into livers perfused with KrebsHenseleit bicarbonate buffer saturated with oxygen/carbon dioxide mixtures (95:5) in a nonrecirculating system. Oxygen uptake was calculated from influent minus effluent oxygen concentration differences, the flow rate, and tissue wet weight. Samples of effluent perfusate were collected and analyzed for metabolites as described under Materials and Methods. Results are expressed as mean ⫾ S.E.M. (n ⫽ 4). Treatment Oxygen Uptake Basal Cotinine (mM) 0.25 0.5 1.25 118 ⫾ 5 125 ⫾ 4 130 ⫾ 3 137 ⫾ 2a Glucose L⫹P 16 ⫾ 3 126 ⫾ 9 133 ⫾ 11 17 ⫾ 2 17 ⫾ 3 18 ⫾ 3 119 ⫾ 9 120 ⫾ 9 116 ⫾ 9 134 ⫾ 13 133 ⫾ 14 123 ⫾ 22 Ketone Bodies mol/g/h a p ⬍ 0.05 for comparison with basal by ANOVA with post hoc comparisons in pair-wise fashion using Tukey’s method. plasma has been attributed to nicotine’s prevalent uptake into many tissues, including liver (Kyerematen and Vesell, 1991). Tissue concentrations 2 to 15 times higher than plasma levels have been reported (Ghosheh et al., 2001). In this study, high doses of nicotine (85– 850 M) produced distinct changes in oxygen uptake and may reflect physiological nicotine concentrations. In vivo studies have shown that acute nicotine exposure increases levels of circulating hormones such as epinephrine (Anderson et al., 1993) and norepinephrine (Grunberg et al., 1988; Anderson et al., 1993). Both of these hormones have been shown to increase hepatic oxygen uptake via adrenergic stimulation (Jakob and Diem, 1975; Yuki and Thurman, 1980). Thus, a hormone-dependent increase in respiration might be involved in the effect of nicotine on hepatic respiration. To exclude confounding factors, isolated perfused livers from naive rats were used in this study. When nicotine was infused into perfused rat livers from fed animals, a stimulation of oxygen uptake was observed indicating that nicotine increases hepatic oxygen uptake directly (Fig. 1A). Furthermore, glucagon, a hormone that stimulates hepatic respiration through a cAMP-dependent mechanism (Williamson et al., 1969), was infused into perfused livers from fed rats prior to the addition of nicotine. Under these conditions nicotine failed to increase oxygen uptake (Fig. 6). Two important points can be drawn from these data. First, nicotine and glucagon most likely stimulate hepatic oxygen uptake by a similar mechanism. Second, the perfused liver model removes confounding factors such as direct hormonal stimulation, which, as these data show, make interpretation of the effects of nicotine on hepatic respiration in vivo difficult, if not impossible to elucidate. Involvement of Mitochondrial Respiration in Nicotine-Induced Increases in Oxygen Uptake. Potassium cyanide, a known inhibitor of cytochrome c in the electron transport chain, blocks mitochondrial respiration (Coburn et al., 1979). Following KCN infusion, nicotine failed to increase oxygen uptake (Fig. 4A). Thus, the increase in oxygen uptake was largely dependent on mitochondrial electron transport and reduction of molecular oxygen (Fig. 7). Mitochondria were incubated with concentrations of nicotine ranging from 0.05 mM to 1.25 mM. No significant changes in state 4 and state 3 respiration were observed (data not shown). Therefore, it was concluded that nicotine had no direct effect upon mitochondrial oxygen uptake. The supply of ADP to the mitochondrial respiratory chain is one important factor in the control of rates of oxidative phosphorylation in mitochondria (Chance and Williams, 1955) and is transported across the mitochondrial membrane via adenine nucleotide translocase (Klingenberg, 1976). In the presence of atractyloside, an adenine nucleotide translocase inhibitor, nicotine produced no significant increase in oxygen uptake (Fig. 4B). From these data it is concluded that the supply of ADP to the mitochondria is involved in the increase in oxygen uptake due to nicotine (Fig. 7). Involvement of Glycolysis in Nicotine-Induced Changes in Respiration. Glucagon and ethanol have both been shown to decrease glycolysis, thereby increasing hepatic oxygen consumption (Cherrington and Exton, 1976; Thurman and Scholz, 1977). Inhibition of glycolysis slows an ATPgenerating process resulting in an increase in mitochondrial oxygen consumption due to higher rates of ATP generation. When nicotine was infused into perfused livers from fed rats, glycolysis significantly decreased while oxygen uptake increased (Table 1). When the increase in oxygen uptake is plotted against the decrease in lactate ⫹ pyruvate production, a positive relationship is observed (Fig. 2). The association between the increase in oxygen uptake and the decrease in lactate and pyruvate production suggests that a possible mechanism by which nicotine stimulates respiration is through an inhibition of glycolysis. Additionally, nicotine failed to stimulate oxygen uptake in perfused livers from Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017 Fig. 6. Glucagon blocks the increase in oxygen uptake due to nicotine in perfused livers from fed rats. Livers from fed rats were perfused with Krebs-Henseleit bicarbonate buffer (pH 7.4) at 37°C. At times indicated by the arrows, glucagon (10 nM) or nicotine (85 and 850 M) dissolved in saline was infused. Oxygen uptake was calculated from the difference in influent minus effluent O2 concentration, flow rate, and the liver weight. Representative oxygen trace from experiments repeated three times. 935 936 Dewar et al. 24-h fasted rats, where there is a shift from carbohydrate metabolism to fatty acid oxidation due to the depletion of glycogen (Krebs and Hems, 1970; Hansen and Bottermann, 1975) (Fig. 1). This observation further supports the involvement of glyocolysis and suggests a required role for liver glycogen. In support of this hypothesis, when glucose was infused into perfused livers from 24-h fasted rats, the effect of nicotine on both changes in oxygen uptake and glycolysis was partially restored (Fig. 3, A and B). In the fed state, when glycogen is a substrate for glycolysis, 1.5 mol of ADP are required for each mole of lactate or pyruvate produced. The data show that in the presence of 850 M nicotine, lactate ⫹ pyruvate production was decreased by 52 mol/g/h. This would result in the availability of 78 mol of ADP/g/h. Conversion of this ADP to ATP by the respiratory chain would increase respiration by 13 mol O2/g/h. The data show that in the presence of 850 M nicotine, oxygen uptake increased 28 mol/g/h. Thus, inhibition of glycolysis by nicotine accounts for about half the total increase in oxygen consumption observed. Other mechanisms, therefore, must account for the remainder. A total of 80% of nicotine is metabolized to cotinine in the liver (Jacob et al., 1988). Furthermore, cotinine concentrations in vivo are nearly 10 times higher than nicotine concentrations, and the half-life of cotinine is nearly 4 times as long (Kyerematen and Vesell, 1991). A concentration of 1.25 mM cotinine significantly increased hepatic oxygen uptake (Table 3). Although cotinine does not appear to have similar effects upon glycolysis, hepatic oxygen uptake is increased. Cotinine, therefore, could account for the rest of the increases in oxygen uptake due to nicotine and may be of greater importance since it lasts much longer in vivo. Mechanism of Nicotine-Induced Increase in Hepatic Respiration. Nicotine causes a rapid increase in intracellular calcium (Fig. 5A). Nicotine is a classical ligand for nicotinic acetylcholine receptors, which permit entry of ions through the channels they form (Stroud et al., 1990). Furthermore, when nicotine was added to hepatocytes incubated Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017 Fig. 7. Working hypothesis depicting how nicotine increases oxygen uptake. Nicotine increases intracellular calcium through interaction with a nicotinic acetylcholine receptor, an effect blocked by d-tubocurarine. This activates a cAMP-dependent protein kinase A pathway that stimulates respiration. Inhibition of key enzymes of glycolysis by cAMP decreases rates of glycolysis, thereby causing a relative increase in ADP supply available to mitochondria through an atractyloside-sensitive adenine nucleotide translocase leading to increases in respiration. The involvement of mitochondrial oxidative phosphorylation is supported by inhibition by potassium cyanide. Tubo, d-tubocurarine; N, nicotine; nAChR, nicotinic acetylcholine receptor; AC, adenylyl cyclase; G-6-P, glucose 6-phosphate; F-6-P, fructose-6-phosphate; F-1,6-P, fructose-1,6-phosphate; ANT, adenine nucleotide translocase. Nicotine and Oxygen Uptake References Anderson K, Eneroth P, and Arner P (1993) Changes in circulating lipid and carbohydrate metabolites following systemic nicotine treatment in healthy men. Int J Obesity 17:675– 680. Ashakumary L and Vijayammal PL (1997) Effect of nicotine on lipoprotein metabolism in rats. Lipids 32:311–315. 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E-mail: [email protected] Downloaded from jpet.aspetjournals.org at ASPET Journals on August 1, 2017 in medium containing d-tubocurarine, a nicotinic acetylcholine receptor antagonist, no increase in intracellular calcium was observed (Fig. 5B). Some forms of adenylyl cyclase are stimulated by calcium to generate cAMP (Mons et al., 1998; Gueorguiev et al., 1999) and cAMP is a known regulator of cAMP-dependent protein kinase A (Exton et al., 1981). In PC-12 cells, nicotine increases intracellular cAMP by a Ca2⫹dependent mechanism that is blocked by d-tubocurarine (Baizer and Weiner, 1985). When hepatocytes were incubated in buffer containing nicotine and H-89, oxygen uptake was not stimulated (Table 2). Furthermore, cAMP-dependent protein kinase A inhibits phosphofructokinase, resulting in a decrease in glycolysis (Kimmig et al.,1983). Based on these data, a Ca2⫹-dependent cAMP pathway for the nicotine-induced increase in oxygen uptake is hypothesized (Fig. 7). Taken together, these data support the hypothesis depicted in Fig. 7. A stimulation of nicotinic acetylcholine receptors by nicotine increases intracellular [Ca2⫹] that activates a cAMP-dependent protein kinase A pathway. Activation of the cAMP-dependent protein kinase A pathway leads to a decrease in glycolysis through inhibition of phosphofructokinase, resulting in a relative increase in ADP supply. This drives the mitochondrial respiratory chain, resulting in increased oxygen consumption. Thus, it is concluded that the inhibition of glycolysis by nicotine increases hepatic oxygen uptake due to an ADP-dependent increase in mitochondrial respiration and may provide a possible mechanism that contributes to weight changes observed in heavy smokers. 937