Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

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 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

Top Abstract Introduction Materials and Methods Results Discussion References

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 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.

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 Krebs-Ringer-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 µM N-[2-(p-bromocinnamylamino)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 µg/ml streptomycin, and sometimes 500 µM d-tubocurarine (Sigma-Aldrich). Fura-2/acetoxymethyl ester (Molecular Probes, Eugene, OR) was added to plates at a final concentration of 5 µM 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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

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 µM nicotine caused an immediate 10% increase in respiration from a basal level of 100 µmol/g/h to a value of 110 µmol/g/h in a perfused liver from a fed rat. Infusion of higher concentrations of nicotine (850 µM) increased oxygen consumption to a maximal value around 124 µmol/g/h. Average basal rates of respiration in perfused livers from fed rats were 105 ± 5 µmol/g/h. When 85 and 850 µM nicotine were infused, average rates were increased to 116 ± 6 and 134 ± 8 µmol/g/h, respectively (Table 1). In contrast, in livers from 24-h fasted rats, basal rates of oxygen consumption (113 ± 6 µmol/g/h) were not altered by any concentration of nicotine studied (Fig. 1A and Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   The effect of nicotine infusion on oxygen uptake and lactate + pyruvate production in perfused livers. Livers were perfused with Krebs-Henseleit 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 µM to 850 µM 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. , rates of lactate + pyruvate production. Each point is a mean from four experiments.

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 µmol/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 µmol/g/h lactate + pyruvate production were lowered significantly to 52 ± 9 µmol/g/h by nicotine (850 µM) (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).

View larger version (10K): [in this window] [in a new window]   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 () show mean data (n = 5) for each concentration of nicotine used in perfusion experiments with livers from fed rats (r = 0.933).

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 µM 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 µmol/g/h (p < 0.05 by ANOVA). Under these conditions, average rates of 140 ± 5 and 148 ± 4 µmol/g/h were achieved with the infusion of 85 and 850 µM nicotine, respectively. Representative data of the effects of nicotine (85 and 850 µM) in perfused livers from fasted rats in the presence of glucose (50 mM) are shown in Fig. 3A.

View larger version (19K): [in this window] [in a new window]   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 µM, was infused for 6 and 8 min, respectively, as indicated by the arrow. Figure depicts representative data (n = 4).

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 µmol O₂/g/h were decreased to 38 ± 3 µmol/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 µmol/g/h after termination of KCN infusion.

View larger version (17K): [in this window] [in a new window]   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 µM, 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 µM) significantly decreased basal rates of O₂ uptake by 13% to a rate of 112 ± 3 µmol/g/h (n = 4, p < 0.05 by ANOVA). When 85 and 850 µM nicotine were infused under these conditions, average rates of oxygen uptake (114 ± 3 and 112 ± 2 µmol/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 µM 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).

View larger version (16K): [in this window] [in a new window]   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 µM d-tubocurarine and then treated with 1 mM nicotine. Figure depicts representative data.

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 infusion of nicotine failed to increase oxygen uptake in perfused livers from fed rats (Fig. 6).

View larger version (15K): [in this window] [in a new window]   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 µM) 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.

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 µmol/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

Top Abstract Introduction Materials and Methods Results Discussion References

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 µM (Henningfield et al., 1993). Rapid removal of nicotine from 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 µM) produced distinct changes in oxygen uptake and may reflect physiological nicotine concentrations.

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.

View larger version (20K): [in this window] [in a new window]   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.

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 ATP-generating 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 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).

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 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).