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Effects of nicotine on body weight and plasma insulin in female and male rats
Life Sciences, Vol. 55, No. 12, pp. 925-931, 1994 1994 ~ e r Science lad Printed in the USA. All rights rc.r,crvcd 002A-3205/94 $6.00 + .(30
Pergamon 0024-3205(94)00217.7
EFFECTS OF NICOTINE ON BODY WEIGHT AND PLASMA INSULIN IN FEMALE AND MALE RATS M. Ibrahim Saah 1, Margarita Raygada 2, and Neil E. Grunberg 3 Department of Medical and Clinical Psychology Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799 (Received in final form July 8, 1994) Summary There is an inverse relationship between nicotine and body weight. It has been proposed that this inverse relationship is partially mediated by insulinergic actions, but the effect of nicotine on insulin only has been reported for male rats. The present experiment was designed to examine the effect of nicotine administration on body weight and plasma insulin in female and male rats. In addition, the effects of anesthesia prior to sacrifice on plasma insulin were examined. Nicotine reduced body weight and plasma insulin in female and male rats. In addition, the use of an anesthetic prior to sacrifice was associated with higher plasma insulin levels for male rats. These findings are consistent with the interpretation that insulinergic mechanisms play a role in the nicotine-body weight relationship. Key Words: nicotine, body weight, plasma insulin, gender
The inverse relationship between cigarette smoking and body weight is well documented; cigarette smokers weigh less than comparably aged, same sex non-smokers, and smokers who quit smoking report weight gain (1-9). The effect of cigarette smoking on body weight has important clinical implications. Most notably, many smokers, particularly women, report that they smoke to control body weight and cite weight gain as a primary influence on their decision to continue smoking (10). The mechanisms underlying the cigarette-smoking/body weight relationship have received some research attention but many questions remain (11-15). Knowledge of these mechanisms is important to formulate smoking cessation programs that address the concern of weight gain. In addition, a better understanding of this relationship offers further insight into the reinforcing properties of cigarette smoking. The effect of cigarettc smoking on body weight also has been demonstrated in animal models with administration of nicotinc (16-19), the primary pharmacologic agent in tobacco. These rat studies have reported that nicotine decreases specific (sweet-tasting) food consumption and body weight gain (16, 20-21), and that cessation of nicotine administration results in the reversal of these l Currently at Department of Psychology, University of Virginia, Charlottesville, VA 2Currently at Gerontology Research Center, National Institute on Aging, Baltimore, MD 3Corresponding author: Neil E. Grunberg, Ph.D., Department of Medical and Clinical Psychology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799
926
Nicotine, Body Weight and Plasma Insulin
Vol. 55, No. 12, 1994
actions (22). With regard to possible mechanisms underlying the inverse relationship between nicotine and body weight, nicotine administration decreases plasma insulin levels and increases hypothalamic insulin levels (22). Because of insulin's actions on fat storage and utilization, glucose metabolism, and glycogen utilization, this hormone has been proposed as one mediator of nicotine's effect on body weight. To date, studies examining nicotine's effect on hypothalamic and plasma insulin have included only male rats. The present study was designed to examine nicotine's effects on plasma insulin levels in female Sprague-Dawley rats in addition to male rats. Animals usually are not anesthetized prior to decapitation in studies that include biochemical assessment. In light of recent concerns to minimize stress in laboratory animals, it is important to determine empirically whether anesthetizing prior to decapitation interferes with critical dependent variables or not. In an attempt to clarify the effect of anesthetizing prior to sacrifice on insulin, half of the animals in cach group were anesthetized prior to sacrifice. Methods Subjects Subjects were 30 nulliparous female and 30 male Sprague-Dawley rats obtained from Charles River Breeding Laboratories (Wilburn, MA). Female rats weighed 200-225 grams and male rats 275-300 grams at the beginning of the study. Animals were individually housed in standard polypropylenc shocbox cages (44 cm x 23 cm x 20 cm) with metal grill lids and a floor covering of absorbent wood Pine-Dri shavings. All cages were placed on four-shelved metal racks in a room maintaincd at approximately 22°C and 50% relative humidity, and a 12 hour light/dark cycle. Standard rat chow pellets and water were continuously available. Drag Admini~tratiQn Alzet miniosmotic pumps (model 2002, Alza Corporation, Palo Alto, CA) were implanted SC to deliver nicotine or saline at a rate of approximately 0.5 I,tl/hr for 14 days. Physiological saline was used to make the nicotine solutions (made from nicotine dihydrochloride) and also was the control solution. Equal numbers of animals received saline, 6 mg nicotine/kg body-weight/day, or 12 mg nicotine/kg body-weight/day. These dosages (expressed as nicotine base) were based on previous studies of nicotine and body weight (16,23). Rats were assigned to one of the three treatment groups in a quasi-random manner, matching for body weight. Procedure After an initial gentling period (3 days), animals wcrc anesthetized with Metofane and Alzet miniosmotic pumps were implanted SC in each rat via a small incision between the shoulders that was closed with 9 mm wound clips. Two weeks after pump implantation, animals were decapitated. Half of the animals received an IP injection of sodium pentobarbital (50 mg/kg body weight) prior to sacrifice and half of the animals did not. Anesthetized animals were sacrificed 3-6 minutes after the sodium pentobarbital injection (the time necessary for the anesthetic to take effect). Trunk blood was collected. All samples were stored at -70°C until assayed for insulin. Insulin was measured by competitive-binding radioimmunoassay procedure using a prepared RIA kit (obtained from Radioassay Laboratories Inc.). Results Body weight. Figure 1 presents mean body weight (grams) for female and male rats on days 2, 6, 7, 8, 9, 13 and 14 of drug administration. Overall, males weighed significantly more than females at all time points measured (F[1,52]=1567.69, 12<0.01). Due to different growth curves, similar aged female and male rats differ in weight. When the effect of mean baseline body weight was removed with analysis of covariance, body weight gain was less for the animals that received nicotine at all time points compared to control animals. Repeated measures ANCOVA revealed a
Vol. 55, No. 12, 1994
Nicotine, Body Weight and Plasma Insulin
280
270
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260 -
.....-"'"i"i
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230 -
Females (12 mg/kg/day) 220
, Baseline
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I
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Males (12 mg/kg/day)
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I
I
I
I
I
I
2
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6
8
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12
14
Time (Days) Fig. 1 Effects of nicotine on body weight in female and male rats
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Nicotine, Body Weight and Plasma Insulin
Vol. 55, No. 12, 1994
significant main effect (12<0.01) for drug at days 2, 6, 7, 8, 9, 13, and 14 (see Table I). Post-hoe analyses (Least Significant Difference _t-tests, 12<0.05) revealed significant differences between saline, 6, and 12 mg/kg/day groups starting at day 7 of drug administration. This drug effect was significant (12<0.05) for males at days 2, 6, 7, 8, 9, 13, and 14 (see "Fable I). Post-hoe analyses (LSD l-tests, 12<0.05) revealed significant differences between saline, 6, and 12 mg/kg/day groups starting at day 7 of drug administration. For females, the drug effect was significant (12<0.05) at days 6, 7, 8, 9, 13, and 14 (see Table I). Post-hoe analyses (LSD t-tests, 12<0.05) revealed significant differences between the saline and 6 mg/kg/day groups, and between the saline and 12 mg/kg/day groups starting at day 6 of drug administration. TABLE 1 F-values for the Main Effects of Nicotine on Body Weight All Malcs Females _F[2,52], F[2,271, Fl2,261, p<0.05 12<0.01 12<0.05 Day 2 11.95 4.27 n.s. Day 6 6.48 4.43 8.1 Day 7 34.11 19.71 5.68 Day 8 33.29 19.17 5.89 Day 9 30.30 20.72 4.71 Day 13 33.27 23.82 5.41 Day 14 27.5 19.2 5.73 Insulin. Figure 2 presents mean plasma insulin values (!alU/ml) for females and males across the three drug groups. Overall, plasma insulin levels were significantly lower in females than in males (_.F[1,47]= 15.41, 12<0.01 ). As predicted based on previous reports (17, 22), nicotine administration decreased plasma insulin levels (F[2,47]=2.92, i2=0.03, one-tailed). Post-hoe analysis (LSD _ttests, 12<0.05) revealed significant differences between the 12 mg/kg/day nicotine group and the saline group. There was no gender by drug interaction. Because plasma insulin values were skewed, a log transformation was applied and rcduccd skewness from 1.23 to 0.15. Analyses of the transformed data confirmed that plasma insulin levels were significantly lower in females than in males (F[2,47]=16.53, I2<0.01) and nicotine decreased plasma insulin levels (F[2,47]=3.31, 12<0.05). Post-hoe analysis (I,SD t-tests, 12<0.05) revealed significant differences between the 12 mg/kg/day nicotine group and the saline grot, p. Them was no gender by drug interaction. []
6O
Male
5(1
g .,
U
.I
40 3o 20 lO
0 Saline
6 mg/kg/day
12 mg/kg/day
Drug Condition Fig. 2 Effects of nicotine on plasma insulin
Vol. 55, No. 12, 1994
Nicotine, Body Weight and Plasma Insulin
929
Anesthesia. Figure 3 presents mean plasma insulin values (~IU/ml) for females and males for animals anesthetized or not prior to sacrifice and plasma sampling. There was a main effect for anesthesia (F[1,47]=4.84, t2<0.05) because this difference held predominantly for the malc rats. As illustrated in Figure 4, the effect of nicotine to decrease plasma insulin was greater for the anesthetized animals (37% decrease for 12 mg nicotine males and 37% decrease for 12 mg nicotine females with respect to the same-sex control animals) than for the non-anesthetized animals (I 1% decrease for the 12 mg nicotine males and 13% for 12 mg nicotine females with respect to the samesex control animals). Analyses of the transformed data revealed a main effect for anesthesia (F[1,47]=4.33, 12<0.05); however, the gender by anesthesia interaction was not statistically significant.
60-
[]
Male
•
Female
5O-
,•
40 -
t-,1
3020-
I
I
No
Yes
Anesthetized Prior to Sacrifice
Fig. 3 Effects of anesthesia on plasma insulin across gender
75
T
[]
No Anesthesia (Male)
[]
No Anesthesia (Female)
•
Anesthesia (Male)
•
Anesthesia (Female)
45-
30-
.~
15-
glq O-
Saline
6 mg/kg/day Drug
12 mg/kg/day
Condition
Fig.4 Effects of anesthesia on plasma insulin across gender and drug
930
Nicotine, Body Weight and Plasma Insulin
Vol. 55, No. 12, 1994
Discussion The present study was designed to examine the effects of nicotine on body weight and plasma insulin in female and male rats. Consistent with previous findings, weight gain was less for female and male animals receiving nicotine compared with controls (23, 24). There was an inverse dose-response relationship between nicotine administration and body weight in the three drug conditions. Overall, plasma insulin levels were lower in female than in male animals. As predicted, nicotine decreased plasma insulin values for females as well as males (17, 22). However, the gender by drug interaction did not reach significance for this dependent measure. The use of an anesthetic prior to animal sacrifice and plasma sampling did have a significant effect on plasma insulin with greater overall mean plasma insulin values in the anesthetized versus the nonanesthetized animals. There was a significant gender by anesthesia interaction because this difference held predominantly for male animals. A critical look at the data may suggest that the effects of nicotine on insulin were largely due to the use of the pentobarbital anesthetic; however, previous reports with non-anesthetized animals have repeatedly shown that nicotine affects plasma insulin levels with an inverse dose-response (17, 22). Therefore, we interpret the results to mean that nicotine decreases plasma insulin, and that this effect is enhanced by the pentobarbital. These findings are consistent with the interpretation that insulinergic mechanisms play a role in the nicotine-body weight relationship in female as well as male rats. However, in order to establish a causal role for insulin in the nicotine-body weight relationship, the effects of nicotine on body weight and food consumption must be examined while independently manipulating insulin (22). Furthermore, the present findings indicate that the use of an anesthetic is a relevant factor to consider when post-sacrifice plasma insulin values ~u-e used as a dependent measure. Acknowledgments The authors wish to thank Kevin Alvares, Stephanie Nespor, and Leslie Wood for their valuable assistance in the laboratory. We also thank Kelly Brown and Laura Klein for their comments on the manuscript. This work was supported by USUHS Protocol No. CO7223. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD, the USUHS, the University of Virginia, or the National Institute on Aging. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. I1.
N.E. G R U N B E R G Advances in Behavioral Medicine Volume 2, E.S. Katkin and S.B. Manuck (eds), 97-129, JAI Press, Connecticut (1986). E.R. GRITZ, R.C. KLESGES, and A.W. MEYERS, Ann. Behav. Med. I 1 1 4 4 - 1 5 3 (1989). R.C. KLESGES, A.W. MEYERS, L.M. KLESGES, and M.E. L A V A S Q U E , Psych. Bull. 106204-230 (1989). R.C. KLESGES, A.W. MEYERS, S.E. WINDERS, and S.N. FRENCH, Ann. Behav. Med. 1._!_1134-143 (1989). J.T. W A C K , and J. RODIN, Amer. J. of Clin. Nut. 3___55366-380 (1982). S.E. WINDERS, and N.E. GRUNBERG, Ann. Behav. Med. 1._!1125-133 (1989). N.E. GRUNBERG, Research Advance~ in Alcohol and Drug Problems, Volume 10, I..T. Kozlowski, et al. (eds), 273-315, Plenum Press, New York (1990). USDHHS, The Health (~on~equences of Smoking: Nicotine Addiction, USI)HHS, PHS, CDC, CCDPHP, OSH. DHHS Publication No. (CDC) 88-8406 (1988). USDHHS, The Health Benefits of Smoking Cessation, USDHHS, PHS, CDC, CCDPHP, OSH. DHHS Publication No. (CDC) 90-8416 (1990). R.C. KLESGES and L.M. KLESGES, Int. J. Eating Disorders. 7_413-419 (1988). N.E. GRUNBERG, M.R.C. G R E E N W O O D , F. COLLINS, L. EPSTEIN, D. H A T S U K A M I , R. NISURA, K. O'CONNELI,, O. POMERLEAU, E. RAVUSSIN, B.
Vol. 55, No. 12, 1994
12. 13. 14. 15. 16 17. 18. 19. 20. 21. 22. 23. 24.
Nicotine, Body Weight and Plasma Insulin
931
ROLLS, J. AUDRIAN,, and M. CODAY, Health Psychol. I 1 4-9 (1992). K.A. PERKINS, L.H. EPSTEIN, B.L MARKS , R.L. STILLER, and R.G. JACOB, New England J. of Med. 32_0 898-903 (1989). K.A. PERKINS, L.H. EPSTEIN, B.L. MARKS, R.L. STILLER, J.E. SEXTON, M.It. FERNSTRON, R.G. JACOB, and R. SOLBERG, Amer. J. of Clin. Nut. 52 228-233 (1990). K.A. PERKINS, L.H. EPSTEIN, and S. PASTOR, J. of Consult. and Clin. Psychol. 58 121-125 (1990). K.A. PERKINS, L.H. EPSTEIN, R.L. STILLER, B.L. MARKS, and R.G. JACOB, Amer. J. of Clin. Nut. 50 545-550 (1989). N.E. GRUNBERG, Addict. Behav. 7__317-331 (1982). N.E. GRUNBERG, The Pharmacology of Nicotine: ISCU Symposium Series, M.J. Rand and K. Thurau (eds), 97-1 I0, IRC Press, Washington, D.C. (1988). N.E. GRUNBERG, K.A. POPP, D.J. BOWEN, S.M. NESPOR, S.E. WINDERS, and S.E. EURY, Life Sci. 4__22161-170 (1988). E.D. LEVIN, M.M. MORGAN, C. GALVEZ, and G.D. ELLISON, Phys. and Behav. 39 441-444. (1987). N.E. GRUNBERG and D.E. MORSE, J. Appl. Soc. Psychol. l_A4310-317 (1984). N.E. GRUNBERG, D.J. BOWEN, V.A. MAYCOCK, and S.M. NESPOR, Psychopharm. 87 198-203 (1985). N.E. GRUNBERG and M. RAYGADA, Advanccs in Pharmacological Sciences: Effects of Nicotine on Biological Systems, 131-142, Birkhauser Verlag, Basel (1991). N.E. GRUNBERG, D.J. BOWEN, and D.E. MORSE, Psychopharm. 83 553-557 (1984). N.E. GRUNBERG, D.J. BOWEN, and S.E. WINDERS, Psychopharm. 90 101-105 (1986).
Pergamon 0024-3205(94)00217.7
EFFECTS OF NICOTINE ON BODY WEIGHT AND PLASMA INSULIN IN FEMALE AND MALE RATS M. Ibrahim Saah 1, Margarita Raygada 2, and Neil E. Grunberg 3 Department of Medical and Clinical Psychology Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799 (Received in final form July 8, 1994) Summary There is an inverse relationship between nicotine and body weight. It has been proposed that this inverse relationship is partially mediated by insulinergic actions, but the effect of nicotine on insulin only has been reported for male rats. The present experiment was designed to examine the effect of nicotine administration on body weight and plasma insulin in female and male rats. In addition, the effects of anesthesia prior to sacrifice on plasma insulin were examined. Nicotine reduced body weight and plasma insulin in female and male rats. In addition, the use of an anesthetic prior to sacrifice was associated with higher plasma insulin levels for male rats. These findings are consistent with the interpretation that insulinergic mechanisms play a role in the nicotine-body weight relationship. Key Words: nicotine, body weight, plasma insulin, gender
The inverse relationship between cigarette smoking and body weight is well documented; cigarette smokers weigh less than comparably aged, same sex non-smokers, and smokers who quit smoking report weight gain (1-9). The effect of cigarette smoking on body weight has important clinical implications. Most notably, many smokers, particularly women, report that they smoke to control body weight and cite weight gain as a primary influence on their decision to continue smoking (10). The mechanisms underlying the cigarette-smoking/body weight relationship have received some research attention but many questions remain (11-15). Knowledge of these mechanisms is important to formulate smoking cessation programs that address the concern of weight gain. In addition, a better understanding of this relationship offers further insight into the reinforcing properties of cigarette smoking. The effect of cigarettc smoking on body weight also has been demonstrated in animal models with administration of nicotinc (16-19), the primary pharmacologic agent in tobacco. These rat studies have reported that nicotine decreases specific (sweet-tasting) food consumption and body weight gain (16, 20-21), and that cessation of nicotine administration results in the reversal of these l Currently at Department of Psychology, University of Virginia, Charlottesville, VA 2Currently at Gerontology Research Center, National Institute on Aging, Baltimore, MD 3Corresponding author: Neil E. Grunberg, Ph.D., Department of Medical and Clinical Psychology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799
926
Nicotine, Body Weight and Plasma Insulin
Vol. 55, No. 12, 1994
actions (22). With regard to possible mechanisms underlying the inverse relationship between nicotine and body weight, nicotine administration decreases plasma insulin levels and increases hypothalamic insulin levels (22). Because of insulin's actions on fat storage and utilization, glucose metabolism, and glycogen utilization, this hormone has been proposed as one mediator of nicotine's effect on body weight. To date, studies examining nicotine's effect on hypothalamic and plasma insulin have included only male rats. The present study was designed to examine nicotine's effects on plasma insulin levels in female Sprague-Dawley rats in addition to male rats. Animals usually are not anesthetized prior to decapitation in studies that include biochemical assessment. In light of recent concerns to minimize stress in laboratory animals, it is important to determine empirically whether anesthetizing prior to decapitation interferes with critical dependent variables or not. In an attempt to clarify the effect of anesthetizing prior to sacrifice on insulin, half of the animals in cach group were anesthetized prior to sacrifice. Methods Subjects Subjects were 30 nulliparous female and 30 male Sprague-Dawley rats obtained from Charles River Breeding Laboratories (Wilburn, MA). Female rats weighed 200-225 grams and male rats 275-300 grams at the beginning of the study. Animals were individually housed in standard polypropylenc shocbox cages (44 cm x 23 cm x 20 cm) with metal grill lids and a floor covering of absorbent wood Pine-Dri shavings. All cages were placed on four-shelved metal racks in a room maintaincd at approximately 22°C and 50% relative humidity, and a 12 hour light/dark cycle. Standard rat chow pellets and water were continuously available. Drag Admini~tratiQn Alzet miniosmotic pumps (model 2002, Alza Corporation, Palo Alto, CA) were implanted SC to deliver nicotine or saline at a rate of approximately 0.5 I,tl/hr for 14 days. Physiological saline was used to make the nicotine solutions (made from nicotine dihydrochloride) and also was the control solution. Equal numbers of animals received saline, 6 mg nicotine/kg body-weight/day, or 12 mg nicotine/kg body-weight/day. These dosages (expressed as nicotine base) were based on previous studies of nicotine and body weight (16,23). Rats were assigned to one of the three treatment groups in a quasi-random manner, matching for body weight. Procedure After an initial gentling period (3 days), animals wcrc anesthetized with Metofane and Alzet miniosmotic pumps were implanted SC in each rat via a small incision between the shoulders that was closed with 9 mm wound clips. Two weeks after pump implantation, animals were decapitated. Half of the animals received an IP injection of sodium pentobarbital (50 mg/kg body weight) prior to sacrifice and half of the animals did not. Anesthetized animals were sacrificed 3-6 minutes after the sodium pentobarbital injection (the time necessary for the anesthetic to take effect). Trunk blood was collected. All samples were stored at -70°C until assayed for insulin. Insulin was measured by competitive-binding radioimmunoassay procedure using a prepared RIA kit (obtained from Radioassay Laboratories Inc.). Results Body weight. Figure 1 presents mean body weight (grams) for female and male rats on days 2, 6, 7, 8, 9, 13 and 14 of drug administration. Overall, males weighed significantly more than females at all time points measured (F[1,52]=1567.69, 12<0.01). Due to different growth curves, similar aged female and male rats differ in weight. When the effect of mean baseline body weight was removed with analysis of covariance, body weight gain was less for the animals that received nicotine at all time points compared to control animals. Repeated measures ANCOVA revealed a
Vol. 55, No. 12, 1994
Nicotine, Body Weight and Plasma Insulin
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270
iT
260 -
.....-"'"i"i
.....-'" .L "'O " C).. .,,[,[•.''.'"
v
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..'± ~ o
TT
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240 Females (Saline) Females (6mg/kg/day)
230 -
Females (12 mg/kg/day) 220
, Baseline
I
I
I
I
I
I
I
2
4
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........~ g
440 -- .
..°o°°"
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i
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(Saline)
~-
Males (6mg/kg/day)
l w
Males (12 mg/kg/day)
I
I
I
I
I
I
I
2
4
6
8
I0
12
14
Time (Days) Fig. 1 Effects of nicotine on body weight in female and male rats
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Nicotine, Body Weight and Plasma Insulin
Vol. 55, No. 12, 1994
significant main effect (12<0.01) for drug at days 2, 6, 7, 8, 9, 13, and 14 (see Table I). Post-hoe analyses (Least Significant Difference _t-tests, 12<0.05) revealed significant differences between saline, 6, and 12 mg/kg/day groups starting at day 7 of drug administration. This drug effect was significant (12<0.05) for males at days 2, 6, 7, 8, 9, 13, and 14 (see "Fable I). Post-hoe analyses (LSD l-tests, 12<0.05) revealed significant differences between saline, 6, and 12 mg/kg/day groups starting at day 7 of drug administration. For females, the drug effect was significant (12<0.05) at days 6, 7, 8, 9, 13, and 14 (see Table I). Post-hoe analyses (LSD t-tests, 12<0.05) revealed significant differences between the saline and 6 mg/kg/day groups, and between the saline and 12 mg/kg/day groups starting at day 6 of drug administration. TABLE 1 F-values for the Main Effects of Nicotine on Body Weight All Malcs Females _F[2,52], F[2,271, Fl2,261, p<0.05 12<0.01 12<0.05 Day 2 11.95 4.27 n.s. Day 6 6.48 4.43 8.1 Day 7 34.11 19.71 5.68 Day 8 33.29 19.17 5.89 Day 9 30.30 20.72 4.71 Day 13 33.27 23.82 5.41 Day 14 27.5 19.2 5.73 Insulin. Figure 2 presents mean plasma insulin values (!alU/ml) for females and males across the three drug groups. Overall, plasma insulin levels were significantly lower in females than in males (_.F[1,47]= 15.41, 12<0.01 ). As predicted based on previous reports (17, 22), nicotine administration decreased plasma insulin levels (F[2,47]=2.92, i2=0.03, one-tailed). Post-hoe analysis (LSD _ttests, 12<0.05) revealed significant differences between the 12 mg/kg/day nicotine group and the saline group. There was no gender by drug interaction. Because plasma insulin values were skewed, a log transformation was applied and rcduccd skewness from 1.23 to 0.15. Analyses of the transformed data confirmed that plasma insulin levels were significantly lower in females than in males (F[2,47]=16.53, I2<0.01) and nicotine decreased plasma insulin levels (F[2,47]=3.31, 12<0.05). Post-hoe analysis (I,SD t-tests, 12<0.05) revealed significant differences between the 12 mg/kg/day nicotine group and the saline grot, p. Them was no gender by drug interaction. []
6O
Male
5(1
g .,
U
.I
40 3o 20 lO
0 Saline
6 mg/kg/day
12 mg/kg/day
Drug Condition Fig. 2 Effects of nicotine on plasma insulin
Vol. 55, No. 12, 1994
Nicotine, Body Weight and Plasma Insulin
929
Anesthesia. Figure 3 presents mean plasma insulin values (~IU/ml) for females and males for animals anesthetized or not prior to sacrifice and plasma sampling. There was a main effect for anesthesia (F[1,47]=4.84, t2<0.05) because this difference held predominantly for the malc rats. As illustrated in Figure 4, the effect of nicotine to decrease plasma insulin was greater for the anesthetized animals (37% decrease for 12 mg nicotine males and 37% decrease for 12 mg nicotine females with respect to the same-sex control animals) than for the non-anesthetized animals (I 1% decrease for the 12 mg nicotine males and 13% for 12 mg nicotine females with respect to the samesex control animals). Analyses of the transformed data revealed a main effect for anesthesia (F[1,47]=4.33, 12<0.05); however, the gender by anesthesia interaction was not statistically significant.
60-
[]
Male
•
Female
5O-
,•
40 -
t-,1
3020-
I
I
No
Yes
Anesthetized Prior to Sacrifice
Fig. 3 Effects of anesthesia on plasma insulin across gender
75
T
[]
No Anesthesia (Male)
[]
No Anesthesia (Female)
•
Anesthesia (Male)
•
Anesthesia (Female)
45-
30-
.~
15-
glq O-
Saline
6 mg/kg/day Drug
12 mg/kg/day
Condition
Fig.4 Effects of anesthesia on plasma insulin across gender and drug
930
Nicotine, Body Weight and Plasma Insulin
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Discussion The present study was designed to examine the effects of nicotine on body weight and plasma insulin in female and male rats. Consistent with previous findings, weight gain was less for female and male animals receiving nicotine compared with controls (23, 24). There was an inverse dose-response relationship between nicotine administration and body weight in the three drug conditions. Overall, plasma insulin levels were lower in female than in male animals. As predicted, nicotine decreased plasma insulin values for females as well as males (17, 22). However, the gender by drug interaction did not reach significance for this dependent measure. The use of an anesthetic prior to animal sacrifice and plasma sampling did have a significant effect on plasma insulin with greater overall mean plasma insulin values in the anesthetized versus the nonanesthetized animals. There was a significant gender by anesthesia interaction because this difference held predominantly for male animals. A critical look at the data may suggest that the effects of nicotine on insulin were largely due to the use of the pentobarbital anesthetic; however, previous reports with non-anesthetized animals have repeatedly shown that nicotine affects plasma insulin levels with an inverse dose-response (17, 22). Therefore, we interpret the results to mean that nicotine decreases plasma insulin, and that this effect is enhanced by the pentobarbital. These findings are consistent with the interpretation that insulinergic mechanisms play a role in the nicotine-body weight relationship in female as well as male rats. However, in order to establish a causal role for insulin in the nicotine-body weight relationship, the effects of nicotine on body weight and food consumption must be examined while independently manipulating insulin (22). Furthermore, the present findings indicate that the use of an anesthetic is a relevant factor to consider when post-sacrifice plasma insulin values ~u-e used as a dependent measure. Acknowledgments The authors wish to thank Kevin Alvares, Stephanie Nespor, and Leslie Wood for their valuable assistance in the laboratory. We also thank Kelly Brown and Laura Klein for their comments on the manuscript. This work was supported by USUHS Protocol No. CO7223. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD, the USUHS, the University of Virginia, or the National Institute on Aging. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. I1.
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