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Animal Physiology

Prenatal Exposure to Nicotine Causes Postnatal Obesity and Altered Perivascular Adipose Tissue Function Yu-Jing Gao,* Alison C. Holloway,† Zhao-hua Zeng,‡ Gareth E. Lim,† James J. Petrik,§ Warren G. Foster,† and Robert M.K.W. Lee*

Abstract GAO, YU-JING, ALISON C. HOLLOWAY, ZHAO-HUA ZENG, GARETH E. LIM, JAMES J. PETRIK, WARREN G. FOSTER, AND ROBERT M.K.W. LEE. Prenatal exposure to nicotine causes postnatal obesity and altered perivascular adipose tissue function. Obes Res. 2005;13: 687– 692. Objective: Recent epidemiological studies have shown that there is an increased risk of obesity and hypertension in children born to women who smoked during pregnancy. The aim of this study was to examine the effect of fetal and neonatal exposure to nicotine, the major addictive component of cigarette smoke, on postnatal adiposity and blood vessel function. Research Methods and Procedures: Female Wistar rats were given nicotine or saline (vehicle) during pregnancy and lactation. Postnatal growth was determined in the male offspring from weaning until 26 weeks of age. At 26 weeks of age, fat pad weight and the function of the perivascular adipose tissue (PVAT) in the thoracic aorta and mesenteric arteries were examined. Results: Exposure to nicotine resulted in increased postnatal body weight and fat pad weight and an increased amount of PVAT in the offspring. Contraction of the aorta induced by phenylephrine was significantly attenuated in the presence of PVAT, whereas this effect was not observed in the aortic

Received for review May 25, 2004. Accepted in final form February 3, 2005. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. *Department of Anaesthesia and †Obstetrics and Gynaecology, McMaster University, Hamilton, Ontario, Canada; ‡Research Unit of Cardiovascular Diseases, Department of Internal Medicine, The First Affiliated Hospital of Guangzhou Medical College, Guangzhou, China; and §Department of Biomedical Sciences, University of Guelph, Ontario, Canada. Address correspondence to Robert M. K. W. Lee, Department of Anaesthesia (HSC-2U3), McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. E-mail: [email protected] Copyright © 2005 NAASO

rings from the offspring of nicotine-exposed dams. Phenylephrine-induced contraction without PVAT was not different between saline- and nicotine-exposed rats. Transfer of solution incubated with PVAT-intact aorta to PVAT-free aorta induced a marked relaxation response in the rats from saline-exposed dams, but this relaxation response was significantly impaired in the rats from nicotine-exposed dams. Discussion: Our results showed that prenatal nicotine exposure increased adiposity and caused an alteration in the modulatory function of PVAT on vascular relaxation response, thus providing insight into the mechanisms underlying the increased prevalence of obesity and hypertension in children exposed to cigarette smoke in utero. Key words: adipose tissue, aorta, contraction, nicotine, vessel relaxation

Introduction Reports from human epidemiological studies suggest that lower birth weight is associated with an increased risk of central obesity and subsequently an increased cardiovascular risk in later life (1). In developed countries, it has been suggested that maternal cigarette smoking during pregnancy is a significant environmental contributor to fetal growth restriction (2– 4). Despite the fact that maternal smoking causes fetal growth retardation, ⬃15% to 20% of all pregnant women smoke during their pregnancies (3,5–9). Recent epidemiological studies have shown that children exposed to cigarette smoke in utero have an increased risk of obesity and hypertension in later life (4,10 –15), although the mechanism(s) underlying this association are unknown. Intrauterine growth restriction in pregnant women who smoke may be a result of fetal hypoxia caused by diffusion of carbon monoxide into the fetal circulation (5). However, mainstream cigarette smoke is estimated to contain as many as 4000 chemicals (16,17), which may act to inhibit fetal growth through direct toxic effects on fetal and placental cells or through alterations in uteroplacental and fetoplacenOBESITY RESEARCH Vol. 13 No. 4 April 2005

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Nicotine Effect on Obesity and Vessel Function, Gao et al.

tal blood flow (9). In rats, prenatal exposure to nicotine alone, the major addictive component of cigarette smoke, was sufficient to result in reduced birth weight (18,19). Furthermore, Newman et al. (18) reported that rats exposed to nicotine in utero were heavier than control animals at 9 weeks of age, and Williams and Kanagasabai (20) reported that fetal nicotine exposure increased body fat in the fetus at gestation day 20 (term 21 days). These data suggest that fetal nicotine exposure may result in increased adiposity of the offspring, but such a correlation remains to be determined. Almost all systemic arteries are surrounded by a layer of perivascular adipose tissue (PVAT).1 In most studies to date, PVAT is routinely removed in functional studies, based largely on the assumption that PVAT may impede the diffusion of test compounds to the vascular tissue in in vitro studies. Therefore, despite the ubiquity of adipose tissue around blood vessels, little is known about the function of PVAT in modulating vascular function. PVAT from the aorta of adult Sprague-Dawley rats is now known to secrete a relaxing factor that inhibits the contractile response of the aorta to angiotensin II, serotonin, or phenylephrine (PE) (21). This substance apparently acts through the activation of potassium channels in vascular smooth muscle cells, but its identity is unknown (21). In spontaneously hypertensive rats, prenatal exposure to nicotine increased blood pressure postnatally (22), and children born to mothers who smoked during pregnancy are at an increased risk of hypertension in later life (11). We, therefore, hypothesize that nicotine exposure can decrease the ability of the relaxation factor secreted from PVAT to cause vessel relaxation. Nicotineinduced changes in the relaxation factor secreted from PVAT might explain, in part, the observed hypertension after fetal nicotine exposure. Therefore, the goal of this study was to examine the role of nicotine exposure in utero and during lactation on 1) the amount of fat, including PVAT, in the offspring and 2) the ability of PVAT-derived relaxation factor to cause vessel relaxation.

Research Methods and Procedures Preparation of Animals The studies were approved by the institutional review board and followed the Canadian Guidelines on Animal Care. Nulliparous 200- to 250-gram female Wistar rats were maintained under controlled lighting (12:12 L:D) and temperature (22 °C) with ad libitum access to food and water. Dams were injected subcutaneously with 1 mg/kg body weight per day nicotine bitartrate (Sigma Aldrich, St. Louis, MO) or saline (vehicle) for 14 days before mating and

1 Nonstandard abbreviations: PVAT, perivascular adipose tissue; PE, phenylephrine; CSA, cross-sectional area; LVW, left ventricle weight; BW, body weight.

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during pregnancy until weaning. They were time mated on Day 0 of pregnancy and littered on Day 22. Pups were weighed after birth, and litter size was reduced to eight to assure uniformity of litter size between treated and control litters. To eliminate any confounding effects of the female reproductive cycle, only male offspring were used for these experiments. After weaning at postnatal Day 21, two offspring per litter were selected randomly and caged as sibling pairs. Pups were weighed weekly from postnatal Day 1 until 26 weeks of age to assess postnatal growth. At 26 weeks of age, the rats were killed, and perirenal, mesenteric, and epididymal fat pads were removed from the animals and weighed. The thoracic aorta and mesenteric arteries were removed and stored in 4 °C oxygenated physiological salt solution with the following composition (in mM): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 22 NaHCO3, 11.1 glucose, and 1.6 CaCl2. The vessels were processed for morphological and functional studies as outlined below. Vessel Preparation for Reactivity Experiments Vessel reactivity was studied in vitro using a conventional organ bath system. Two pairs of aortic rings (⬃3 to 4 mm long) were cut consecutively from the middle part of the thoracic aorta. In each pair, PVAT was left intact on one ring (Fat⫹), whereas PVAT was removed in the other ring (Fat⫺). After 1 hour of equilibration under a resting tension of 3 grams at 37 °C, the Fat⫹ and Fat⫺ rings were contracted with 1 ␮M PE (an ␣-adrenoceptor agonist: SigmaAldrich) to a submaximal tension (70% to 80% of the maximal). When the contraction in response to PE had reached a plateau, 3 mL of the solution incubated with Fat⫹ ring (donor) was transferred to the Fat⫺ ring (recipient). The relaxation of the recipient artery (Fat⫺) was expressed as a percentage of the precontracted tension. Transferring solution between Fat⫺ rings served as a control. The cumulative concentration-contraction curve to PE was expressed as a percentage of the contraction to KCl (60 mM), which induces contraction through membrane depolarization. Morphological Study The heart, a segment of thoracic aorta, and a second-order branch of mesenteric arteries were fixed in 10% buffered formalin. Cross-sections of the blood vessels were made and stained with Gomori’s trichrome or hematoxylin and eosin. Cross-sectional areas (CSAs) of PVAT, the media, and lumen diameter of the aorta and mesenteric arteries were measured using a light microscope equipped with a camera lucida and tracing digitizing pad (23). Blood vessels from seven rats in each group (nicotine or saline) were prepared for histological measurements, but PVAT was not preserved properly during processing in some rats; therefore, tissues from only four to five animals were measured.


Statistical Analysis The results are expressed as mean ⫾ SE, where n represents the number of rats. Data were checked for normality and equal variance. Statistical analysis was performed by one-way ANOVA or unpaired Student’s t test; p ⱕ 0.05 was considered significant.

Results Body weight of the nicotine-treated rats began to increase significantly in comparison with that of the control rats at 10 weeks of age (Figure 1). At 26 weeks, values for body weight, left ventricular weight, and fat pad weight of epididymal, mesenteric, and perirenal tissues were significantly higher in the rats exposed to nicotine than controls (Table 1). However, the increased weight of the left ventricle (LVW) in the nicotine-exposed offspring seems to have been caused by an overall increase in body weight (BW), because the LVW/BW ratio was not different between the two groups. The CSA of PVAT around the aorta and mesenteric arteries was also increased in the rats exposed to nicotine compared with controls. CSAs of the media in the aorta and mesenteric arteries were similar between the two groups of rats. Contraction of the aorta from control rats in response to PE was significantly higher in the Fat⫺ vessels compared with Fat⫹ vessels (Figure 2A), whereas in the aorta from rats exposed to nicotine prenatally, the contractile response to PE was not different between Fat⫹ and Fat⫺ rings (Figure 2B). In the presence of PVAT, aorta from nicotine-exposed rats showed a higher contraction to PE than the vessels from control rats (Figure 2C). This difference was not observed when PVAT was removed (Figure 2D). The tension generated in response to 60 mM KCl was not different between Fat⫹ and Fat⫺ rings within each group nor between nicotine and control groups (Figure 2E). Transfer of bathing solution from Fat⫹ to Fat⫺ vessels caused a small relaxation response in the recipient aorta from nicotine-exposed rats (Figure 3A), and this relaxation response was significantly reduced in the nicotine-exposed rats compared with the control rats (Figure 3, B and C). The same transfer procedure between Fat⫺ rings within each group did not induce relaxation (data not shown).

Discussion We have shown in this study for the first time, to our knowledge, that nicotine exposure throughout pregnancy and lactation increased the amount of body fat, including PVAT, in the offspring and decreased the efficacy of the relaxation factor released from PVAT to cause vessel relaxation. These findings may have significant implications for the control of blood pressure in these animals as they age and may explain, in part, the increased prevalence of hypertension and obesity in children born to mothers who smoke during pregnancy.

Figure 1: Body weight at different ages of Wistar rat offspring from dams exposed to nicotine or saline. * p ⬍ 0.05, ** p ⬍ 0.01 vs. respective saline-exposed rats.

Recent epidemiological studies have shown that there is an increased risk of obesity and hypertension in children born to women who smoked during pregnancy (4,10 –15). Maternal smoking during pregnancy is an important risk factor for overweight and adiposity in children between 5 and 7 years of age, and the effect of maternal smoking to increase obesity in the offspring has been shown to persist even after adjustment for a wide range of confounding factors, including birth weight, socioeconomic status, and maternal diet (4,10,13,15). Similarly, maternal cigarette smoking has been associated with increased blood pressure in the children of smokers at 6 years of age, an effect that is not wholly attributable to the effects of maternal smoking on birth weight or the child’s current weight (11). Taken together, these studies suggest that it may be a direct effect of intrauterine exposure to the chemicals in cigarette smoke that accounts for the increased risk of obesity and hypertension in the offspring of women who smoke during pregnancy. The health consequences of obesity are numerous because obesity is a significant etiologic factor for many diseases including type 2 diabetes, cardiovascular disease, and cancer (24 –27). It is difficult to assess mechanism(s) by which maternal cigarette smoking can alter postnatal health because there are as many as 4000 chemicals present in cigarette smoke (16,17). However, results from this study suggest that fetal and neonatal exposure to nicotine, the addictive component of cigarette smoke, may be important in the development of postnatal obesity in the offspring. Prenatal and lactational exposure to nicotine caused a significant increase in postnatal growth, an effect that became obvious after only 9 weeks of age. At 26 weeks of age, the nicotine-treated offspring were significantly heavier than the saline-treated controls. Similarly, Newman et al. (18) showed that nicotine exposure for 19 days prenatally and 16 days postnatally resulted in significantly heavier body OBESITY RESEARCH Vol. 13 No. 4 April 2005

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Table 1. BW, LVW, fat pad weight, and PVAT (thoracic aorta and mesenteric branch) at the age of 26 weeks (n ⫽ 4 –16 rats)

BW (g) LVW (mg) LVW/BW ratio (mg/g) Fat pad (g) Epididymal Mesentery Perirenal Total Thoracic aorta Area of PVAT (mm2) CSA of media (mm2) Mesenteric artery (the second-order branch) PVAT CSA (mm2) CSA of media (mm2)

Control (saline)

Nicotine

p

576.5 ⫾ 14.4 (n ⫽ 15) 987.8 ⫾ 40.7 (n ⫽ 6) 1.71 ⫾ 0.2 (n ⫽ 6)

653.9 ⫾ 10.4 (n ⫽ 16) 1120.1 ⫾ 40.3 (n ⫽ 7) 1.72 ⫾ 0.3 (n ⫽ 6)

0.0002 0.042 0.79

6.88 ⫾ 0.59 (n ⫽ 15) 6.68 ⫾ 0.67 (n ⫽ 15) 9.27 ⫾ 0.56 (n ⫽ 15) 22.83 ⫾ 1.44 (n ⫽ 15)

10.2 ⫾ 10.24 (n ⫽ 16) 8.84 ⫾ 0.52 (n ⫽ 16) 13.40 ⫾ 0.87 (n ⫽ 16) 32.48 ⫾ 1.94 (n ⫽ 16)

0.002 0.016 0.0005 0.0005

7.66 ⫾ 0.73 (n ⫽ 5) 0.71 ⫾ 0.40 (n ⫽ 5)

11.20 ⫾ 1.27 (n ⫽ 4) 0.80 ⫾ 0.27 (n ⫽ 4)

0.038 0.09

2.65 ⫾ 0.24 (n ⫽ 5) 0.021 ⫾ 0.003 (n ⫽ 5)

3.89 ⫾ 0.25 (n ⫽ 5) 0.023 ⫾ 0.004 (n ⫽ 5)

0.009 0.73

weights in the offspring after weaning. Pausova et al. (22) reported that prenatal exposure to nicotine did not result in any weight gain in either the spontaneously hypertensive rats or normotensive Brown Norway rats between salineand nicotine-exposed animals at 9 weeks of age. In this study, we have shown that the accelerated postnatal growth of the nicotine-exposed offspring was not apparent until after 9 weeks of age. It is, therefore, possible that a difference in body weight may have occurred in the spontaneously hypertensive rats and Brown Norway rats if these animals had been allowed to age further. In addition to an overall weight gain, nicotine exposure during fetal and neonatal life resulted in a significant increase in fat pad weights and PVAT at 26 weeks of age. The concomitant increase in fat pad weight and PVAT suggests that in utero and lactational exposure to nicotine results in increased weight gain as a result of an overall increase in adiposity in the offspring. Results from our study have shown for the first time, to our knowledge, that exposure to nicotine during fetal and neonatal life can alter the modulatory action of PVAT on vessel contraction in animals during adult life, based on the following observations. Aortic rings from the control rats contracted less in response to PE in the presence of PVAT than without PVAT, suggesting that PVAT was releasing a relaxation factor that attenuated the contractile response. In the nicotine group, the effect of PVAT on PE-induced contraction was not significant compared with that present in the control rats, indicating that the relaxation factor released by PVAT in the nicotine group was less effective. 690


This is confirmed by the transfer experiments where we found that incubating the Fat⫺ vessel with the bathing solution from the Fat⫹ vessel caused only a small relaxation response in the nicotine treatment group compared with the control group. Interestingly, the diminished secretion or action of the relaxation factor in nicotine-treated animals occurred despite an increased amount of PVAT surrounding the aorta. The change in contractility of aorta from the nicotine group in the presence of PVAT was not caused by an inherent change in vessel contractility because the contraction in response to PE, which operates through the activation of ␣-adrenergic receptors, was similar between the control and nicotine groups after removal of PVAT. Furthermore, the response to KCl, which operates through the depolarization of the cell membrane, was similar between the Fat⫹ and Fat⫺ rings in the control and nicotine groups, and there was no difference between the aorta from control and nicotine groups in the absence of PVAT. This is not surprising because this relaxation factor apparently acts by tyrosine kinase-dependent activation of K⫹ channels (21), so that depolarization of the cell membrane with KCl will render the relaxation factor ineffective. Taken together, these results suggest that in utero and lactational exposure to nicotine can alter the function of PVAT in the adult animal. Furthermore, we propose that diminished secretion of the relaxation factor from the PVAT in nicotine-exposed animals may result in impaired vessel relaxation in vivo and may ultimately lead to hypertension in the offspring. This is supported by our findings that neonatal exposure to nicotine using a similar protocol induced the development of hyper-


Figure 2: (A-D) Concentration-contraction response to phenylephrine of Fat⫹ and Fat⫺ aortic rings from the offspring of Wistar rats exposed prenatally to nicotine or saline. The results are expressed as a percentage of KCl contraction. * p ⬍ 0.05 vs. (A) Fat⫹ rings or (C) rings from saline-exposed rats. (E) Contraction of aortic rings to 60 mM KCl, expressed in absolute value.

tension in the offspring of normotensive Wistar rats and two genetic strains of hypertensive rats (Yu-Jing Gao, Alison Holloway, and Robert Lee, unpublished results). In conclusion, we have found that fetal and neonatal exposure to nicotine resulted in an accelerated postnatal weight gain and increased visceral adiposity as well as an increase in the amount of fat surrounding the vasculature. Moreover, to our knowledge, this is the first study to show that nicotine exposure during fetal and neonatal life results in an attenuation of vessel relaxation in adult life in response to a factor from the PVAT, probably because of an alteration in the nature or a decrease in the amount of the relaxation factor secreted. We speculate that, if the role of PVAT in humans is similar to our animal model, altered deposition and/or function of the PVAT may contribute to impaired vessel relaxation in the offspring of women who smoked during pregnancy, which may explain, in part, the increased prevalence of hypertension in these children. Furthermore, increased adiposity as a result of fetal and neonatal nicotine exposure may explain the increased prevalence of obesity in children exposed to cigarette smoke in utero.

Figure 3: (A and B) Typical tracing showing the relaxation caused by the transfer of bathing solution incubated with Fat⫹ aortic ring to Fat⫺ rings in the presence of 1 ␮M PE in arteries from salineor nicotine-exposed offspring. (C) Summary of the relaxation induced by the transfer of bathing solution from Fat⫹ ring to Fat⫺ ring, expressed as a percentage of precontraction value by PE. ** p ⬍ 0.01 vs. saline-exposed rats.

Acknowledgments We thank Sandra Stals, Nina Li, and the staff of the Central Animal Facility of McMaster University for assistance with the animal studies. This research was supported by the Heart and Stroke Foundation of Ontario. References 1. Oken E, Gillman MW. Fetal origins of obesity. Obes Res. 2003;11:496 –506. 2. Kramer MS. Socioeconomic determinants of intrauterine growth retardation. Eur J Clin Nutr. 1998;52(Suppl 1):S29 – 32. 3. Robinson JS, Moore VM, Owens JA, McMillen IC. Origins of fetal growth restriction. Eur J Obstet Gynecol Reprod Biol. 2000;92:13–9. 4. Wideroe M, Vik T, Jacobsen G, Bakketeig LS. Does maternal smoking during pregnancy cause childhood overweight? Paediatr Perinat Epidemiol. 2003;17:171–9. OBESITY RESEARCH Vol. 13 No. 4 April 2005

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5. Andres RL, Day MC. Perinatal complications associated with maternal tobacco use. Semin Neonatol. 2000;5:231– 41. 6. Cnattingius S, Lambe M. Trends in smoking and overweight during pregnancy: prevalence, risks of pregnancy complications, and adverse pregnancy outcomes. Semin Perinatol. 2002;26:286 –95. 7. Faiz AS, Ananth CV. Etiology and risk factors for placenta previa: an overview and meta-analysis of observational studies. J Matern Fetal Neonatal Med. 2003;13:175–90. 8. Nordentoft M, Lou HC, Hansen D, et al. Intrauterine growth retardation and premature delivery: the influence of maternal smoking and psychosocial factors. Am J Public Health. 1996; 86:347–54. 9. Shiverick KT, Salafia C. Cigarette smoking and pregnancy I: ovarian, uterine and placental effects. Placenta. 1999;20:265–72. 10. Bergmann KE, Bergmann RL, Von Kries R, et al. Early determinants of childhood overweight and adiposity in a birth cohort study: role of breast-feeding. Int J Obes Relat Metab Disord. 2003;27:162–72. 11. Blake KV, Gurrin LC, Evans SF, et al. Maternal cigarette smoking during pregnancy, low birth weight and subsequent blood pressure in early childhood. Early Hum Dev. 2000;57: 137– 47. 12. Morley R, Leeson PC, Lister G, Lucas A. Maternal smoking and blood pressure in 7.5 to 8 year old offspring. Arch Dis Child. 1995;72:120 – 4. 13. Toschke AM, Koletzko B, Slikker W Jr, Hermann M, Von Kries R. Childhood obesity is associated with maternal smoking in pregnancy. Eur J Pediatr. 2002;161:445– 8. 14. Vik T, Jacobsen G, Vatten L, Bakketeig LS. Pre- and post-natal growth in children of women who smoked in pregnancy. Early Hum Dev. 1996;45:245–55. 15. Von Kries R, Toschke AM, Koletzko B, Slikker W Jr. Maternal smoking during pregnancy and childhood obesity. Am J Epidemiol. 2002;156:954 – 61. 16. Rustemeier K, Stabbert R, Haussmann HJ, Roemer E, Carmines EL. Evaluation of the potential effects of ingredi-

692


17.

18.

19.

20.

21.

22.

23.

24.

25. 26.

27.

ents added to cigarettes. Part 2: chemical composition of mainstream smoke. Food Chem Toxicol. 2002;40:93–104. Swauger JE, Steichen TJ, Murphy PA, Kinsler S. An analysis of the mainstream smoke chemistry of samples of the U.S. cigarette market acquired between 1995 and 2000. Regul Toxicol Pharmacol. 2002;35:142–56. Newman MB, Shytle RD, Sanberg PR. Locomotor behavioral effects of prenatal and postnatal nicotine exposure in rat offspring. Behav Pharmacol. 1999;10:699 –706. Evereklioglu C, Alasehirli B, Sari I, Cengiz B, Bagci C. Effect of nicotine exposure during gestation on neonatal rat crystalline lenses. Eye. 2004;18:67–73. Williams CM, Kanagasabai T. Maternal adipose tissue response to nicotine administration in the pregnant rat: effects on fetal body fat and cellularity. Br J Nutr. 1984;51:7–13. Lohn M, Dubrovska G, Lauterbach B, Luft FC, Gollasch M, Sharma AM. Periadventitial fat releases a vascular relaxing factor. FASEB J. 2002;16:1057– 63. Pausova Z, Paus T, Sedova L, Berube J. Prenatal exposure to nicotine modifies kidney weight and blood pressure in genetically susceptible rats: a case of gene-environment interaction. Kidney Int. 2003;64:829 –35. Dukacz SA, Feng MG, Yang LF, Lee RM, Kline RL. Abnormal renal medullary response to angiotensin II in SHR is corrected by long-term enalapril treatment. Am J Physiol Regul Integr Comp Physiol. 2001;280:R1076 – 84. Abate N. Obesity and cardiovascular disease. Pathogenetic role of the metabolic syndrome and therapeutic implications. J Diabetes Complications. 2000;14:154 –74. Bray GA. The underlying basis for obesity: relationship to cancer. J Nutr. 2002;132(11 Suppl):3451S–5S. Ravussin E, Smith SR. Increased fat intake, impaired fat oxidation, and failure of fat cell proliferation result in ectopic fat storage, insulin resistance, and type 2 diabetes mellitus. Ann N Y Acad Sci. 2002;967:363–78. Sowers JR. Obesity and cardiovascular disease. Clin Chem. 1998;44:1821–5.