Maternal smoking and risk of
metabolic syndrome in the
offspring: Evidence from
animal studies
Dr. Alison Holloway
Department of Obstetrics and Gynecology
Reproductive Biology Division
McMaster University
October 1st 2014
What is metabolic syndrome?
Metabolic syndrome is the name for a group of risk factors that
raises your risk for heart disease and other health problems, such
as diabetes and stroke.
These include:
Central obesity
Elevated serum triglycerides
(≥ 150 mg/dL (1.7 mmol/L))
Reduced HDL cholesterol (< 40 mg/dL (1.03 mmol/L*) males and < 50
mg/dL (1.29 mmol/L*)
Raised blood pressure (systolic BP ≥ 130 or diastolic BP ≥ 85 mm Hg)
Raised fasting plasma glucose (≥ 100 mg/dL (5.6 mmol/L), or type 2 diabetes
Why worry?
• The prevalence of metabolic syndrome continues to grow unabated
• Worldwide it is estimated that there are 1 billion overweight and 300 million
obese adults (World Health Organization)
•Overweight and obesity are associated with increased mortality and morbidity
(including an increased risk of chronic diseases such as diabetes, hypertension, asthma,
heart disease, and cancer)
Estimated prevalence of obesity among all Canadians by province, 2000-2012
15-19%
20-24%
25-29%
30-34%
≥35%
2000
2003
2005
2007
2008
2009
2010
2011
2012
Sources: Gotay C, Katzmarzyk P, Janssen I, Dawson M, Aminoltejari K, Bartley N (2013). Updating the Canadian obesity maps: An epidemic in progress. Canadian Journal of
Public Health 104(1). Retrieved from http://journal.cpha.ca/index.php/cjph/article/view/3513. Adults with BMI >30kg/m2 in each province calculated from the self-reported height
and weight surveys conducted by the CCHS and corrected to account for misreporting of height and weight.
Statistics Canada. Table 105-0501 - Health indicator profile, annual estimates, by age group and sex, Canada, provinces, territories, health regions (2012 boundaries) and peer
groups, occasional, CANSIM (database). Accessed: 2013-10-20 . Permalink: http://www5.statcan.gc.ca/cansim/a05?lang=eng&id=1050501
Source: WHO Diabetes Programme (http://www.who.int/diabetes/facts/en/)
Does Metabolic Syndrome Begin in the Womb?
The Barker Hypothesis- original version
Maternal Undernutrition
Fetal Growth Retardation
Coronary Heart Disease In Later Life
Death from coronary heart disease
before age 65 according to birthweight
(Hertfordshire population)
1.6
P for trend=0.001
1.4
Hazard ratio
1.2
1.0
0.8
0.6
0.4
0.2
0.0
≤5.5
5.5-6.5
6.5-7.5
7.5-8.5
Birthweight
Barker et al.,1989 Lancet 2:577
8.5-9.5
≥9.5
The Barker Hypothesis
Also known as:
1) Fetal origins of adult disease hypothesis
2) Fetal programming hypothesis
3) Developmental origins of health and disease hypothesis
Adverse environments in fetal life and
early childhood establishes an increased
risk of disease in adult life
Causes of low birthweight in humans
and animal models
Prenatal glucocorticoid exposure
Uteroplacental insufficiency/hypoxia
Maternal / Fetal undernutrition
Intrauterine Growth Restriction
Hypertension
Obesity
Type 2 Diabetes
Causes of low birthweight in humans
and animal models
Prenatal glucocorticoid exposure
Uteroplacental insufficiency/hypoxia
Maternal / Fetal undernutrition
Maternal smoking
Intrauterine Growth Restriction
Hypertension
Obesity
Type 2 Diabetes
Maternal cigarette smoking
• Associated with preterm
labour, low birthweight
babies and other adverse
obstetrical outcomes
• Approximately 15-20% of all
pregnant women smoke
during their pregnancies
22.7% of pregnant women at McMaster
reported smoking during pregnancy (Foster
et al., 2005. Am J Obstet Gynecol 193:1900-1907)
Q: What component of cigarette
smoke is important for increased risk
of postnatal disease?
A: Nicotine?
Why is nicotine important?
• Nicotine addiction is primary reason women do not quit
smoking during pregnancy
• Nicotine replacement therapy (NRT) has been suggested as
an acceptable therapy for pregnant women who can’t quit
smoking by other means (OMA, 1999)
•Nicotine administration alone during pregnancy in rats causes
a reduction in birthweight
(Newman et al., 1999 Behav Pharmacol 10:699-706)
Locomotor behavioral effects of prenatal and postnatal
nicotine exposure in rat offspring.
Abstract
The purpose of this study was to determine if prenatal/postnatal nicotine exposure results in
hyperactive offspring. Rat offspring were exposed to nicotine, through implantation of osmotic
minipumps in dams, at levels of 0.75, 1.5 and 3.0 mg/kg/day, for 19 days prenatally and 16 days
postnatally. Offspring were measured for gestation length, body weight, litter size, sex difference
and locomotor activity. No significant effects were shown for gestation length, litter size or male to
female pup ratio. However, higher percentage of pup deaths resulted from nicotine-exposed dams
than from control dams. Significantly less litter body weight was shown in nicotine-exposed
offspring on postnatal day 1 when compared to controls. However, these offspring surpassed the
control groups in litter body weight on postnatal day 14 and 21. Hyperactivity was shown in
offspring exposed to prenatal/postnatal nicotine at levels of 0.75 and 3.0 mg/kg/day on postnatal
day 14, but not on postnatal day 21 or at the 1.5 mg/kg/day condition. Results are consistent with
the hypothesis that rat offspring are susceptible to the neurochemical and neurobehavioral effects
of prenatal/postnatal nicotine exposure.
Behav Pharmacol 1999 Nov;10(6-7):699-706
Effect of nicotine on fetal growth
7
Saline
Nicotine
*
6
*
5
Weight (g)
4
3
2
1
0
d15
Gruslin et al., 2009 Reprod Sci 16: 875-882
d18
d21
PND1
Hypothesis
Nicotine exposure in utero will cause
metabolic syndrome in the offspring
Specific aims
To assess the effect of in utero
exposure to nicotine on:
• Postnatal growth and adiposity
• Markers of cardiovascular disease
• Glucose homeostasis
Introduction: Animal Model
2 weeks
3 weeks
3 weeks
Mating
Pregnancy
Lactation
Nicotine (1.0 mg/kg/d)
Saline (Vehicle)
Birth
(Postnatal
Day 1)
Weaning
(Postnatal
Day 21)
23 weeks
Outcome measures to determine
changes related to:
Obesity
Hypertension
T2DM
Endpoint
26 weeks
Birthweight
7.5
Birthweight (g)
7.0
*
6.5
6.0
5.5
5.0
4.5
4.0
saline
nicotine
Fetal exposure to nicotine reduced average birthweight approximately 20%. There was no effect on litter size
OBESITY
Fetal and neonatal exposure to nicotine
increases body weight
*
700
Body weight (g)
600
nicotine
saline
500
*
*
400
300
200
100
0
0
5
10
15
20
Age (weeks)
25
30
Fetal and neonatal exposure to
nicotine increases fat pad weight
3 weeks of age
WAT (% of bodyweight)
+67%
26 weeks of age
6
5
+35%
4
3
2
1
0
(Somm et al., 2008 Endocrinology 149:6289-6299)
*
saline
nicotine
*P<0.05
Perivascular fat tissue (PVFT) at 26
weeks of age
J Endocrinol. 2008 Apr;197(1):55-64
PVFT area
(mm2)
Saline
Nicotine
Mesenteric
artery
7.7 ± 0.73
11.2 ± 1.27*
Thoracic
artery
2.6 ± 0.24
3.9 ± 0.25*
*P<0.05
Nicotine-exposed animals have dyslipidemia
Age
7 weeks
Serum triglyceride
concentration
mg/dL
Body weight (g)
SALINE
NICOTINE
SALINE
NICOTINE
71.6 ± 10.56
95.6 ± 6.29
264.2 ± 7.94
264.7 ± 1.43
p=0.052
15 weeks
115.9 ± 3.44
126.8 ± 2.89*
487.5 ± 11.65
545.0 ± 6.24*
26 weeks
149.5 ± 5.56
177.2 ± 16.48*
576.5 ± 14.44
653.9 ± 10.43*
*P<0.05
What causes the increased weight gain
in nicotine-exposed animals?
Mechanisms of Programming?
Nicotine (or other insults)
PHYSIOLOGICAL SYSTEMS
•Reorganisation of organ structure
•Altered organ function
•Altered cell number
•Altered intracellular organization
DNA
•altered cell specific gene regulation
•altered DNA binding proteins
•changes in mitochondrial DNA
EPIGENETIC CHANGES
•altered DNA methylation
Increased adipocyte differentiation
(Somm et al., 2008 Endocrinology 149:6289-6299)
*P<0.05
Food intake (g/rat/day)
Increased food consumption and
decreased activity
*
32
28
24
20
16
saline
nicotine
*P<0.05
HYPERTENSION
Blood pressure
Mean arterial pressure (mm Hg)
170
160
Systolic BP
150
140
Diastolic BP
130
BP increased by
14-17 weeks of age
120
Same changes seen
in WKY & SHR rats
110
100
saline
nicotine
Blood pressure at 26 weeks of age in saline- and nicotine-exposed male offspring (n=15 per
group).
What causes the increased blood
pressure in nicotine-exposed animals?
• Altered renal development?
Elevated blood pressure does not
appear to be related to changes in
renal structure or function
Saline
Nicotine
Kidney weight (g)
2.84 ± 0.05
2.93 ± 0.07
Kidney weight
(% body weight)
0.69 ± 0.01
0.66 ± 0.01
Nephron density
(per mm3)
108.2 ± 1.80
93.2 ± 5.04
Nephron number
(x1000 per kidney)
65.4 ± 1.95
68.83 ± 3.99
Glomerulus area
(µm2)
7298 ± 609
8115 ± 858
Urinary albumin
(ng /mg creatinine)
0.38 ± 0.066
0.38 ± 0.063
Gao YJ et al., unpublished data
What causes the increased blood
pressure in nicotine-exposed animals?
• Altered renal development?
• Altered vascular reactivity?
Vascular reactivity
Contraction (% of KCl)
200
*
Nicotine
*
150
Saline
100
50
0
8
7
6
5
Phenylephrine (- log M)
4
What causes the increased blood
pressure in nicotine-exposed animals?
• Altered renal development?
• Altered vascular reactivity?
Conclusion
Fetal and neonatal exposure to
nicotine results in postnatal
hypertension
TYPE 2 DIABETES
Fasting glucose
*
3200
3000
2800
AUC
Fasting serum glucose (mmol/l)
9.0
2600
2400
2200
nicotine
2000
SV
NV
7.5
saline
6.0
4.5
0
5
10
15
20
25
30
Age (weeks)
Basal fasting glucose at 4, 7, 15 and 26 weeks of age (n=15 per group). Measurements were made on the
same animals at each age. The inset panel represents the total area under the curve (* p<0.05)
Oral Glucose Tolerance Test
•Animals fasted overnight
•Fasting blood sample taken at t=0
•Glucose challenge (2g/kg) by gavage
•Serial blood samples collected at 30’ and 120’
to assess peak glucose and ability to clear the
glucose load respectively
•Total glucose response to the challenge (area
under the curve) calculated
Nicotine-exposed offspring are dysglycaemic
by 26 weeks of age
2 Weeks
3 Weeks
3 Weeks
Mating
Pregnancy
Lactation
23 Weeks
Nicotine (1.0 mg/kg/d)
Saline (Vehicle)
Birth
Endpoint
(26 weeks)
Weaning
(3 weeks)
*
14
1400
*
*
12
1300
nicotine
AUC
Serum glucose (mmol/l)
1500
10
saline
8
0
20
1100
1000
900
2g/kg glucose
6
1200
40
60
80
100
Time (minutes)
120
140
800
saline
nicotine
Conclusion
Fetal and neonatal exposure to
nicotine results in aberrant
glucose control in adulthood
What causes nicotine-induced
dysglycemia?
• Reduced beta cell mass?
Beta cell mass
12
10
*
saline
Beta cell mass (mg)
8
6
2
nicotine
*
4
*
0
0
5
10
15
20
25
30
Age (weeks)
Beta cell mass from birth to 26 weeks of age measured by immunohistochemical staining for insulin and
morphometric analysis at 26 weeks of age (n=5 per group). Data are presented as mean ± SEM (*p<0.05).
What causes nicotine-induced
dysglycemia?
• Reduced beta cell mass?
• Reduced insulin effect at target tissues?
Insulin resistance
Insulin response to OGTT
*
AUC insulin
300
250
200
150
100
50
0
saline
nicotine
Increased insulin response was not sufficient
to normalize glucose response to OGTT .
Insulin resistance
Insulin response to OGTT
400
*
300
250
300
200
ROD
AUC insulin
Insulin receptor expression
(skeletal muscle)
150
200
100
100
*
50
0
0
saline
nicotine
saline
nicotine
What causes nicotine-induced
dysglycemia?
Reduced beta cell mass?
Reduced insulin effect at target tissues?
Reduced beta cell function?
Beta cell function: Glucose stimulated
insulin secretion (islets)
*
3.3 mmol glucose (basal)
16.7 mmol glucose (stimulated)
Islet cells from nicotine-exposed animals are unable to release
insulin in response to a glucose stimulus
What causes nicotine-induced
dysglycemia?
Reduced beta cell mass?
Reduced insulin effect at target tissues?
Reduced beta cell function?
Does in utero exposure to cigarette
smoke affect postnatal health in
humans?
• Increased risk of obesity
Ino T. 2010. Maternal smoking during pregnancy and offspring obesity: Meta-analysis. Pediatrics International 52:
94-99
• Increased risk of hypertension
Power C et al. 2010. Maternal smoking in pregnancy, adult adiposity and other risk factors for cardiovascular
disease. Atherosclerosis 211: 643-648.
• Increased risk of type 2 diabetes
Montgomery SM and Ekbom A. 2002. Smoking during pregnancy and diabetes mellitus in a British longitudinal
birth cohort. BMJ 324: 26-27.
Summary of studies assessing maternal smoking and diabetes risk in the offspring
1
Conclusions
Fetal and neonatal exposure to nicotine
results in increased postnatal body weight
and adiposity
The metabolic phenotype of nicotineexposed animals is consistent with what is
seen in children born to women who
smoked during pregnancy
What about other smoking cessation drugs?
Two non-NRTs approved for use
for smoking cessation
•Varenicline
•Bupropion
(Champix®)
http://www.medix24.com/french/
images/champix-box.jpg
(Zyban®)
http://www.tristatemeds.com/upl
oad/zyban_box1.jpg
Smoking cessation pharmacotherapies
nAChR
Nicotine
Full agonist
Varenicline
Partial agonist
Bupropion
Antagonist
Beta cell function: Glucose stimulated
insulin secretion (INS-1E cells)
Insulin release
(% of control at 3.3 mM glucose)
300
3.3 mmol glucose (basal)
*
16.7 mmol glucose (stimulated)
250
200
150
100
50
0
Control
Nicotine
(1µM)
Beta cell function: Glucose stimulated
insulin secretion (INS-1E cells)
3.3 mmol glucose (basal)
Insulin release
(% of control at 3.3 mM glucose)
300
16.7 mmol glucose (stimulated)
*
250
200
150
100
50
0
Control
Nicotine
(1µM)
Varenicline
(1µM)
Beta cell function: Glucose stimulated
insulin secretion (INS-1E cells)
Insulin release
(% of control at 3.3 mM glucose)
300
3.3 mmol glucose (basal)
*
16.7 mmol glucose (stimulated)
250
200
150
100
50
0
Control
Nicotine
(1µM)
Varenicline
(1µM)
Bupropion
1µM
Beta cell function: Glucose stimulated
insulin secretion (INS-1E cells)
3.3 mmol glucose (basal)
Insulin release
(% of control at 3.3 mM glucose)
300
16.7 mmol glucose (stimulated)
*
*
250
200
150
100
50
0
Control
Nicotine
(1µM)
Varenicline
(1µM)
Bupropion
1µM
Mecamylamine
100µM
Bupropion use in pregnancy
Bupropion is the most commonly prescribed non-SSRI
antidepressant for use during pregnancy and during the
post-partum period
Andrade et al., 2008 Am J Obstet Gynecol 198: 194.e1-194.e5
% of pregnancies
6
5
4
3
2
1
0
SSRI
TCA
bupropion
other
Effect of fetal and neonatal exposure to
bupropion (Zyban®)
Saline
Bupropion (5 or 10mg/kg/d)
2 wks
Mating
3 wks
3 wks
Parturition
(PND1)
Outcome measures to determine
changes related to:
Weaning
(Week 3)
Obesity
T2DM
6 months:
Endpoint
Bupropion exposure does not increase
adiposity in the offspring
.
Outcome measure
Control
Bupropion
Bupropion
5mg/kg
10mg/kg
P-value
Body weight (g)
563.6 ± 13.1
607.6 ± 11. 9
574.7 ± 1 5.0
P=0.06
Mesenteric fat pad weight (g)
6.8 ± 0. 6
7.9 ± 0.6
7.2 ± 0.7
P=0.43
Perirenal fat pad weight (g)
13.0 ± 0. 6
15.5 ± 1.0
Epididymal fat pad weight (g)
7.3 ± 0.9
8.8 ± 0.6
7.6 ± 1. 2
P=0.46
Total fat pad weight (g)
27.0 ± 1. 8
32.2 ± 1.7
28.8 ± 3.6
P=0.33
Total fat (% of body wt)
4.8 ± 0.2
5.3 ± 0.3
4.9 ± 0.5
P=0.58
Data are presented as mean ± SEM.
Reprod Sci. 2013 Oct;20(10):1156-61
14.0 ± 1.8
P=0.3 5
Glucose homeostasis
GTT
22
Control
Bupropion 5mg/kg/d
Bupropion 10mg/kg/d
20
Glucose (mmol/l)
18
16
14
12
10
8
6
4
0
20
40
60
Time (minutes)
80
100
120
140
Conclusion
Bupropion exposure during fetal and
neonatal development did not affect
metabolic homeostasis in the offspring
Fetal and neonatal nicotine exposure may
adversely affect the health of the offspring
and raise concerns regarding the safety of
NRT use during pregnancy
What next?
25
*
20
15
10
5
0
Control
Nicotine
Experimental Groups
N Ma, DB Hardy and AC Holloway, unpublished data
Hepatic FAS: -actin mRNA Levels
Hepatic triglyceride levels (mg/g)
Examine why nicotine-exposed animals
have dyslipidemia
FAS
8
*
6
4
2
0
Control
Nicotine
Experimental Groups
Acknowledgements
Students and Staff
Dr. Jenny Bruin
Amanda Woynillowicz
Bryce Poirier
Jillian Hyslop
Nicole DeLong
Edward Hadzocos
Igal Raizman
Gareth Lim
Lisa Kellenberger
Alex Petre
Sandra Stals
Bart Hettinga
Staff of the CAF
Collaborators
Dr. Yu-Jing Gao
Dr. Jim Petrik
Dr. Hertzel Gerstein
Dr Dan Hardy
Dr Robert Lee
Dr Katherine Morrison
Dr Sandeep Raha
FUNDING
Bupropion?
nAChR
Nicotine
Full agonist
Bupropion
(Zyban®)
Antagonist