Maternal smoking and risk of metabolic syndrome in the offspring: Evidence from
animal studies
Dr. Alison HollowayDr. Alison HollowayDepartment of Obstetrics and GynecologyDepartment of Obstetrics and Gynecology
Reproductive Biology DivisionReproductive Biology DivisionMcMaster UniversityMcMaster University
October 1October 1stst 2014 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 < 50mg/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)
2003
2007
2005
2008 2009
2010
2000
2011 2012
Estimated prevalence of obesity among all Canadians by province, 2000-2012
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
15-19% 20-24% 25-29% 30-34% ≥35%
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
≤5.5 5.5-6.5 6.5-7.5 7.5-8.5 8.5-9.5 ≥9.50.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
P for trend=0.001
Birthweight
Death from coronary heart disease before age 65 according to birthweight
(Hertfordshire population)H
azar
d ra
tio
Barker et al.,1989 Lancet 2:577
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
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
Intrauterine Growth Restriction
Hypertension
Obesity
Type 2 Diabetes
Causes of low birthweight in humans and animal models
Maternal smoking
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?
• 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)
Why is nicotine important?
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
d15 d18 d21 PND1
0
1
2
3
4
5
6
7SalineNicotine
Wei
ght (
g)
**
Gruslin et al., 2009 Reprod Sci 16: 875-882
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
2 weeks
Birth(Postnatal
Day 1)
3 weeks
Pregnancy Lactation
3 weeks
Mating
Weaning(Postnatal Day 21)
23 weeks
Endpoint26 weeks
Nicotine (1.0 mg/kg/d)
Saline (Vehicle)
Introduction: Animal Model
Outcome measures to determine changes related to:
ObesityHypertension
T2DM
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Bir
thw
eig
ht (
g)
saline nicotine
*
Birthweight
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
Age (weeks)
0 5 10 15 20 25 300
100
200
300
400
500
600
700
Bo
dy w
eig
ht (
g)
nicotine
saline
*
**
Fetal and neonatal exposure to nicotine increases fat pad weight
(Somm et al., 2008 Endocrinology 149:6289-6299)
3 weeks of age
saline
nicotine
*P<0.05
0
1
2
3
4
5
6
*
WA
T (
% o
f b
od
ywei
gh
t)
26 weeks of age
+67% +35%
Perivascular fat tissue (PVFT) at 26 weeks of age
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 J Endocrinol. 2008 Apr;197(1):55-64
Nicotine-exposed animals have dyslipidemia
653.9 ± 10.43*576.5 ± 14.44177.2 ± 16.48*149.5 ± 5.5626 weeks
545.0 ± 6.24*487.5 ± 11.65126.8 ± 2.89*115.9 ± 3.4415 weeks
264.7 ± 1.43264.2 ± 7.9495.6 ± 6.29p=0.052
71.6 ± 10.567 weeks
NICOTINESALINENICOTINESALINE
Body weight (g)Serum triglyceride concentration
mg/dL
Age
*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
Increased food consumption and decreased activity
saline nicotine
16
20
24
28
32
Foo
d in
take
(g/
rat/d
ay) *
*P<0.05
HYPERTENSION
Blood pressure
saline nicotine100
110
120
130
140
150
160
170
Mea
n ar
teria
l pre
ssur
e (m
m H
g) Systolic BP
Diastolic BP
Same changes seen in WKY & SHR rats
Blood pressure at 26 weeks of age in saline- and nicotine-exposed male offspring (n=15 per group).
BP increased by14-17 weeks of age
What causes the increased blood pressure in nicotine-exposed animals?
• Altered renal development?
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
Elevated blood pressure does not appear to be related to changes in
renal structure or function
Gao YJ et al., unpublished data
What causes the increased blood pressure in nicotine-exposed animals?
• Altered renal development?
• Altered vascular reactivity?
Phenylephrine (- log M)
0
50
100
150
200
8 7 6 5 4
Nicotine
Saline
**
Con
trac
tion
(% o
f K
Cl)
Vascular reactivity
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 glucoseF
astin
g s
eru
m g
luco
se (
mm
ol/l)
SV NV2000
2200
2400
2600
2800
3000
3200 *
0 5 10 15 20 25 304.5
6.0
7.5
9.0
AU
C
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)
saline
nicotine
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 (areaunder the curve) calculated
Birth
Pregnancy LactationMating
Weaning(3 weeks)
Endpoint(26 weeks)
Nicotine (1.0 mg/kg/d)
Saline (Vehicle)
3 Weeks 3 Weeks2 Weeks 23 Weeks
* *
0 20 40 60 80 100 120 140
6
8
10
12
14
2g/kg glucose
Ser
um
glu
cose
(m
mol
/l)
Time (minutes)
saline nicotine
AU
C
800
900
1000
1100
1200
1300
1400
1500
*
nicotine
saline
Nicotine-exposed offspring are dysglycaemic by 26 weeks of age
Conclusion
Fetal and neonatal exposure to nicotine results in aberrant
glucose control in adulthood
What causes nicotine-induced dysglycemia?
• Reduced beta cell mass?
0 5 10 15 20 25 30
0
2
4
6
8
10
12
Age (weeks)
Bet
a ce
ll m
ass
(m
g)
*
*
*
Beta cell mass
saline
nicotine
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?
0
50
100
150
200
250
300 *
saline nicotine
AU
C in
sulin
Insulin resistance
Insulin response to OGTT
Increased insulin response was not sufficient to normalize glucose response to OGTT .
0
50
100
150
200
250
300 *
saline nicotine
AU
C in
sulin
Insulin resistance
RO
D
saline nicotine0
100
200
300
400
*
Insulin response to OGTT Insulin receptor expression(skeletal muscle)
Reduced beta cell mass?
Reduced insulin effect at target tissues?
Reduced beta cell function?
What causes nicotine-induced dysglycemia?
Beta cell function: Glucose stimulated insulin secretion (islets)
Islet cells from nicotine-exposed animals are unable to release insulin in response to a glucose stimulus
3.3 mmol glucose (basal)
16.7 mmol glucose (stimulated)
*
Reduced beta cell mass?
Reduced insulin effect at target tissues?
Reduced beta cell function?
What causes nicotine-induced dysglycemia?
Does in utero exposure to cigarette smoke affect postnatal health in
humans?
• Increased risk of obesityIno T. 2010. Maternal smoking during pregnancy and offspring obesity: Meta-analysis. Pediatrics International 52: 94-99
• Increased risk of hypertensionPower 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 nicotine-exposed animals is consistent with what is seen in children born to women who smoked during pregnancy
What about other smoking cessation drugs?
http://www.medix24.com/french/images/champix-box.jpg
http://www.tristatemeds.com/upload/zyban_box1.jpg
Two non-NRTs approved for use for smoking cessation
•Varenicline (Champix®)
•Bupropion (Zyban®)
Smoking cessation pharmacotherapies
Varenicline
Nicotine
Full agonist
nAChR
Partial agonist
Bupropion
Antagonist
Control Nicotine(1µM)
0
50
100
150
200
250
300 3.3 mmol glucose (basal)
16.7 mmol glucose (stimulated)*
Insu
lin r
elea
se
(% o
f con
trol
at 3
.3 m
M g
luco
se)
Beta cell function: Glucose stimulated insulin secretion (INS-1E cells)
Varenicline(1µM)
0
50
100
150
200
250
300
Control Nicotine(1µM)
*
Insu
lin r
elea
se
(% o
f con
trol
at 3
.3 m
M g
luco
se)
3.3 mmol glucose (basal)
16.7 mmol glucose (stimulated)
Beta cell function: Glucose stimulated insulin secretion (INS-1E cells)
Bupropion1µM
0
50
100
150
200
250
300
Control Nicotine(1µM)
*
Insu
lin r
elea
se
(% o
f con
trol
at 3
.3 m
M g
luco
se)
3.3 mmol glucose (basal)
16.7 mmol glucose (stimulated)
Beta cell function: Glucose stimulated insulin secretion (INS-1E cells)
Varenicline(1µM)
Bupropion1µM
0
50
100
150
200
250
300
Control Nicotine(1µM)
*
Insu
lin r
elea
se
(% o
f con
trol
at 3
.3 m
M g
luco
se)
3.3 mmol glucose (basal)
16.7 mmol glucose (stimulated)
Beta cell function: Glucose stimulated insulin secretion (INS-1E cells)
Varenicline(1µM)
Mecamylamine100µM
*
Bupropion use in pregnancy
Bupropion is the most commonly prescribed non-SSRI antidepressant for use during pregnancy and during the post-partum periodAndrade et al., 2008 Am J Obstet Gynecol 198: 194.e1-194.e5
SSRI TCA bupropion other0
1
2
3
4
5
6
% o
f pr
egna
ncie
s
2 wks
Mating Parturition(PND1)
Weaning(Week 3)
Saline
Bupropion (5 or 10mg/kg/d)
6 months:Endpoint
3 wks 3 wks Outcome measures to determine changes related to:
ObesityT2DM
Effect of fetal and neonatal exposure to bupropion (Zyban®)
Bupropion exposure does not increase adiposity in the offspring
.
Data are presented as mean ± SEM.
Outcome measure
C ontrol Bupropion
5mg/kg
Bupropion
10mg/kg
P- value
Body weight (g)
563.6 ± 13.1 607.6 ± 11.9 574.7 ± 15.0 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 14.0 ± 1.8 P=0.35
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
Reprod Sci. 2013 Oct;20(10):1156-61
0 20 40 60 80 100 120 1404
6
8
10
12
14
16
18
20
22 Control
Bupropion 10mg/kg/d Bupropion 5mg/kg/d
Glu
cose
(mm
ol/l
)
Time (minutes)
GTT
Glucose homeostasis
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?
Examine why nicotine-exposed animals have dyslipidemia
FAS
Control Nicotine0
2
4
6
8
*
Experimental Groups
Hep
atic
FA
S:
-a
ctin
mR
NA
Lev
els
Control Nicotine0
5
10
15
20
25
*
Experimental Groups
Hep
atic
tri
gly
ceri
de
leve
ls (
mg
/g)
N Ma, DB Hardy and AC Holloway, unpublished data
AcknowledgementsStudents and Staff
Dr. Jenny Bruin Amanda Woynillowicz Bryce PoirierJillian HyslopNicole DeLongEdward HadzocosIgal Raizman Gareth LimLisa KellenbergerAlex Petre
Sandra StalsBart HettingaStaff of the CAF
CollaboratorsDr. Yu-Jing Gao Dr. Jim PetrikDr. Hertzel GersteinDr Dan HardyDr Robert LeeDr Katherine MorrisonDr Sandeep Raha
FUNDING
Bupropion?
Nicotine
Full agonist
nAChR
Bupropion(Zyban®)
Antagonist