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