Frederick vom Saal Division of Biological Sciences

Frederick vom Saal Division of Biological Sciences

DEVELOPMENTAL FACTORS IN ADULT
OBESITY: DIET, ENVIRONMENTAL CHEMICALS
AND IMPORTANCE OF ANIMAL MODELS
Frederick vom Saal
Division of Biological Sciences
University of Missouri-Columbia, USA
Bisphenol A
Funded by research grants from the
National Institutes of Health (NIH)
IUGR
MACROSOMIA
(Premature) (Maternal Type II diabetes)
(Reduced placental blood flow)
Proposed factors influencing induction of IUGR
λλ
Babies that are overweight at birth are at risk for adult
obesity and metabolic disease.
Some babies with intrauterine growth restriction (IUGR)
are at risk for a rapid period of postnatal weight gain,
adult obesity and metabolic disease.
THRIFTY PHENOTYPE HYPOTHESIS
Body weight
λλ
Macrosomia
IUGR
Normal
IUGR
Birth
EXPERIMENTAL APPROACHES FOR
ANIMAL RESEARCH ON THE FETAL
BASIS OF METABOLIC SYNDROME
Treatment of Pregnant Females
™ Caloric restriction
™ Protein restriction
™ High fat diet
™ Exercise
™ Dexamethasone administration
™ Knockout mice (e.g. 11ß-HSD)
™ Environmental chemicals - PLASTIC
Even et al. 1994
J. Reprod. Fertil.
Uterine Placental
Position Blood RADIOLABELED MICROSPHERES
Flow
O1
O2
O3
M-2
M-1
M0
M1
M2
C3
C2
C1
31.5
33.1
20.6
20.8
9.3
18.8
27.3
29.7
31.3
43.5
36.6
Kirkpatrick et al
Unpublished
Grams
MALE MOUSE BODY WEIGHT AT BIRTH
2.5
2
1.5
1
0.5
0
<5
1
52550752
3
4
5 >95%
6
25
50
75
95
Birth Weight Percentile
Percent
PERCENT INCREASE IN BODY WEIGHT
DURING THE 7 DAYS AFTER WEANING
IN MALE MICE
100
80
60
40
20
0
<5
1
52550752
3
4
5 >95%
6
25
50 75 95
Birth Weight Percentile
N = 294 pups
Percent
PERCENT INCREASE IN BODY WEIGHT
DURING THE 7 DAYS AFTER WEANING
IN FEMALE MICE
100
80
60
40
20
0
<5
550751
2 253
4
5 >95%
6
25 50 75
95
Birth Weight Percentile
N = 287 pups
Glucose clearance by Birth Weight Category
GLUCOSE TOLERANCE IN MALE MICE
Glucose Concentration (mg/dL)
290
265
240
215
190
165
140
115
90
0
20
IUGR
40
Macrosomia
60
80
100
Time (min)
Coe et al. unpublished
COULTER COUNTER ANALYSIS OF FAT CELL DIAMETER IN MALE MICE
Male-6
Male- 14
450
450
400
400
High
Low
350
16000
14000
250
250
12000
200
Total count
300
Count
300
200
10000
8000
Adipoc yte s ize , um
6000
4000
163
149
135
121
107
93.6
79.7
65.9
52.1
38.3
24.5
10.6
163
149
135
0
121
0
107
2000
0
93.6
50
79.7
50
65.9
100
H
High
Adipoc yte s ize , um
L
Low
1 2 0
Adipocyte diameter, 8 m
52.1
100
38.3
150
24.5
150
10.6
Count
350
Total number of cells
1 0 0
a
*
Low BW at Brith
8 0
a
*
6 0
4 0
*a
High BW at Brith
2 0
H ig h
L o w
0
10%
25%
50%
Percentage group
*a: indicates difference between High and Low birth wt groups (P<0.05)
75%
90%
Coe et al.
unpublished
ADULT EPIDIDYMIDAL ADIPOCYTE GENE EXPRESSION:
Genes affected in IUGR compared to Macrosomic animals
COMPARISON
OF MALES CATEGORIZED WITH IUGR
AND MACROSOMIA AT BIRTH
180
160
Number of genes affected
140
120
100
p<0.05
80
60
40
20
0
Up-Regulated
Taylor et al. unpublished
Down-Regulated
Fold difference in gene expression
ADULT EPIDIDYMIDAL
GENE EXPRESSION:
AdiposeADIPOCYTE
tissue gene expression
COMPARISON OF MALES CATEGORIZED WITH IUGR AND
2.5
MACROSOMIA AT BIRTH
∗∗
2
Glucose
Metabolism
Macro
*
IUGR
Ubiquitinization
1.5
1
Cell
Cycle
Lipid
Metabolism
∗∗
*
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗
0.5
Taylor et al. unpublished
Fads3
Vldlr
Gsk3b
Slc2a10
Slc12a7
Insl6
Hyal1
Cbl
Ccne2
Ccnd2
0
IUGR and Macrosomia Result in
Differences in Adult Obesity
Hundreds of Genes in Epididymidal Adipocytes
Differ in Activity
Affymetrix Mouse Genome 430 Array
High risk - Heavy
Optimum
High risk - Light
Optimum
High risk - Heavy
EXPERIMENTAL DESIGN
Randomly assign adult CD-1 mice
to be fed different mouse feeds
1. Purina 5008: Pregnancy and lactation chow
Purina 5001: Post-weaning chow
- soy based rodent feeds
- high levels of phytoestrogens:
genistein and daidzein
2. Purina 5K96C
- casein based feed
- undetectable phytoestrogens (MCF-7 cells)
Examine offspring at different ages
SERUM ESTRADIOL
Casein-based 5K96C
Low Phytoestrogens
Stimulates endogenous
Estradiol
100
Serum Estradiol (pg/ml)
90
80
70
60
50
40
30
20
10
Ruhlen et al.
Submitted
0
5008 5K96C
Maternal
MOTHER
5008 5K96C
Female pup
FEMALE FETUS
5008 5K96C
Male pup
MALE FETUS
EFFECT OF FEED ON BODY WEIGHT IN FEMALE MICE
45
PND 90
41
37
33
29
Body Weight (g)
19
1
PND 26
2
17
15
13
13
1
PND 20
2
1
PND 1
2
12
11
Ruhlen et al.
Submitted
10
1.8
1.7
1.6
1.5
1.4
1.3
1.2
5008/1
1
5K96C
2
GONADAL FAT PAD RENAL FAT PAD
3000
FEMALE
MALE
FEMALE
MALE
Weight (mg)
2500
2000
1500
1000
500
0
1
2
3
4
5008 5K96 5008 5K96
5001
5001
Ruhlen et al. Submitted
5
6
7
8
9
5008 5K96 5008 5K96
5001
5001
PURINA FEEDS
ng/ml
SERUM LEPTIN IN ADULT MICE
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
FEMALE
1
5008
5001
Ruhlen et al.
Submitted
2
5K96C
MALE
3
4
5008
5001
PURINA FEEDS
5
5K96C
PPM Genistein Equivalents
ESTROGENIC ACTIVITY IN DIFFERENT LOTS OF
CASEIN-BASED PURINA 5K96C FEEDS
MCF-7 CELL BIOASSAY
60
CV = 84%
50
Purina 5008
40
HIGH
30
20
LOW
10
0
1
2
Welshons and Taylor
unpublished
3
4
5
6
7
Purina 5K96C Lots
8
9
2-MONTH OLD CD-1 FEMALE MICE
MALE FAT PADS
3 MONTH OLD
SERUM LEPTIN
Serum Leptin (ng/ml)
Weight (mg)
2000
GONADAL FAT
1800
1600
1400
1200
1000
800
600
400
16
14
12
5K96C
5001
10
8
6
4
2
200
0
Gonadal
NC
Cont Fat 5008/1
5K96C
0
Adrenal Fat
NC
Cont
Females
5K96C
5008/1
BISPHENOL A LEACHES FROM NEW
POLYCARBONATE BABY BOTTLES
Taylor, vom Saal, Welshons and Rottinghaus (unpublished)
NATURAL AND MANMADE ESTROGENS
POLYCARBONATE PLASTIC & RESIN
Bisphenol A
NATURAL HORMONE
Estradiol
ESTROGENIC DRUG
Diethylstilbestrol (DES)
HO
OH
INTRAUTERINE POSITION INFLUENCES SERUM ESTRADIOL
AND TESTOSTERONE IN MALE MOUSE FETUSES
2M
E2 = 78 pg/ml
T = 5.7 ng/ml
1M
E2 = 94 pg/ml
T = 4.6 ng/ml
2F
E2 = 101 pg/ml
T = 4.2 ng/ml
F. vom Saal
J. Anim. Sci.
67:1824-1840, 1989
WEAN WEIGHT (G)
PRENATAL BISPHENOL A EXPOSURE INCREASES BODY WEIGHT
AT WEANING IN MICE WITH ELEVATED FETAL ESTROGEN LEVELS
A
13
12 P<0.001
11
10
9
8
7
6
C
P<0.001
P<0.001
WEAN WEIGHT (G)
5
13
12 P<0.001
11
10
9
8
7
6
5
C BPA
All Pups
Maternal BPA Oral Dose:
2 µg/kg/day, GD 11-17
P<0.01
P<0.01
C BPA
2F
C BPA
1MF
C BPA
2M
High Medium Low
Fetal Estradiol
Howdeshell et al.
Nature, 1999
Bisphenol A
activates cAMP Responsive Element Binding
Protein (CREB) at Human Exposure Levels (95%
of Americans)
Ca
New
Receptor
K
p- CREB
J.P. Myers
Calafat et al. 2005; Zhang et al. 2003; Quesada et al. 2003
BPA
Gene in differentiating fat cell
Promoter
C/EBP
β
mRNA
production
site
DNA sequence
aP2 (FABP)
C/EBPα
SCD1
P-CREB
PPARγ
GLUT4 (glucose uptake)
Ob (leptin) gene
Weight (Grams)
EMBRYONIC EXPOSURE TO BISPHENOL A INCREASES
POSTNATAL GROWTH RATE IN MICE
16
14
12
10
8
6
Wean Weight
2
1.8
1.6
1.4
1.2
1
1 Birth Weight 2
~4 pups / litter
CONTROL
1
Takai et al.. 2000
Biochem. Biophys. Res. Comm.
BISPHENOL
A
2
1 nM (228 pg/ml)
BISPHENOL A STIMULATES INSULIN
SECRETION IN PANCREATIC ß CELLS IN
IN MICE
Plasma Insulin (ng/ml)
0.3
0.2
0.1
0
Alonso-Magdalena et al.
Env.Health Pers. 2006
Ctl
E2
BPA
10 µg/kg
All Manmade Chemicals and Bisphenol A:
Production Parallels the Global Obesity Epidemic
6543210-
Modified from:
Baillie-Hamilton, 2002
Bisphenol A (BPA) Production
Chemical Production
♦
% Overweight Adults
Total
Chemicals
BPA
♦
Overweight
& Obesity
♦
Years
Percent Overweight Adults
7-
Synthetic Chemical Production
(billion kg/annum)
Bisphenol A Production
(Billions of pounds)
♦
BISPHENOL A
(as of December 2007)
EVIDENCE FOR BIAS DUE TO SOURCE OF
FUNDING IN RELATION TO REPORTED
OUTCOME IN RESEARCH WITH ANIMALS
SOURCE OF
FUNDING
Government
Chemical Corporations
STUDY OUTCOME
HARM
NO HARM
153 (92%) 14 (8%)
0 (0%)
153
vom Saal and Hughes, 2005
Environ. Health Perspect.
13 (100%)
23
167
13
180
Also > 180 studies of
moleculear mechanisms
BIASED OUTCOME DUE TO SOURCE OF FUNDING
IN LOW-DOSE BISPHENOL A RESEARCH
(as of December 2005)
STUDIES THAT USED THE ESTROGEN-INSENSITIVE
CHARLES RIVER SPRAGUE-DAWLEY (CD-SD) RAT
SOURCE
OF FUNDING
REPORTED STUDY OUTCOME
HARM
NO HARM TOTAL
Government
0 (0%)
7 (100%)
7
Chemical Corporations
0 (0%)
3 (100%)
3
0 (0%)
10 (100%)
10
vom Saal and Hughes, 2005
Environ. Health Perspect.
DOCUMENT PROVIDED TO THE CALIFORNIA ASSEMBLY
ŅReported low-dose effects have not
been replicated in repeat studies
conducted in independent laboratories.
The vast majority of available data
shows no low dose effect whatsoever.Ó
Bisphenol A Safety Overview
American Chemistry Council
Document for California Assembly
December 2005
Environmental Science and Technology
February 22, 2006
THE WEINBERG PROPOSAL
Paul D. Thacker
A scientific consulting firm says that it aids
companies in trouble, but critics say that it
manufactures uncertainty and undermines science.
“We will harness, focus and involve the
scientific and intellectual capital of our
company with one goal in mind —
creating the outcome our client desires.”
Letter from the FDA to the California
Assembly, April, 2005
The FDA sees no reason to ban bisphenol A.
The FDA approved bisphenol A for use in food
and beverage containers in 1963. The standard
was that bisphenol A was Generally Regarded
As Safe (GRAS), since it had been in use for a
few years and there was no evidence of harm.
George Pauli, Ph.D.
Associate Director for Science and Policy
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
PUBLISHED STUDIES SINCE 1997 REPORTING
LOW-DOSES EFFECTS OF BISPHENOL A IN
MICE AND RATS DURING DEVELOPMENT
♦ 17=Altered brain structure & chemistry
21=Altered behavior
♦ 5=Increased prostate size and cancer
9=Lowered sperm production
♦ 6=Altered adult hormone levels
♦ 7=Abnormal mammary glands
3=Early puberty in females
♦ 7=Increased subsequent body weight
♦ 2=Altered immune system
SHOULD BISPHENOL A (BPA )
BE BANNED IN BABY PRODUCTS AND FOOD
AND BEVERAGE CONTAINERS?
Hundreds of published scientific studies show:
1) High exposure is occurring: fetuses - adults.
2) Developing fetuses and babies are more
sensitive to BPA than adults.
3) BPA is an endocrine disruptor that causes
a wide range of abnormalities in animals.
4) Alternative plastics that are much more
stable and safer than BPA are available.
STUDENTS AND COLLABORATORS
STUDENTS
λ Brittany Angle
λ Benjamin Coe
λ James Kirkpatrick
λ Maren Bell
λ Davide Ponzi
COLLEAGUES
λ Wade Welshon
λ Barry Timms
λ Paola Palanza
λ Stefano Parmigiani
λ Huidong Shi
λ Steve Yang
RESEARCH ASSOCIATES
λ Jiude Mao
λ Julia Taylor