Join Sharon Ladyman, PhD and Vicki Vieira-Potter, PhD as they present applications of rodent metabolic phenotyping with a focus on the effects of hormones and pregnancy on daily activity in mice. A reduction in voluntary physical activity during pregnancy in mice is mediated by prolactin Sharon Ladyman, PhD Pregnancy is an energetically demanding challenge for the mother and as such, pregnant females undergo numerous metabolic adaptations to meet these demands, including increased food intake and a rapid lowering of energy expenditure and physical activity levels. A particularly striking example is a profound reduction in voluntary running wheel activity (RWA) that occurs as soon as mice become pregnant. We hypothesized that prolactin, one of the first hormones to increase in response to mating in rodents, drives the pregnancy-induced suppression of physical activity levels. Neuronal and Metabolic Pathways Influenced by Sex Hormones Vicki Vieira-Potter, PhD Estrogen-sufficient females are more physically active than males and are protected against adipose tissue and systemic metabolic dysfunction. The mechanisms are not fully understood, but we demonstrate that ovarian removal causes significant mRNA changes in the nucleus accumbens (NAc) brain region (i.e., the reward center), which correlate significantly with physical inactivity. We hypothesize that sex differences in the NAc may help explain differences in physical activity and metabolism.

1. From Pregnancy to Menopause:
Studies of Physical Activity,
Behavior, and Energy Balance
in Mice
Victoria Vieira-Potter, PhD
Associate Professor
Department of Nutrition & Exercise Physiology
University of Missouri, Columbia
Sharon Ladyman, PhD
Senior Research Fellow,
Department of Anatomy
University of Otago

2. From Pregnancy to Menopause:
Studies of Physical Activity,
Behavior, and Energy Balance
in Mice
Experts present applications of rodent metabolic
phenotyping with a focus on the effects of
hormones and pregnancy on daily activity in
mice.

3. Sharon Ladyman, PhD
Senior Research Fellow,
University of Otago
A reduction in voluntary
physical activity during
pregnancy in mice is mediated
by prolactin
Copyright 2020 S. Ladyman and InsideScientific. All Rights Reserved.

4. Energy demands during pregnancy and lactation
127000kcal (530000 kJ)
pregnancy plus 6 months
of lactation
Growth and development of
fetus + placenta
Increase mass of maternal tissue
- mammary gland development
- increase blood volume
- increase fluid retention
- fat mass

5. Energy demands during pregnancy and lactation
127000kcal (530000 kJ)
pregnancy plus 6 months
of lactation
- Increased energy intake
- Changes in energy partitioning?
Decreased energy utilized for non-essential functions?

6. One way ANOVA, t-test, C57BL/6J female mice n=6
humans mice
Forsum and Lof 2007
Energy expenditure during pregnancy

7. Suppression of non-essential functions, ‘sparing energy for pregnancy’?
humans mice
Do physical activity
levels decrease
during pregnancy?
One way ANOVA,
t-test, C57BL/6J
female mice n=6Forsum and Lof 2007

8. Pregnancy-induced suppression of running wheel activity
nonpregnant
Day of pregnancy
Ladyman et al 2018
Physiology and behaviour
Repeated measures
ANOVA, t-test,
C57BL/6Jfemale mice n=6

9. Early hormonal changes in pregnancy: plasma “lactogen” levels
Hypothesis: early increases in prolactin drive reduction in running wheel
activity and physical activity during pregnancy
ParturitionProlactin surges Placental lactogen
dominance
early mid late
pregnancy

10. Acute prolactin treatment decreases running wheel activity
Two-way ANOVA, interaction time x treatment P<0.0001
*Sidak’s multiple comparison test P<0.05, paired t-test, C57BL/6Jfemale mice n=12

11. Acute prolactin treatment and
physical activity
T-test, repeated measures ANOVA with *Sidak’s multiple comparison test P<0.05,
paired t-test, C57BL/6Jfemale mice n=8-12 per group
• No effect on distance travelled on
elevated plus maze or open field test
• Minor reduction in home cage
ambulation and slight increase in fine
movements in home cage
• No major change in home cage
behaviours

12. Specific deletion of Prlr from forebrain neurons
Exon 4 Exon 5 Exon 6
lox66
lox71
GFP
Prlrlox/lox
Exon 4 Exon 5GFP
Prlrlox/lox + Cre recombinase
X
Prlrlox/lox
=
Prlrlox/lox/CamK-IIa-Cre
Forebrain neuron specific
deletion of prolactin receptors
CamK-IIa-Cre
Exposure to bacteriophage recombinase Cre results in Cre mediated inversion of the Prlr gene to
delete exons 6-10 and turn on expression of green fluorescent protein (GFP)

13. Central Prlr required for pregnancy-induced suppression of RWA
mating
RWA for each mouse is expressed as a percentage of their individual average daily levels in the virgin state and analysis by mixed effects model analysis. Forebrain neuron specific
deletion of Prlr: Two-way ANOVA with Sidak’s multiple comparison post hoc virgin vs early pregnancy: Prlrlox/lox: P=0.0002, Prlrlox/lox/CamK-Cre: P=0.3899

14. Central Prlr required for pregnancy-induced suppression of RWA
mating
RWA for each mouse is expressed as a percentage of their individual average daily levels in the virgin state and analysis by mixed effects model analysis. GABA neuron
specific deletion of Prlr: Two-way ANOVA with Sidak’s multiple comparison post hoc virgin vs early pregnancy: Prlrlox/lox: P=0.0007, Prlrlox/lox/vGat-Cre: P=0.7888

15. What are the Prlr responsive neuron populations mediating this effect?
Implicated in running wheel activity
Has prolactin receptors
?
Prolactin

16. Prolactin-induced pSTAT5 in kisspeptin neurons
Araujo-Lopes et al 2014 Endocrinology
Prlr and kisspeptin mRNA colocalization
Brown et al 2019
Endocrinology
Padilla et al 2019
Current Biology
Neurons involved in prolactin-induced suppression of RWA during pregnancy: arcuate nucleus kisspeptin neurons

17. Neurons involved in prolactin-induced suppression of RWA during pregnancy: arcuate nucleus kisspeptin neurons
mating
RWA activity for each mouse is expressed as a percentage of their individual average daily levels in the virgin state and analysis by mixed effects model analysis.
Kisspeptin neuron specific deletion of Prlr: Two-way ANOVA with Sidak’s multiple comparison post hoc early pregnancy: Prlrlox/lox vs Prlrlox/lox/kiss1-Cre: P=0.0.162

18. Neuron specific Prlr KO compared to kisspeptin specific Prlr KO
Kisspeptin specific Prlr KO
Neuron specific Prlr KO

19. Neurons involved in prolactin-induced suppression of RWA during pregnancy: medial preoptic
area of hypothalamus (MPOA)
MPOA deletion of Prlr leads
to pup abandonment
Increased RWA is associated
with pup abandonment
MPOA has prolactin-sensitive
projections to the VTA
Brown et al 2017 PNAS
Brown et al 2017 PNAS
Ladyman et al 2018
Physiology and behaviour

20. MPOA specific deletion of prolactin receptor
AAV-Cre AAV-cre injectioncontrol injection
GFP GFP
pSTAT5 pSTAT5
GFP: indicates where
the Prlr gene has
undergone
recombination
pSTAT5: intracellular
signaling molecule
phosphorylated in
response to prolactin
Prlrlox/lox

21. What are the Prlr responsive neuron populations mediating this effect?
mating
RWA activity for each mouse is expressed as a percentage of their individual average daily levels in the virgin state and analysis by mixed effects model analysis. MPOA
neuron specific deletion of Prlr: Two-way ANOVA with Sidak’s multiple comparison post hoc virgin vs early pregnancy: Control : P=0.01, AAV-Cre: P=0.9687
Interaction treatment x time
P<0.0001

22. Suppression of running wheel activity during pregnancy
Running wheel activity as a behavioural output
• Exercise, ‘suppression of non-essential functions’
• Reward
• Thermoregulation
Major rapid change in a biological system as soon as
mice become pregnant that requires prolactin
receptors in the brain
Running wheels in the wild
Meijer and Robbers 2014 Proc Biol Sci

23. Acknowledgements
Prof. Dave Grattan
Kirsten Carter
Zin Khant Aung
Pene Knowles
Dr. Rosie Brown
Dr. Teodora Georgescu
Abigail Fergus

24. Victoria Vieira-Potter, PhD
Associate Professor
Department of Nutrition & Exercise Physiology
University of Missouri, Columbia
Neuronal and Metabolic
Pathways Influenced by
Sex Hormones
Copyright 2020 V. Vieira-Potter and InsideScientific. All Rights Reserved.

25. Sex differences in obesity and metabolism
Is it our head or our hormones?

26. Obesity prevalence in higher among women across ages.
• NHANES data indicate
more women are obese.
• Despite this, young
women are protected
metabolically.
• Risk reverses following
menopause!
• What explains this?
(I.e., role of estrogen?)

27. ↑ Metabolic Syndrome
abdominal obesity
insulin resistance
inflammation
dyslipidemia
hypertension
↑ Type 2 Diabetes
↑ Cardiovascular Disease
Menopause
What protects women prior to menopause?
• We think it involves
estrogen’s effects on
adipose tissue and
brain.
• We use rodent models
to investigate these
questions.

28. 0
5
10
15
20
25
30
35
FEMALE MALE
Grams
Body Weight
*
0
10
20
30
FEMALE MALE
Grams
*
Lean Mass
0
5
10
15
FEMALE MALE
Grams
% Body Fat
0
0.5
1
FEMALE MALE
0
0.2
0.4
0.6
0.8
1
1.2
FEMALE MALE
ADIPO-IRHOMA-IR
*
*
12-16 wk-old male and female WT C57BL6 mice; n=10/grp; *P<0.05
Ovary-intact, young female rodents are more insulin sensitive than males.

29. PGAT depot, 12-16 wk-old male and female WT C57BL6 mice; n=10/grp; *P<0.05
What role might physical activity play in
these sex differences?
Increased UCP1
(i.e., beige fat)
0
1
2
3
4
5
6
7
leptin MCP1 TNFa UCP1
mRNAFoldDifference
MALE WT
FEMALE WT
* ** *
ND in
males
Females have lower adipose tissue inflammation and greater UCP1 expression
compared to males.

30. Females rodents are also more physically active!
0
100
200
300
400
500
Meters
light dark total
Cage Physical Activity
*
*
*
12-16 wk-old male and female WT C57BL6 mice; n=10/grp; *P<0.05
What is the role of ovarian hormones (e.g., estrogen)??
Ovarian hormone loss
Obesity/adipose
tissue dysfunction
Insulin resistance

31. Ovarian hormone loss induces obesity and physical inactivity.
“OVX”SHM
Week 17 Week 24
SHM
OVX
Are HCR rats protected?
HIGH FIT RATS
LOW FIT RATS
Vieira-Potter et al. and Greenberg: Adipose tissue
inflammation and reduced insulin sensitivity in OVX
mice occurs in the absence of increased adiposity,
Endocrinology 2012

32. 0
20
40
60
80
100
120
140
160
1 2 3 4 5 6 7 8 9 10 11
Runningdistance(km/wk)
Weeks
HCR SHM HCR OVX
LCR SHM LCR OVX
“OVX”SHM
Are HCR rats protected?
Vieira-Potter VJ et al. 2015 AJP Reg
HIGH FIT RATS
LOW FIT RATS
Ovarian hormone loss induces obesity and physical inactivity.

33. Postmenopausal obesity is due to reduced physical activity
MONET study (102 women followed for 5 years from pre-post menopause) found:
1. Total energy expenditure decreased over time in postmenopausal women
due mostly to lower physical activity
2. Time spent in moderate physical activity decreased and the time spent sedentary increased
Duval, K et al (2013). Effects of the menopausal
transition on energy expenditure: a MONET
Group Study, EJCN 67, 407-411.
Why does this happen???
Menopause-associated obesity is due to physical inactivity (not dietary changes)!

34. Dopamine (DA) signaling in the nucleus
accumbens (NAc) brain region drives reward-
based motivation.
This key brain region is known to
drive motivation for physical activity.
Youngmin Park, PhD
Effects of intrinsic aerobic capacity and ovariectomy on voluntary wheel running
and nucleus accumbens dopamine receptor gene expression

35. High running associates with greater dopamine receptor expression in the NAc
Rats bred to engage in high levels of
physical activity also have greater
net positive dopaminergic (DA)
signaling in NAc.
0
2
4
6
8
10
12
14
16
18
HCR LCR
km/week
Wheel Running Distance
0
0.2
0.4
0.6
0.8
1
1.2
HCR LCR
excitatoryDrd/InhibitoryDrdmRNA
Net Positive DA Gene Expression
(NAc Brain Region)
* *
Park et al. Physiol Behav 2016

36. Park et al. Physiol Behav 2016
OVX reduces dopamine receptor expression, correlating with reduced wheel
running

37. HYPOTHESIS:
⬇︎Reduced estrogen (E2) → ⬇ dopamine signaling in the NAc → ⬇motivated physical activity
➢ Physical activity and EE
➢ Obesity/adipose tissue assessment
➢ Brain (NAc) RNAseq analysis
Male (n=8) and
Female (n=11)
Aromatase
Knockout (ArKO)
Male (n=10) and
Female (n=9)
Wild Type (WT)

38. Dusti Shay, MS
ArKO WT
Changes in nucleus accumbens gene expression
accompany sex-specific suppression of spontaneous
physical activity in aromatase knockout mice
NAc RNAseq
analysis →
Pathway analyses
performed

39. • Aromatase deletion increases adiposity in
both sexes.
• Aromatase deletion also increases visceral
fat and insulin resistance (not shown here).
• What is responsible for the increase in
adiposity?
Shay et al. 2020
RESULTS:
Percent Body Fat
Changes in nucleus accumbens gene expression accompany sex-specific
suppression of spontaneous physical activity in aromatase knockout mice
What’s happening in the ?

40. 23 25
Pts
Gldn
Cryab
Nxpe4
DEGs in Males
(KO v. WT)
DEGs in Females
(KO v. WT)
6-Pyruvoyltetrahydropterin Synthase
(Pts):
Catalyzes the second and irreversible step
in the formation of tetrahydrobiopterin
(BH4), an essential cofactor in
catecholamine (i.e., dopamine,
norepinephrine) biosynthesis.
Dopamine synthesis
Inflammatory responses
Heat shock proteins
Nervous system development
Shay et al. 2020
RNA Sequencing and Pathway Analysis

41. Pts gene expression correlates strongly with the CLOCK gene, Per3, in NAc
across sexes!
R² = 0.6057
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5 2 2.5
Pts
Per3
r = 0.778
P = 0.001
N = 14
(Circadian regulation gene)
Pts, essential
cofactor in
dopamine
biosynthesis, was a
top DEG in ArKO vs.
WT in both sexes.
Are SLEEP patterns affected
by estrogen?

42. • Sleep assessment via custom designed
macros allows us to assess how brain-
specific changes associate with sleep, in
addition to other physiological measures.
• Estrogen loss adversely affected sleep
patterns, especially in female mice.
• This associated with circadian rhythm gene
changes in the NAc.
Shay et al. 2020

43. NAc Pts gene expression correlates negatively with abdominal obesity
r = -0.539
P = 0.047
N = 14
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
1.3
0 1 2 3
Pts
PGAT (g)
Connection?

44. Might increased brain Pts activity due to estrogen help explain sex differences
in adipose tissue UCP1?
Pts
NAc Pts gene expression correlates
negatively with abdominal obesity and
positively with white adipose tissue
(WAT) UCP1!
WAT UCP1/NAc Pts:
r = .626
P = 0.017, N = 14
UCP1

45. The Take-Away Points…
• Prior to menopause, females are metabolically protected; the mechanism may
involve estrogen’s positive effects on adipose tissue and brain.
• Females have FIT fat! (e.g., more relative BAT and UCP1
expression, less inflammation, greater insulin sensitivity)
• Prior to estrogen loss, females are also more physically active.
• Estrogen loss causes weight gain and reduces energy expenditure,
leading to insulin resistance.
• Sex differences in pathways associated with fat metabolism,
dopamine signaling, and circadian regulation in the Nac brain
region may help explain sex differences in behavior and metabolism.

46. Many thanks to the PhIT-FAT lab:
Becky Welly, MS (former MS student, now PhIT-FAT lab manager)
Young-Min Park, PhD (former PhD student, now assistant professor at Incheon National University)
Terese Zidon, PhD (former PhD student, now teaching professor at Central Methodist University)
Stephanie Clookey, MS (former MS student, in medical school at MU)
Dusti Shay (current doctoral student)
Laura Clart (former MS student)
Candace Rowles (former UG/IMSD fellow)
Julia Hatzigeorgiou (former UG/NEP research fellow)
Eric Queathem (current UG/McNair scholar)
Elyse Fraizer (current UG/IMSD fellow)
Acknowledgements
Vicki Vieira-Potter, Ph.D.
Email: vieirapotterv@missouri.edu
Associate Professor
Department of Nutrition & Exercise Physiology
University of Missouri, Columbia
Collaborators:
Cheryl Rosenfeld, PhD (Mizzou)
Jaume Padilla, PhD (Mizzou)
Steve Britton, PhD (Michigan)
Lauren Koch, PhD (Michigan)
Frank Booth, PhD (Mizzou)
Matt Will, PhD (Mizzou)
John Thyfault, PhD (KUMC)
Jill Kanaley, PhD (Mizzou)
R. Scott Rector, PhD (Mizzou)
Dennis Lubahn, PhD (Mizzou)
Kevin Fritsche, PhD (Mizzou)
Scott Givan, PhD (Mizzou)
Nathan Bivens, PhD (Mizzou)
Funding:
MU Research Council Grants to V Vieira-Potter
MU Core Facility Grant to D Shay
MU-CBIS Pilot Grant P50AT006273
Chris Hardin, PhD
NEP Department Chair

47. References
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48. Victoria Vieira-Potter, PhD
Associate Professor
Department of Nutrition & Exercise Physiology
University of Missouri, Columbia
Sharon Ladyman, PhD
Senior Research Fellow,
Department of Anatomy
University of Otago
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