Dr. Reilly Enos and Dr. Eran Levin discuss estrogen's metabolic impact and how isotopic labeling and 13C-labeled nutrients can be used for animal physiology and nutrition research. Reilly Enos, PhD – Harnessing the power of estrogen to regulate metabolic processes Dr. Reilly Enos’ research focuses on the role that sex steroids and their receptors play in regulating metabolic processes, particularly in the setting of obesity. In this webinar, Dr. Enos will discuss his research on tissue-specific fluctuations of sex steroids throughout the estrous cycle in mice, provide insights into the importance of the quantity of estrogen necessary to impact physiological processes, as well as an understanding of the central versus peripheral effects of estrogen action. Eran Levin, PhD – Unlocking Insights: Utilizing 13C Labeled Nutrients for Cutting-Edge Physiology and Nutrition Research Dr. Eran Levin will discuss the potential of using 13C-labeled nutrients in physiology and nutrition research in animal models. Specifically, he will share practical tips for designing and conducting experiments using isotopic labeling techniques and demonstrate how they can provide unprecedented insights into metabolic pathways, nutrient utilization, and behaviors in both vertebrate and invertebrate models including insects, reptiles, and mammals. Key Topics Include: - The role that estrogen plays in regulating metabolic and behavioral processes in males and females - The tissue-specific fluctuations of sex steroids throughout the estrous cycle - Insight into the importance of tissue-specificity in developing hormonal therapies - The importance of estrogen quantity in regulating physiological processes - Understand the diverse range of 13C labeled nutrients available - Specific applications of labeled amino acids in studies of protein metabolism, cellular signaling, and typical nutrient utilization - How to integrate 13C labeling techniques with respirometry for a comprehensive assessment of metabolic processes, energy expenditure, and substrate utilization in animal models - How to calculate metabolic rates in free-flying animals using 13C bicarbonate

2. EXPLORING ESTROGEN’S ROLE
IN METABOLISM
Reilly Enos, PhD
Assistant Professor
University of South Carolina, School of Medicine

3. WEBINAR OUTLINE
• Brief overview of sex steroids including androgens and
estrogens
• Clinical morbidities linked to estrogen deficiency
• Overview of some questions that elude scientists with respect to
estrogen and how we are attempting to shed light on
unanswered questions using animal models
• Translational relevance of basic science research

4. STEROIDS
Taken from PMID: 25846979
Aromatase

5. SITES OF STEROID SYNTHESIS
Predominant Sites
• Gonads
• Ovaries
• Testes
• Adrenal Glands
• Cortex
• Adipose Tissue
• Brain
• Skeletal Muscle
• Kidney
• Etc.
Secondary Sites
Taken from PMID: 25846979

6. SEX STEROIDS
ANDROGENS (Primary)
1. Testosterone
2. Dihydrotestosterone (DHT)
3. Androstenedione
4. Androstenediol
Dehydroepiandrosterone
Sulfate (DHEAS)
5. Dehydroepiandrosterone
(DHEA)
ESTROGENS
1. Estrone (E1) (primary
estrogen during post-
menopause in humans)
2. 17β-Estradiol (E2) (primary
estrogen during pre-
menopause)
3. Estriol (E3) – high during
pregnancy
4. Estetrol (E4) – derived from
fetus

7. ESTROGEN THROUGHOUT THE LIFESPAN
Taken from PMID: 19002143

8. CLINICAL MORBIDITIES ASSOCIATED WITH
ESTROGEN DEFICIENCY
Taken from PMIDs: 18332882 & 31997311

9. WHAT ARE SOME OF THE QUESTIONS THAT ELUDE
SCIENTISTS?
• How is E2 protective against obesity and associated metabolic
dysfunction – are the primary effects central or peripheral?
• What levels of E2 are necessary to elicit certain physiological
effects? Why is this important?
1. First, we must understand the natural cyclicity of E2
• What animal models can we use to answer these questions?

10. HOW DO WE STUDY ESTROGEN
DEFICIENCY IN A MOUSE MODEL?

11. OVARIECTOMY OR AROMATASE KNOCKOUT
• Benefits
• Similarities to humans with respect
to endocrine changes
1. Increases in FSH
2. Interrupted cycling
3. Dramatic reduction/non-detectable
levels of plasma estradiol
• Pre-menopausal oophorectomy is
performed in approximately 300,00 females/year
in USA
• Drawbacks
• No peri-menopause
• Short duration of sexual maturity
(depending on age at which OVX occurs)

12. THE IMPORTANCE OF AROMATASE
Taken from PMID: 25846979
Aromatase

13. HOW IS E2 PROTECTIVE AGAINST OBESITY AND
ASSOCIATED METABOLIC DYSFUNCTION – ARE
THE PRIMARY EFFECTS CENTRAL OR
PERIPHERAL?

14. CENTRAL VS. PERIPHERAL EFFECTS OF ESTROGEN
Central
• Impacts food intake
• Impacts physical activity
Peripheral
• Deletion or blockade of the
primary estrogen receptor
(ERα) has been shown to
impact metabolic processes
in various tissues
Mauvais-Jarvis et. al. (PMID 23460719)

15. TWO MODELS OF ESTROGEN
DEFICIENCY
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0
10
20
30
40
50
Female Body Weight Data
Weeks of Diet
Grams
F WT LFD
F AROM KO LFD
F WT HFD
F AROM KO HFD
OVX HFD
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0
5
10
15
20
25
Female Body Weight Δ
Weeks of Diet
Grams
F WT LFD
F AROM KO LFD
F WT HFD
F AROM KO HFD
OVX HFD
a
b
c
d
ME: Diet
ME: Genotype
✱
Pay Attention Here

16. E2 DEFICIENCY & ITS CENTRAL EFFECTS
Promethion Metabolic Cages

17. HOW DO YOU TEASE OUT CENTRAL
VS. PERIPHERAL EFFECTS OF E2?

21. • No benefit with respect to glucose metabolism
• But adipose tissue inflammation….
IMPACT ON GLUCOSE METABOLISM AND ADIPOSE
TISSUE INFLAMMATION

22. INTERVENTION STUDY

24. WHAT ABOUT ESTROGEN LEVELS?

25. #1 PROBLEM SEX STEROID RESEARCHERS FACE
•Reliable and Accurate Measurement of Sex
Steroids, Particularly Estrogens
Liquid Chromatography-Mass
Spectrometry for Sex Steroids
at Picogram Levels Using
Derivatization (1-Methylimidazole-2-
sulfonyl chloride) in Plasma & Tissue*
*See references PMIDs 25543003, 36039699, & 37421340
LOQ in 200 uL of charcoal stripped fetal bovine serum:
E2: 0.1 pg
Testosterone: 0.5 pg
Androstenedione: 0.5 pg
Progesterone: 1.0 pg
For further reading regarding mass spectrometry
and estrogens read the work of: David
Handelsman (University of Sydney), Claes
Ohlsson (University of Gothenburg), Kiran Soma
(University of British Columbia), and Ruth Andrew
(University of Edinburgh) to name a few…

26. Female Prevention
Female Intervention

27. FOLLOW-UP STUDY IN MALES
PMID: 37421340

28. METABOLIC CAGE DATA

29. CONCLUSIONS
• Different levels of E2 impact different physiological processes
A. Important for Hormone Replacement Therapy?

30. • WHAT LEVELS OF E2 ARE NECESSARY TO ELICIT
CERTAIN PHYSIOLOGICAL EFFECTS? WHY IS THIS
IMPORTANT?
First, we must understand the natural
cyclicity of E2

31. HUMANS VS RODENTS
Humans Mice

32. UNCERTAINTY IN THE FIELD?

33. THE STUDY (UNPUBLISHED)
Diestrus 1
Proestrus Estrus

34. ESTRADIOL (E2) DATA

36. CLINICAL RELEVANCE
(HORMONE REPLACEMENT THERAPY)
• Modes of Hormone Replacement in the Clinic
1. Nasal
2. Pills
3. Skin gel
4. Skin patches
5. Vaginal Creams
6. Vaginal Ring
• Combinational Therapy
1. Estrogen + Progesterone
• Potential Side Effects Due to “whole body” Effects
1. Rare increase of absolute breast cancer risk in some cases
2. Jury is out regarding heart disease – may depend on timing of when therapy is
initiated

37. CONCLUSIONS/THOUGHTS/FUTURE DIRECTIONS
• Brain is king
• Inducible overexpression of aromatase in other tissues
A. The desire would be to determine the role(s) that estrogen plays in
different tissues and the amount necessary to elicit beneficial effects
• Targeted estrogen therapies in the future
A. What are the needs of the patient?
B. Need to think about mode of delivery, type of estrogen, dose of
delivery, when to administer, combinational therapies

38. ACKNOWLEDGEMENTS
Current & Former Lab Team
Ahmed Aladhami, MS, PhD
Christian Unger, MS
Trey Hope, MS
Cassidy Socia (Undergrad)
Cam Rice (Undergrad)
Aarushi Bhasin (Undergrad)
Faculty Mentors/Collaborators
Angela Murphy (USC)
Kandy Velazquez (USC)
Fiona Hollis (USC)
Holly LaVoie (USC)
Bill Cotham (USC)
Deborah Clegg (TTUHSC)
Owen McGuinness (Vanderbilt)
Kendall Nettles (Scripps, Florida)
Priscilla Furth (Georgetown)
John McCarthy (Kentucky)
Funding
ASPIRE
K01AT010348
K01AT010348-03S1
P20GM103641-10S1
P20-GM109091-10S1
U01 – CA272977-01
INBRE Pilot Project
USC - Magellan

39. THANKS!
WE ARE HAPPY TO COLLABORATE!
Reilly Enos
Assistant Professor
enosr@email.sc.edu

40. Use of 13C-Labeled Nutrients for Advanced
Animal Physiology and Nutrition Research
Eran Levin, PhD, The Nutritional Ecology Lab, School of Zoology, Tel-Aviv University

41. The truth is out there…

42. Cavity Ring-Down Spectroscopy (CRDS)
• Portable, simple and (relatively) cheap CO2 analyzers
• 13C/12C ratio in exhaled CO2
• 13C/12C ratio in tissues
1.8
m
δ13C

43. 13C labeled nutrients
• ~ 10,000 13C enriched nutrients are commercially available
• Detection and tracking of a specific nutrient (macro and micro
nutrients)
~98.9% ~1.1% ~10-12%

44. Real time respirometry and δ13C detection
CO2 free air
CO2
Analyzer
O2
Analyzer
δ13C
Analyzer
RER = CO2 eliminated / O2 consumed
Exhaled air
Temperature controlled
metabolic chamber
(reference)
δ13C (‰)
VCO2
.

45. Real time respirometry and δ13C detection
CO2 free air
δ13C
Analyzer
Exhaled air
Temperature controlled
metabolic chamber δ13C (‰)
VCO2
.

46. 1-13C
Tissue
12CO2
12CO2
12CO2
13CO2
13CO2
12CO2
12CO2
13CO2
CRDS
Combustion chamber
δ13C
13C/12C ratio in tissues

47. Carbohydrates (simple and complex)
• Labeling of different positions on mlecule
• Tracking specific metabolic pathways

48. RER= VCO2/VO2
Prizinger et al. 1992
Voigt et al. 1999

50. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
200 300 400 500 600 700 800 900
RER
Time (sec)
RER for a fed M. sexta is around 1.7

51. RER for a fed monarch butterfly
(Danaus plexippus) is also high
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
5500 5550 5600 5650 5700 5750 5800 5850 5900 5950 6000
RER
Time (s)

52. RER for a fed bee is usually 1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 50 100 150 200 250 300 350 400 450 500
RER
Time (sec)

53. Where is the “extra” CO2 is coming from?
PPP- Pentose Phosphate Pathway

54. Feeding labeled sugar to a hawk moth
RER δ13C

55. RER= VCO2/VO2
Levin et al. 2017 Science
Manduca sexta
● ●
RER

56. RER= VCO2/VO2
Levin et al. 2017 Science
Manduca sexta
● ●
RER
RER

57. Complex sugars

58. 13C–chitin synthesis
13C-U glucose
Chitin extraction
13C labeled chitin (complex sugar)
Bouchebti et al. 2023 Entomologia Generalis
Zophobas morio
CRDS

59. Only larvae can digest chitin!
CO2 CO2
(non labeled chitin) (non labeled chitin)
Bouchebti et al. 2023 Entomologia Generalis

60. Ethanol
13
12

62. Rate of ethanol metabolism by honey bee and Oriental hornet
Bouchebti et al. in prep

63. Fatty acids

64. Absorbance, oxidation and allocation of 1-13C Labeled fatty
acids in diet
13
13
13
MUFA
PUFA
SFA

65. Dubiner et al. JEB 2023

66. CO2
Fat body Respiration
Seltzer et al. Journal of Insect Physiology 2023

67. Modification of fatty acids in the tissue
Domer et al. in prep

68. Amino acids and proteins
GABA
β-Alanine
Taurine

71. Bodner et al., 2021 Insect Biochemistry and Molecular Biology

72. Dietary proline is oxidized as metabolic fuel
Bodner et al., 2021 Insect Biochemistry and Molecular Biology

73. 1-13C-Leu
Labeled protein
+
10 days
1-13C-Leu
muscle protein
Can adult hornets digest protein?
Bodner et al., 2022 Biology

74. a
a
a
ab
a
b
b
b
c
Two-way ANOVA, p < 0.0001
Queens
(n=30)
Males
(n=34)
Workers
(n=35)
‫בה‬
‫בה‬
Caste dependent protein digestion
δ
13
C
(‰)
Bodner et al., 2022 Biology
13C
12C

75. Digestive efficiency/dependence
a
a
a
ab
a
b
b
b
c
Two-way ANOVA, p < 0.0001
Queens
(n=30)
Males
(n=34)
Workers
(n=35)
δ
13
C
(‰)
Bodner et al., 2022 Biology
13C
12C 13C+larvae

76. Free flight metabolic rate
Green Rose Chafer (Cetonia aurata)
Mango stem borer (Batocera rufomaculata)

77. Free flight metabolic rate
Sodium bicarbonate (13C)
13
Urca et al. 2021. Proceedings of the Royal Society B

78. Free flight metabolic rate
Urca et al. 2021. Proceedings of the Royal Society B

79. Endless applications for the use of 13C
labeled macronutrients!
• Whole organism
• Specific tissues
• In the field
• In the lab
• Physiology
• Metabolism
• Behavior
• Nutrient flow

80. THANK YOU!
The Nutritional
Ecology lab
Levona Bodner
Dr. Sofia Bouchebti
Dr. Tali Magory
Ariel Drabkin
Shaher Dubiner
Dr. Rya Zeltzer
Dr. Adi Domer
Nitzan Cohen
Mika Volov
Roy Ben Bezalel
Dr. Reut Vardi