MATERNAL AND FETAL HEPATIC ENDOCANNABINOID SYSTEM … · Overview of the development of NAFLD in...
1
RESEARCH POSTER PRESENTATION DESIGN © 2011 www.PosterPresentations.com MATERNAL AND FETAL HEPATIC ENDOCANNABINOID SYSTEM (ECS) AND MATERNAL FAT CONSUMPTION. Introduction 1Dept. of Obstetrics and Gynecology, Texas Tech University Health Sciences Center at the Permian Basin, Odessa, TX, USA , 2Texas Pregnancy and life Course Health Center, Texas Biomedical Research Institute San Antonio Texas, San Antonio, TX, USA 3Department of Animal Science, University of Wyoming, Laramie, WY, USA, 4Department of Pharmacodynamics and Molecular Pharmacology, Collegium Medicum Nicolaus Copernicus University of Poland, Torun, Poland, Poland 5Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, USA, 6Department Pathology, University of Tennessee, Memphis, TN, USA 7Texas Biomedical Research Institute, San Antonio, TX, USA, 8Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA The authors would like to thank the personnel of the Texas Biomedical Institute and Center for Pregnancy and Newborn Research (UTHSC—San Antonio) for their help. This study was partially supported by Texas Biomedical Research Institute Grant C06 RR013556 and NIH grant HD21350 to Dr. Peter Nathanielsz (UTHSC—San Antonio), NIH NCRR grant P51 RR013986 to the Southwest National Primate Research. This work was supported by the TTUHSC start- up funds to N.S-L. The endocannabinoid system (ECS) is comprised of endocannabinoids (ECB’s), the product of dietary fatty acids, and G-protein coupled cannabinoid receptors 1 and 2 (CBR1 and CBR2). The endogenous ECB ligands, for the cannabinoid receptors are arachidonoyl ethanolamide (anandamide or AEA) and 2-arachidonoylglycerol (2-AG). The CB1 receptor is expressed in brain and peripheral tissues, while the CB2 receptor is expressed in microglia, immune and hematopoietic tissues. 1. M. Li, et al. Developmental programming of nonalcoholic fatty liver disease: the effect of early life nutrition on susceptibility and disease severity in later life (2015), BioMed Research International, vol. 2015, Article ID 437107. 2. Mechoulam, R., Hanuš, L.O., Pertwee, R., Howlett, A.C. Early phytocannabinoid chemistry to endocannabinoids and beyond (2014) Nature Reviews Neuroscience, 15 (11), pp. 757-764. 3. Rodriguez-Sanchez, I.P., et al., The endocannabinoid system in the baboon (Papio spp.) as a complex framework for developmental pharmacology, Neurotoxicol Teratol (2016), http://dx.doi.org/10.1016/j.ntt.2016.06.006. Objective Results Discussion and Conclusion References Acknowledgement Materials and Methods Figure 1. Structure of endogenous ECBs 2-AG and AEA) (Mechoulam, 2014). Baboons (Papio spp) were fed a diet of 45% fat, called high fat diet (HFD) while controls (CTR) ate a 12% fat diet from at least 9 months prior to conception through pregnancy until 0.9 gestation. Six HF and eight CTR, including archived baboon samples, livers of male and female fetuses, as well as the maternal liver, were evaluated using immunostaining. Commercially available CB1R (CB1 monoclonal primary antibody, Immunogenes; Budakeszi, Hungary. Cat# Img-CB1r-mab001) and CB2R (CB2 mouse monoclonal primary antibody, Novus Biologicals; Littleton, Co, USA. Cat# H00001269-M01) antibodies were applied for immunohistochemistry, and the secondary antibody was included in the Vectastain ABC kit (Vector laboratories; Burlingame, CA. Cat# PK 4002) The slides were scanned using the NanoZoomer SQ (Hamamatsu; Middlesex, NJ), quantification was performed using ImageScope™ v11.1.2.752 by Aperio (Leica Biosystems; Buffalo Grove, IL). Western blot was also performed with the same primary antibodies and β- Actin antibody (Monoclonal Anti β-Actin peroxidase antibody clone AC-15. St. Louis, MO, USA. Cat# A3854). The dilution used for the CB1 primary antibody was 1:1000 in 5% BSA (3 hour incubation at 40C), for the CB2 primary antibody 1:2000 in 5% BSA (3 hour incubation at 40C), and β-Actin primary antibody 1:20,000 in 5% BSA. Previous data showed a direct role of ECB’s in alcoholic and obesity-related non-alcoholic fatty liver disease (NAFLD) (Fig.2). Fatty liver disease is the significant public health issue, being the major cause of liver transplantation in 21 st century. Especially alarming is the fact, that 74% of obese individuals eventually develop NAFLD and NASH. The disturbing fact is that fatty liver is programmed in utero, contributing to epidemic of childhood metabolic disorders, however, the interventional strategies and prevention are limited. Despite the fact, that ECBs has been a pharmacological target for obesity and NAFLD treatment, no data exists regarding ECBs pharmacology in pregnancy and obesity. Figure 4. A. Western blot. B and C. Predicted CNR1-ORF and CNR2-ORF from the baboon (Papio spp.) (Dr. J. Guindon, in Rodriquez-Sanchez et al 2016) with the specific epitopes, recognized by antibodies. Figure 2. Endocannabinoid pathway in the pathogenesis of NAFLD (FA =fatty acid). 0 5 10 15 20 25 30 35 40 45 50 CTR HFD Histoscore Fetal hepatic CB1R protein expression in female fetuses of mothers fed HF (n=5) and CTR (n=4) diet. 0 10 20 30 40 50 60 70 80 CTR HFD Histoscore Fetal hepatic CB2R protein expression in female fetuses of mothers fed HF (n=5) and CTR (n=4) diet. 0 10 20 30 40 50 60 70 CTR HFD Histoscore Fetal hepatic CB1R protein expression in male fetuses of mothers fed HF (n=7) and CTR (n=6) diet * p<0.05 0 10 20 30 40 50 60 70 CTR HFD Histoscore Fetal hepatic CB2R protein expression in male fetuses of mothers fed HF (n=6) and CTR (n=9) diet 0 10 20 30 40 50 60 70 80 CTR HFD Histoscore Maternal hepatic CB1R protein expression in mothers, carrying female fetuses, fed HF (n=5) and CTR (n=5) diet. 0 10 20 30 40 50 60 70 80 90 CTR HFD Histoscore Maternal hepatic CB2R protein expression in mothers, carrying female fetuses, fed HF (n=5) and CTR (n=5) diet. 0 10 20 30 40 50 60 70 80 90 CTR HFD Histoscore Maternal hepatic CB1R protein expression in mothers, carrying male fetuses, fed HF (n=5) and CTR (n=8) diet. 0 10 20 30 40 50 60 CTR HFD Histoscore Maternal hepatic CB2R protein expression in mothers, carrying male fetuses, fed HF (n=5) and CTR (n=7) diet. Results CB1 CB2 A C B Maternal high fat diet programs offspring metabolism and intake contributes to the development of Nonalcoholic steatohepatitis (NASH) in adult offspring via the mechanism of up-regulated hepatic lipogenesis. From the other hand the activation of CB1R leads to lipogenesis and reduced fatty acid oxidation in hepatocytes. In general, increased expression of CB1R in liver of experimental, diet-induced or genetically modified model of NAFLD is a well described phenomenon in non-pregnant animals. Our funding regarding increased CB1R expression in the male offspring is in line with the results obtained in experimental models of maternal high fat diet, showing increased adiposity and hepatic steatosis in male, but in the female offspring. Our data suggests that lipogenesis, but not inflammatory responses, might be the major pathways of in utero programming of NAFLD by maternal consumption of high fat diet. Figure 5 . Overview of the development of NAFLD in the context of developmental programming. (Li, 2015) Figure 3. Blue arrows indicate bile duct, red arrows-artery, yellow arrows-portal vein and green arrows-lipid droplet. To evaluate the expression of CB1 and CB2 receptors in maternal and fetal liver in baboon (Papio spp) model of high fat diet. Our data is highly relevant in development of prevention strategies to counteract the gender- dependent effect of maternal obesity and prevent devastating effect of maternal obesity on the offspring health. Marcel Chuecos 1 , Cun Li 2,3 , Maira Carrillo 1 , Kushal Gandhi 1 , Cezary Skobowiat 4,5 , Gary Ventolini 1 , Patrick Joseph 6 , Edward Dick 7 , Gene Hubbard 8 , Peter Nathanielsz 2,3 , Natalia E. Schlabritz-Loutsevitch 1* ([email protected])
Transcript of MATERNAL AND FETAL HEPATIC ENDOCANNABINOID SYSTEM … · Overview of the development of NAFLD in...
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MATERNAL AND FETAL HEPATIC ENDOCANNABINOID SYSTEM (ECS) AND
MATERNAL FAT CONSUMPTION.
Introduction
1Dept. of Obstetrics and Gynecology, Texas Tech University Health Sciences Center at the Permian Basin, Odessa, TX, USA , 2Texas Pregnancy and life Course Health Center, Texas Biomedical Research Institute San Antonio Texas, San Antonio, TX, USA
3Department of Animal Science, University of Wyoming, Laramie, WY, USA, 4Department of Pharmacodynamics and Molecular Pharmacology, Collegium Medicum Nicolaus Copernicus University of Poland, Torun, Poland, Poland
5Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, USA, 6Department Pathology, University of Tennessee, Memphis, TN, USA
7Texas Biomedical Research Institute, San Antonio, TX, USA, 8Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
The authors would like to thank the personnel of the Texas Biomedical Institute and Center
for Pregnancy and Newborn Research (UTHSC—San Antonio) for their help. This study was
partially supported by Texas Biomedical Research Institute Grant C06 RR013556 and NIH
grant HD21350 to Dr. Peter Nathanielsz (UTHSC—San Antonio), NIH NCRR grant P51 RR013986
to the Southwest National Primate Research. This work was supported by the TTUHSC start-
up funds to N.S-L.
The endocannabinoid system (ECS) is comprised of endocannabinoids (ECB’s), the product
of dietary fatty acids, and G-protein coupled cannabinoid receptors 1 and 2 (CBR1 and
CBR2). The endogenous ECB ligands, for the cannabinoid receptors are arachidonoyl
ethanolamide (anandamide or AEA) and 2-arachidonoylglycerol (2-AG). The CB1 receptor is
expressed in brain and peripheral tissues, while the CB2 receptor is expressed in microglia,
immune and hematopoietic tissues.
1. M. Li, et al. Developmental programming of nonalcoholic fatty liver disease: the effect of early life nutrition on
susceptibility and disease severity in later life (2015), BioMed Research International, vol. 2015, Article ID 437107.
2. Mechoulam, R., Hanuš, L.O., Pertwee, R., Howlett, A.C. Early phytocannabinoid chemistry to endocannabinoids and
beyond (2014) Nature Reviews Neuroscience, 15 (11), pp. 757-764.
3. Rodriguez-Sanchez, I.P., et al., The endocannabinoid system in the baboon (Papio spp.) as a complex framework for
developmental pharmacology, Neurotoxicol Teratol (2016), http://dx.doi.org/10.1016/j.ntt.2016.06.006.
Objective
Results
Discussion and Conclusion
References
Acknowledgement
Materials and Methods
Figure 1. Structure of endogenous ECBs 2-AG and AEA) (Mechoulam, 2014).
Baboons (Papio spp) were fed a diet of 45% fat, called high fat diet (HFD) while controls (CTR) ate a 12% fat
diet from at least 9 months prior to conception through pregnancy until 0.9 gestation. Six HF and eight CTR,
including archived baboon samples, livers of male and female fetuses, as well as the maternal liver, were
evaluated using immunostaining. Commercially available CB1R (CB1 monoclonal primary antibody,
Immunogenes; Budakeszi, Hungary. Cat# Img-CB1r-mab001) and CB2R (CB2 mouse monoclonal primary
antibody, Novus Biologicals; Littleton, Co, USA. Cat# H00001269-M01) antibodies were applied for
immunohistochemistry, and the secondary antibody was included in the Vectastain ABC kit (Vector
laboratories; Burlingame, CA. Cat# PK 4002) The slides were scanned using the NanoZoomer SQ
(Hamamatsu; Middlesex, NJ), quantification was performed using ImageScope™ v11.1.2.752 by Aperio (Leica
Biosystems; Buffalo Grove, IL). Western blot was also performed with the same primary antibodies and β-
Actin antibody (Monoclonal Anti β-Actin peroxidase antibody clone AC-15. St. Louis, MO, USA. Cat# A3854).
The dilution used for the CB1 primary antibody was 1:1000 in 5% BSA (3 hour incubation at 40C), for the CB2
primary antibody 1:2000 in 5% BSA (3 hour incubation at 40C), and β-Actin primary antibody 1:20,000 in 5%
BSA.
Previous data showed a direct role of ECB’s in alcoholic and obesity-related non-alcoholic
fatty liver disease (NAFLD) (Fig.2). Fatty liver disease is the significant public health
issue, being the major cause of liver transplantation in 21st century. Especially alarming is
the fact, that 74% of obese individuals eventually develop NAFLD and NASH. The
disturbing fact is that fatty liver is programmed in utero, contributing to epidemic of
childhood metabolic disorders, however, the interventional strategies and prevention are
limited. Despite the fact, that ECBs has been a pharmacological target for obesity and
NAFLD treatment, no data exists regarding ECBs pharmacology in pregnancy and obesity.
Figure 4. A. Western blot. B and C. Predicted CNR1-ORF and CNR2-ORF from the baboon
(Papio spp.) (Dr. J. Guindon, in Rodriquez-Sanchez et al 2016) with the specific epitopes,
recognized by antibodies.
Figure 2. Endocannabinoid pathway in the pathogenesis of NAFLD (FA =fatty acid).
0
5
10
15
20
25
30
35
40
45
50
CTR HFD
His
tosc
ore
Fetal hepatic CB1R protein expression in
female fetuses of mothers fed HF (n=5) and
CTR (n=4) diet.
0
10
20
30
40
50
60
70
80
CTR HFD
His
tosc
ore
Fetal hepatic CB2R protein expression in
female fetuses of mothers fed HF (n=5) and
CTR (n=4) diet.
0
10
20
30
40
50
60
70
CTR HFD
His
tosc
ore
Fetal hepatic CB1R protein expression in male
fetuses of mothers fed HF (n=7) and CTR (n=6)
diet
* p<0.05
0
10
20
30
40
50
60
70
CTR HFD
His
tosc
ore
Fetal hepatic CB2R protein expression in
male fetuses of mothers fed HF (n=6) and
CTR (n=9) diet
0
10
20
30
40
50
60
70
80
CTR HFD
His
tosc
ore
Maternal hepatic CB1R protein expression
in mothers, carrying female fetuses, fed HF
(n=5) and CTR (n=5) diet.
0
10
20
30
40
50
60
70
80
90
CTR HFD
His
tosc
ore
Maternal hepatic CB2R protein expression
in mothers, carrying female fetuses, fed
HF (n=5) and CTR (n=5) diet.
0
10
20
30
40
50
60
70
80
90
CTR HFD
His
tosc
ore
Maternal hepatic CB1R protein expression
in mothers, carrying male fetuses, fed HF
(n=5) and CTR (n=8) diet.
0
10
20
30
40
50
60
CTR HFD
His
tosc
ore
Maternal hepatic CB2R protein expression in
mothers, carrying male fetuses, fed HF (n=5)
and CTR (n=7) diet.
Results
CB1
CB2
A
C
B
Maternal high fat diet programs offspring
metabolism and intake contributes to the
development of Nonalcoholic steatohepatitis
(NASH) in adult offspring via the mechanism of
up-regulated hepatic lipogenesis. From the
other hand the activation of CB1R leads to
lipogenesis and reduced fatty acid oxidation in
hepatocytes. In general, increased expression
of CB1R in liver of experimental, diet-induced
or genetically modified model of NAFLD is a
well described phenomenon in non-pregnant
animals. Our funding regarding increased
CB1R expression in the male offspring is in
line with the results obtained in experimental
models of maternal high fat diet, showing
increased adiposity and hepatic steatosis in
male, but in the female offspring. Our data
suggests that lipogenesis, but not
inflammatory responses, might be the major
pathways of in utero programming of NAFLD
by maternal consumption of high fat diet.
Figure 5 . Overview of the
development of NAFLD in
the context of
developmental
programming. (Li, 2015)
Figure 3. Blue arrows indicate bile duct, red arrows-artery, yellow arrows-portal vein and green arrows-lipid droplet.
To evaluate the expression of CB1 and CB2 receptors in maternal and fetal liver in baboon (Papio spp)
model of high fat diet.
Our data is highly relevant in development of
prevention strategies to counteract the gender-
dependent effect of maternal obesity and prevent
devastating effect of maternal obesity on the
offspring health.
Marcel Chuecos1, Cun Li2,3, Maira Carrillo1, Kushal Gandhi1, Cezary Skobowiat4,5, Gary Ventolini1, Patrick Joseph6, Edward Dick7, Gene Hubbard8, Peter Nathanielsz2,3, Natalia E. Schlabritz-Loutsevitch1*
