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BABOON PLACENTAL ENDOCANNABINOID RESPONSES TO MATERNAL HIGH FAT DIET

Introduction

1OB/GYN - Research, TTUHSC-Permian Basin, Odessa, Texas, United States. 2Animal Science, University of Wyoming, Laramie, Wyoming, United States. 3Exp. Physiology, SNPRC, San Antonio, Texas, United States. 4Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacology, The Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland.

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.

Objective

Discussion and Conclusion

References

Acknowledgements

1. Brocato, B., … Schlabritz-Loutsevitch, N. (2013). Endocannabinoid crosstalk between placenta and maternal fat in a baboon model (Papio spp.) of obesity.

Placenta 34(11), 983-989. 2. Cota, D., et al. (2003). The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral

lipogenesis. J Clin Invest, 112(3), 423-431. 3. Fugedi, G., et al. (2014). Increased placental expression of cannabinoid receptor 1 in preeclampsia: an observational

study. BMC Pregnancy Childbirth, 14, 395. 4. Russo, E. B. et al. (2004). Clinical endocannabinoid deficiency (CECD): can this concept explain therapeutic benefits

of cannabis in migraine, fibromyalgia, irritable bowel syndrome and other treatment-resistant conditions? Neuro Endocrinol Lett, 25(1-2), 31-39. 5. Pardo, F., et al.

(2015). Human supraphysiological gestational weight gain and fetoplacental vascular dysfunction. Int J Obes (Lond), 39(8), 1264-1273. 6. Qiao, L., et al. (2015).

Maternal High-Fat Feeding Increases Placental Lipoprotein Lipase Activity by Reducing SIRT1 Expression in Mice. Diabetes, 64(9), 3111-3120. 7. Schlabritz-

Loutsevitch, N., et al(2016). Fetal Syndrome of Endocannabinoid Deficiency (FSECD) In Maternal Obesity. Med Hypotheses, 96, 35-38. 8. Spradley, F. T., et al.

(2015). Increased risk for the development of preeclampsia in obese pregnancies: weighing in on the mechanisms. Am J Physiol Regul Integr Comp Physiol, 309(11),

R1326-1343. 9. Stanley, C. P., et al. (2016). The endocannabinoid anandamide causes endothelium-dependent vasorelaxation in human mesenteric arteries.

Pharmacol Res, 113(Pt A), 356-363.

Materials and Methods 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.

Eleven HF and nine CTR placental samples, from male and female fetuses, 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, secondary antibody (Jackson

Immuno Research Laboratories, Inc. USA. Cat. #: 715-035-150), 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.

The TRIzol method was used to isolate RNA from tissue samples (Life Technologies, USA), and cDNA

was synthesized according to the manufacturer’s instructions (Applied Biosystems/ Roche, USA).

qPCR was performed using Fast start Essential DNA Probe Master Mix (Roche, USA), and TaqMan

Gene Expression Assay Probes (Life Technology, USA) for CB1. The TaqMan probes used were CB1

(Hs01038522). The housekeeping gene used for these genes was 18S (Hs99999901). The CB2 gene

was detected using FastStart Essential DNA Green Master Mix (Roche, USA).

Maternal obesity (MO) affects fetal development, which in turn, influences individual’s life

trajectory. Specifically, MO has been linked to autism spectrum disorders, inflammatory bowel

syndrome, fibromyalgia, asthma, non-alcoholic fatty liver diseases (NAFLD), diabetes and obesity

in the offspring. While the suggested mechanisms, linking obesity to the offspring health, are

varying from the oxidative stress, inflammation, fatty acid transport and stress-related pathways,

the common mechanisms remain obscure. Endogenous cannabinoid (eCB) system (ECS) is involved

in the appetite regulation and lipogenesis (Cota et al., 2003). While the spectrum of offspring

health conditions, programmed by MO, is broad, it could be brought under the umbrella of Clinical

Endocannabinoid deficiency syndrome (CECDS), described by Russo in 2004 (Russo, 2004;

Schlabritz-Loutsevitch, German, Ventolini, Larumbe, & Samson, 2016).

ECS is the family of the biologically active lipids – derivatives of omega-3 fatty acids, which

regulate vascular tone, metabolic rate, neurogenesis, inflammatory and stress responses – all

hallmarks of MO. We recently suggested the existence of Fetal Syndrome of Endocannabinoid

deficiency in MO, based on the preliminary data, obtained in human population (Schlabritz-

Loutsevitch et al., 2016) and in natural model of obesity in the baboons (Papio spp.)(B. Brocato et

al., 2013). The different patterns of MO: pre-pregnancy obesity vs. pregnancy-related weight gain,

over-eating vs high-fat –high calorie diet (HFD)-are making the studies of the mechanism of

developmental programming by MO challenging. Animal models represent the opportunity to

dissect specific mechanisms of the dietary patterns and provide important data for development of

interventional strategies in humans.

Figure 1. Protein expression level of CB1R and CB2R using IHC in placenta samples. A and B panels show the placenta sections

from male fetuses in CTR and HFD animals. C and D panels show the placenta sections from female fetus in CTR and HFD

animals. E and F panels show the placenta sections from male fetuses in CTR and HFD animals. G and H panels show the

placenta sections from female fetuses in HFD and CTR animals. In all panels blue arrows point to fetal endothelium, green

arrows point to Cytotrophoblast, and pink arrows point to Syncytiotrophoblast. Negative control figures are shown in small boxes

in all panels (upper right corner). I to L panels show the quantification of IHC figures in HFD and CTR groups. The figures were

taken using a 20 X magnification. Scale bar, 20 μm. Data were represented as the mean ± SEM.

[CTR: Control; HFD: High-Fat Diet; MF: Male Fetus; FF: Female Fetus; IHC: Immunohistochemistry]

To evaluate placental expression of ECS receptors CB1R and CB2R in an experimental model of a

maternal high fat diet.

Marcel Chuecos, BS1, Cun Li, MD2,3, Kushal Gandhi, PhD1, Stacy Martinez, MS1, Cezary Skobowiat, DVM, Phd4, Maira Carrillo, PhD1, Moss Hampton, MD1, Suraparaju Raju, MBBS1,

Elihu Arzate, BA1, Gary Ventolini, MD1, Peter Nathanielsz, MD, PhD2,3 and Natalia Shlabritz-Loutsevich, MD, PhD1* (natalia.schlabritz-lutsevich@ttuhsc.edu).

Figure 2. A) Expression of mRNA for cannabinoid receptors (CB1 and CB2) by Reverse Transcription Real Time quantitative

PCR analysis (q RT-PCR) using specific primers, in maternal liver (n = 3 to 6), fetal liver (n = 4 to 6), and placenta (n = 4 to

6). Results were shown as fold change (2^(-ΔΔCt) method), normalized to levels of control 18S mRNA expression. Data were

represented as the mean ± SEM.

B) Protein expression for cannabinoid receptors (CB1 and CB2) by western blot (n = 4 to 6). Graphs depict relative band

intensity, quantified using Image J software, and normalized to control β-actin expression. Data were represented as mean

± SEM, p < 0.05 was considered as statistically significant difference between the groups (*p < 0.05).

C) Representative images of western blot analysis of CB1R and CB2R expressions detected in placenta (n = 4 to 6).

Results

A B

C

Our finding regarding increased CB2 expression in the placenta of animals fed HFD is

in agreement with our previous observation in the placenta of naturally obese

baboons (Brocato, B., et al., 2013) and placental macrophages infiltration in obesity

(Challier et al., 2008).

The endothelial dysfunction is the hallmark of pregnancies complicated by maternal

obesity and excessive gestational weight gain (Pardo et al., 2015; Spradley, Palei, &

Granger, 2015). Location of CB1R in the fetal placental endothelium is in agreement

with previously published data in the baboon (B. Brocato et al., 2013) and human

placenta (Fugedi et al., 2014). In the present study the expression of CB1R was

mostly located in the fetal vascular endothelium of CTR animals and maternal

feeding with the HFD shifted this expression toward ST.

Endothelium-dependent CB1R-mediated vascular relaxation is the phenomenon

described in mesenteric arteries (Stanley, Hind, Tufarelli, & O'Sullivan, 2016). The

CB1R expression in the outer layer of the ST might be associated with increased

Lipoprotein lipase activity in the ST in maternal obesity (Qiao et al., 2015), since LPL

activity is regulated by eCBs through CB1R related mechanism (Cota et al., 2003).

Thus in HFD eCB effect could be shifted from vascular to metabolic responses. Figure 3. Graphic representation of a transverse section of a

chorionic villus exposed to a control diet. Figure 4. Graphic representation of a transverse section of a

chorionic villus exposed to a high fat diet.

Interested about this study? See poster F-103 for more information