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Stress During Lactation Affects Expression of Components of the Endocannabinoid System in Pancreas of Adult Mice (Chile)

University of Michigan investigator(s) and unit: Betsy Lozoff, M.D., Center for Human Growth and Development; Pediatrics.
International colleague(s) and unit: Miguel Llanos S. Unit of Nutrition and Metabolic Regulation. Institute of Nutrition and Food Technology (INTA). University of Chile. Santiago.Chile.
General Purpose: To elucidate the possible involvement of the endocannabinoid system in adult mice overweight and associated metabolic consequences due to a mild nociceptive (painful or noxious) stress during lactation.
Rationale: Stress during lactation induces metabolic disturbances in overweight in adult mice. These metabolic alterations may be reversed with a type 1 cannabinoid/endocannabinoid receptor (CB1ER) antagonist, suggesting hyperactivity of these receptors in tissues involved in energy homeostasis leading to metabolic disruptions due to early stress. Hyperactive CB1ER may be a direct consequence of its elevated expression, or indirectly by decreased fatty acid amide hydrolase activity (FAAH; the enzyme able to inactivate endocannabinoid agonists, mainly arachidonoylethanolamide or anandamide).
Specific Objective: To evaluate the effects of a nociceptive stress during lactation in pancreas CB1ER expression of adult mice together with evaluation of FAAH expression and activity.
Study design and methods: Twelve-hour-old, CD-1 male mice pups are randomly distributed for maternal crossfostering. During lactation, mice are stressed or not with a daily subcutaneous injection of saline solution in the back (1 μl/g of body weight). Control mice are those cross-fostered but not stressed. Adult 140-day-old mice are sacrificed and pancreas extracted to evaluate mRNA and protein expression of CB1ER and FAAH. Additionally, pancreatic FAAH enzymatic activity is also determined by its ability to hydrolyze 3H-[Anandamide] at 37ºC.
Anticipated undergraduate/graduate student activities on project: It is expected that pancreatic tissue will be already available to perform subsequent experiments to evaluate pancreatic expression of CB1ER and FAAH in stressed and control animals (n=5). Thus, the student will carry out experiments to determine mRNA and protein expression of CB1ER and FAAH. According to availability of time, the student could also evaluate FAAH enzymatic activity present in pancreas previously extracted from stressed and control mice.
Techniques/methods students should become familiar with in advance: It is desirable that student could be familiarized with RT-PCR and Western blot techniques to evaluate mRNA and protein expression of CB1ER and FAAH. To evaluate FAAH activity, the student should learn about isotope (tritium) quantification, lipid extraction and thin layer chromatography techniques.
Suggested readings (minimum of 3-5 articles):
1. Di Marzo, V. (2006). A brief history of cannabinoid and endocannabinoid pharmacology as inspired by the work of British scientists. Trends Pharmacol Sci. 27: 134-40.
2. Loizzo, A. et al. (2006). Overweight and metabolic and hormonal parameter disruptions are induced in adult male mice by manipulations during lactation period. Pediatr Res 59: 111-115.
3. Valenzuela, C. Aguirre C, Castillo V, Ronco AM, Llanos M (2009). The endocannabinoid CB1 receptor antagonist SR141716A reverses adult male mice overweight and metabolic alterations induced by nociceptive stress during lactation. Obesity (Silver Spring). 2010 Jun 17 (in press) [Epub ahead of print].
4.Valenzuela C, Aguirre C, Castillo V, Ronco AM, Llanos M (2010) Participación del Sistema Endocanabinoide en el Desarrollo de Obesidad (A Role for the Endocannabinoid System in Obesity). Rev Med Chile 138(5):621-629 (Spanish)
5. Juan-Picó P, Fuentes E, Bermúdez-Silva FJ, Javier Díaz-Molina F, Ripoll C, Rodríguez de Fonseca F, et al. (2006) Cannabinoid receptors regulate Ca(2+) signals and insulin secretion in pancreatic beta-cell. Cell Calcium 39: 155-62.
6.Vilches-Flores A, Delgado-Buenrostro NL, Navarrete-Vázquez G, Villalobos-Molina R. (2010). CB1 cannabinoid receptor expression is regulated by glucose and feeding in rat pancreatic islets. Regul Pept. 163(1-3):81-7