A Pharmacologically Potentiated Stress during Lactation Alters the Hepatic Endocannabinoid System in Adult Mice: Association with Development of Insulin Resistance
Authors: Llanos Miguel N. , Castillo Valeska A. , Ponzano Paula A. , Ronco Ana Marìa 
Published: November 2023

We have previously shown that stress during lactation induces metabolic alterations in adult mice (1). These effects were abolished by treatment with SR141716A, a type 1 cannabinoid receptor (CB1R) antagonist, suggesting involvement of the Endocannabinoid System (ECS; 1). The ECS is formed in part by CB1R, its agonist the endocannabinoid anandamide (AEA), and the enzyme fatty acid amide hydrolase (FAAH) able to hydrolase AEA. The ECS is involved in stress responses since activation of CB1R mediates the inhibitory feed‐back mechanism produced by elevated levels of corticosterone in the hypothalamus (2). Moreover, liver CB1R is required to develop insulin resistance (IR; 3). In the present study, we evaluate whether a pharmacologically potentiated stress‐due to simultaneous treatment with a CB1R antagonist (AM‐251) ‐ during early lactation, is able to cause a long term disruption of the hepatic ECS. In addition, we analyze the presence of abnormal tyrosine nitration and/or phosphorylation of proteins involved in hepatic insulin signaling.

In early lactation (days 1–10), male pups were daily treated with an oral dose of AM‐251 (3μg/g) and 1h later subjected to stress with a subcutaneous injection of saline in the back (main AMST group; with three other appropriate controls). Maintenance, treatments and euthanasia of mice were conducted according to FASEB Statement of Principles for the use of animals in research and education. Adult, 140 days old mice were subjected to an insulin sensitivity test to evaluate IR. At 150 days animals were sacrificed, and gene/protein expressions of hepatic RCB1, (qPCR and Western Blot) and FAAH activity were evaluated. Tyrosine nitration of hepatic IRβ, IRS1/2, total Akt and phosphorylated Akt‐Ser473/Thr308 were evaluated by immunoprecipitation with magnetic beads linked to specific antibodies. Circulating levels of corticosterone were also measured.

The AMST group showed higher concentrations of circulating corticosterone and developed IR when adults. In addition, a higher significant abundance of hepatic RCB1 protein compared to controls, concomitant to a lower abundance and activity of FAAH were observed. qPCR results showed no changes in CB1R and FAAH mRNA. An imbalance in basal and insulin‐stimulated phosphorylation of AktSer473/Thr308 with a high basal pAkt‐Thr308 abundance, was also obtained. Moreover mice from the AMST group showed higher level of NO2Tir‐Akt and NO2Tir‐pAktSer473/Thr308. Tyrosine nitration was not observed in IRβ, IRS1 and IRS2.

A pharmacologically potentiated stress by means of AM‐251 treatment during the first half of lactation, alter the hepatic SEC of adult mice, increasing abundance of RCB1 and decreasing amount and activity of FAAH. These results suggest, as a consequence, that a concerted mechanism leads to overactivity of RCB1. In addition, CB1R overactivity is associated with an imbalance in the phosphorylation of Akt‐Ser473/Thr308 and a greater NO2Tir‐Akt, leading first to hepatic IR which is subsequently evidenced at systemic level.

Support or Funding Information

Financial Support: FONDECYT‐CHILE, Grant 1130106.