The effects of anandamide and N-acylethanolamine hydrolysis on insulin secretion in rat isolated islets of Langerhans

Richard Anderson, Michael Randall, Sue Chan

University of Nottingham, Nottingham, UK

Endocannabinoid signalling in peripheral tissues is of growing interest given the known associations with obesity; however, little is known about the influence of cannabinoids on endocrine pancreas function and the possible links to type 2 diabetes mellitus (Scheen et al., 2006). This study examined the effects of the endocannabinoid anandamide (AEA) on insulin secretion in rat isolated islets of Langerhans.

Islets of Langerhans were isolated from male Wistar rats (body weight 240-350g) and used in static incubation secretory studies. Batches of three islets were incubated in 200μl of bicarbonate buffered physiological saline solution with test reagents at 37ºC, for 1 hour. Samples of the incubation media were taken and insulin content was determined by radioimmunoassay. 4mM and 20mM glucose controls were used in each experiment to establish islet viability and these controls also included ethanol to account for possible vehicle effects. n represents the number of individual experiments (using islets isolated from n number of rats). Data shown are means ± SEM.

AEA (1μM) was found to inhibit insulin secretion at 20mM glucose by 32.7 ± 10.3% (P<0.01, ANOVA; n=10) but did not affect basal (4mM) or intermediate (8mM) levels of glucose-stimulated insulin secretion. Concentration-response curves constructed at 20mM glucose revealed two distinct responses to AEA but insulin release was always inhibited at 100μM. In the “non-responsive” group, AEA was without effect at concentrations ≤10μM (n=7). In the “responsive” group, AEA inhibited insulin secretion with a pIC50 of 6.02 ± 0.29 (n=6). Methanandamide, a non-hydrolysable analogue of AEA, was found to cause a similar concentration-dependent inhibition of insulin secretion to that of the “responsive” AEA group with a pIC50 of 6.35 ± 0.35 (n=5) at 20mM glucose. These results suggest that inter-experimental variations in rates of AEA hydrolysis may account for the differences between the “responsive” and “non-responsive” groups. As fatty acid amide hydrolase (FAAH) is believed to be the main enzyme responsible for the hydrolysis of AEA, islets were pre-incubated with 10μM URB597, a FAAH specific enzyme inhibitor, prior to the addition of 10μM AEA at 20mM glucose. URB597 did not affect the level of inhibition of insulin release mediated by AEA in either responsive or non-responsive islets.

In summary, AEA inhibits insulin secretion in a glucose and concentration-dependent manner. The responses to AEA were variable between tissues but this was not the case for methanandamide. The variation in responses to AEA could be due to non-FAAH mediated metabolism. Further investigations into the mechanism(s) of inhibition of insulin secretion by AEA are currently being performed. Advancement in the understanding of the role of the endocannabinoid system in the islets of Langerhans may offer novel therapeutic avenues in type 2 diabetes mellitus.

Scheen, A.J. et al. (2006). Lancet 368, 1660-72