Effects of endocannabinoids on feed intake, stress response and whole-body energy metabolism in dairy cows

Endocannabinoids, particularly anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are instrumental in regulating energy homeostasis and stress response. However, little is known about the endocannabinoid system (ECS) in ruminants, although EC could improve dairy health and productivity, at least by increasing feed intake. In this study, we report if intraperitoneal (i.p.) AEA and 2-AG administration affects feed intake, whole-body macronutrient metabolism, isolation and restraint stress, and whether diet composition modulates circulating endocannabinoid concentrations in cows. Twenty Simmental cows in late lactation were fed a grass silage and a corn silage based diet. On each diet, cows received daily i.p. injections with either AEA (5 µg/kg; n = 7), 2-AG (2.5 µg/kg; n = 6) or saline (n = 7) for 8 days. Endocannabinoid administration for 5 days under free-ranging (non-stressed) conditions had no effect on feed intake or energy balance, but attenuated the stress-induced suppression of feed intake when housing changed to individual tie-stalls without social or tactile interaction. Endocannabinoids increased whole-body carbohydrate oxidation, reduced fat oxidation, and affected plasma non-esterified fatty acid concentrations and fatty acid contents of total lipids. There was no effect of endocannabinoids on plasma triglyceride concentrations or hepatic lipogenesis. Plasma AEA concentrations were not affected by diet, however, plasma 2-AG concentrations tended to be lower on the corn silage based diet. In conclusion, endocannabinoids attenuate stress-induced hypophagia, increase short-term feed intake and whole-body carbohydrate oxidation and decrease whole-body fat oxidation in cows.Landwirtschaftliche Rentenbank http://dx.doi.org/10.13039/501100018686Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Forschungsinstitut für Nutztierbiologie (FBN) (2113)Peer Reviewe

Investigations into the endocannabinoid system and endocannabinoid-mediated control of energy homeostasis in late-lactating dairy cows

The endocannabinoid system (ECS) is a comprehensive and multifaceted system within the body of all vertebrates. It consists of G protein-coupled cannabinoid receptors, their endogenous ligands, the so-called endocannabinoids, and the corresponding synthesizing and degrading enzymes. In recent years, intensive research has provided insights into the key elements of the ECS and its vast involvement in many regulatory processes in non-ruminants, primarily rodents and humans. Several mechanisms of endocannabinoid action have been identified in terms of energy homeostasis with the regulation of feed intake and appetite, as well as the stress response and lipid and glucose metabolism. Nevertheless, numerous questions remain unanswered, especially with regard to ruminant species. In dairy cows, targeted activation of the ECS may represent a promising therapeutic approach to overcome problems associated with the periparturient period, but the current understanding of the ECS in ruminants is insufficient. Therefore, the aim of the present work was to further characterize the fundamentals of the ECS in dairy cows and to investigate its involvement in the regulatory processes of energy homeostasis by administering the two major endocannabinoids N-arachidonoylethanolamide (anandamide, AEA) and 2-arachidonoylglycerol (2-AG). To achieve this aim, a series of experiments were conducted with non-pregnant, late-lactating Simmental cows. A subset of experiments within this thesis was conducted for a better understanding of the dynamics of the circulatory endocannabinoid tone in dairy cows. Dairy cows were subjected to: (i) short-term feed deprivation; (ii) stress exposure; (iii) different diet compositions (low and high n6/n3 fatty acid ratio); and (iv) i.p. injections of AEA and 2-AG, with subsequent analysis of plasma endocannabinoid concentrations. Analyses revealed that both feed deprivation and stress exposure increase plasma 2-AG levels to varying degrees in ruminants, similar to nonruminates, whereas plasma AEA levels remained constant. It seems that in dairy cows, 2-AG appears to be more involved in the systemic metabolic response to feed deprivation and stress exposure than AEA. Contrary to expectations, feeding a high n6/n3 diet did not increase plasma endocannabinoid concentrations in dairy cows. Systemic administration of AEA increased circulatory AEA concentrations 2.5 h after injection, while administration of 2-AG did not change plasma endocannabinoid concentrations 2.5 h after injection. The apparent differences in the bioavailability of injected AEA and 2-AG and possible accumulation of AEA, require more detailed studies in the future. To investigate the effects of i.p. administered endocannabinoids on feed intake and hypothalamic orexigenic signaling in dairy cows, feed intake was continuously measured by electronic registration devices and analyzed in different intervals. Analyses revealed that i.p. administered AEA and 2-AG at doses of 5 μg/kg and 2.5 μg/kg, respectively, mainly affected feed intake in the short-term within the first hour after treatment and had no long-term effect. At the end of the trial, cows were slaughtered 2 to 3 hours after i.p. endocannabinoid injection, and brain tissue was collected for immunohistochemical analysis of agouti-related peptide (AgRP), orexin A (OX-A), and cannabinoid receptor 1 (CB1) expressing neurons, and for PCR analysis of AgRP, neuropeptide Y (NPY), and genes related to the ECS. The immunohistological investigation of hypothalamic orexigenes revealed, however, no neuromodulatory effect of either treatment on immunoreactivity or c-Fos activation. The mRNA gene expression of ECS-related genes in the arcuate (ARC) and paraventricular (PVN) nuclei of dairy cows was also not different between treatment groups. However, NPY and AgRP mRNA abundances were downregulated in the ARC of AEA-treated animals, which might indicate a possible counter-regulatory mechanism. To test whether cows may benefit from injected endocannabinoids during stress in terms of stress-induced alterations in feed intake, feed intake was compared with and without exposure to stress stimuli such as social and tactile isolation, and tethering. In fact, AEA and 2-AG administration seemed to attenuate stress-induced hypophagia. To further understand the influence of administered endocannabinoids on the level of whole-body metabolism, cows were kept in respiration chambers twice for three days, and gas exchange was recorded every 6 minutes for detailed analysis. Treatment with AEA and 2-AG increased whole-body carbohydrate oxidation (COX) and metabolic heat production (HP) and decreased whole-body fat oxidation (FOX), mirroring the endocannabinoid-mediated changes in feed intake mentioned above. An analysis independent of feed intake showed that COX was not different between treatment groups, whereas FOX remained lower with endocannabinoid treatment, suggesting that effects extend beyond individual differences in feed intake and that AEA and 2-AG indeed suppress whole-body fat catabolism and/or support lipogenesis. To gain an initial understanding of the effects of administered endocannabinoids on lipid metabolism, plasma samples were collected before and after 9 days of daily treatment and analyzed for plasma non-esterified fatty acids (NEFA) and specific plasma lipid concentrations. We demonstrated that injected endocannabinoids attenuated the increase in plasma free fatty acid concentration associated with stress-induced hypophagia. Furthermore, the analyses suggest that possibly with endocannabinoid application, free fatty acids are used to an increased extent for triglyceride synthesis in adipocytes. In conclusion, the herein discussed results offer new and significant insights into the ECS in dairy cows. This field of research is still in its early stages; thus, much remains to be discovered including the potential for beneficial therapeutic intervention. However, this thesis and the studies published within provide a direction and serve as a foundation for future research