In the present study, repeated administration of AEA was applied to increase plasma AEA concentrations in early-lactating cows. Activation of the ECS was expected to increase dry matter intake and decrease lipolysis, as already described in studies with rodents17,29−33.
Effect of AEA administration on the plasma concentration of endocannabinoids and endocannabinoid-like compounds
In our study, we were able to successfully increase the plasma AEA concentration in early-lactation by repeated daily i.p. injections of 3 µg AEA /kg BW in the first four weeks of lactation. The treatment was specific to AEA, because it did not affect the plasma concentration of other endocannabinoids or endocannabinoid-like compounds, except for EPEA, which tended to be elevated on d 14 p.p. To date, no study has investigated, if AEA administration influences plasma EPEA concentration. In rodents, diet composition can affect EPEA levels in plasma and adipose tissue34, but all animals in our study were fed the same diet and consumed comparable amounts of feed. Thus, it remains elusive whether AEA administration or an unknown influencing factor caused this temporary increase in EPEA. The latter is a CNR1 and CNR2 agonist3 and possess anti-inflammatory properties35; however, further studies are needed to evaluate the role of EPEA on early-lactating cows.
Effect of AEA on energy balance and hepatic gene expression in the first three weeks after parturition
Because we observed major differences in energy metabolism between weeks 1 to 3 p.p. and weeks 4 to 5 p.p., and that the transition period is defined 3 weeks before until 3 weeks after calving36, the discussion distinguishes between these two p.p. periods. Despite elevated plasma AEA concentrations, we did not observe an increase in feed intake during the first three weeks after calving in the AEA group. This result is in contrast to a study in rodents, which used a comparable low AEA dosage, but non-lactating animals30. Furthermore, AEA administration at similar dosage increased feed intake in mid- and late-lactation cows, although this effect was limited to 1 h after i.p. injection and to 10 h after intracerebroventricular injection14,15,27. The authors of these studies explained this short-term effect with the short half-life of AEA, because it is rapidly inactivated by re-uptake and degrading enzymes37. It is conceivable that the AEA treatment in the present study also caused a short-term increase in feed intake but this effect could not be detected on the daily basis. Furthermore, it has to be taken into account that the endocrine status differs substantially between early, late and non-lactating animals. Another important factor is that the sensitivity towards various hormones regulating feed intake, i.e., leptin, and perhaps also AEA, is diminished in early-lactation.
Administration of AEA had also no effect on milk yield, ECM yield, and milk constituents, which together with the unaltered feed intake resulted in a comparable energy balance between the groups in the first 3 weeks p.p. This finding is underlined by comparable plasma NEFA and β-hydroxybutyrate concentrations between the groups. A previous study reported also no changes in plasma NEFA and β-hydroxybutyrate concentrations after i.p. AEA administration to late-lactating cows15. However, a reduction in milk yield after intracerebroventricular injection was observed in mid- and late-lactating cows16.
Previous studies in rodents, humans, and cows reported the involvement of the ECS in the regulation of lipolysis17,38,39. In early-lactating cows, fat mobilisation occurs due to the negative energy balance, resulting in a loss of adipose tissue mass and increase of plasma NEFA concentration. To our knowledge, the present study is the first to investigate the influence of AEA administration on fat depots in vivo. In our study, we did not observe a clear effect of AEA treatment on various fat depots and fat layer thicknesses, although there was a trend towards more omental adipose tissue in week 2 p.p. and significantly more omental adipose tissue in week 3 p.p. in the AEA group. The difference in lipomobilisation between the investigated fat depots can be explained by a different depot-specific responsiveness to lipolytic signals40,41.
The plasma NEFA concentration is a marker for fat mobilisation42. The plasma NEFA concentration were not different between the first 3 weeks p.p. Moreover, we did not observe any effect on the norepinephrine-stimulated in vitro lipolysis in week 2 p.p. This finding is consistent with a previous study showing no changes in isoproterenol-induced lipolysis after CNR1 receptor agonist administration39. Overall, our and earlier results indicate that AEA has no or only a limited effect on lipomobilisation in early-lactation. This finding supports the hypothesis proposed by Myers et al. 39, who suggested a resistance to ECS activation in adipose tissue of early-lactating cows39. The authors reported no effect of CNR1 receptor activation on the lipolysis rate in adipose tissue explants from cows obtained 1 to 3 weeks p.p.39.
Lipid mobilisation and the resulting increased influx of NEFA into the liver can exceed the hepatic capacity for oxidizing fatty acids, thus leading to lipid accumulation in liver of cows during early-lactation43. In rodents, the activation of the ECS increases fatty acid synthesis and lipid deposition in the liver20. In the present study, we showed that i.p. AEA administration decreased hepatic GPR55 mRNA expression. Downregulation of G-protein coupled receptors is induced, among others, by prolonged exposure to an agonist44,45. Therefore, the diminished GPR55 expression in the present study is likely due to chronic exposure of AEA, a GPR55 agonist46. To date, the physiological role of GPR55 in the liver of ruminants is unresolved. In humans and rodents, GPR55 is involved in the regulation of hepatic lipid metabolism47,48 and insulin signalling49. Thus, the lower GPR55 mRNA abundance suggests that administered AEA alters hepatic lipid metabolism through GPR55 in cattle. Indeed, AEA treated cows showed reduced DGAT1 and DGAT2 mRNA abundance. Inhibition of DGAT1 has been shown to result in less lipid droplet accumulation and TG concentration in primary calf hepatocytes after incubation with fatty acids, whereas DGAT2 inhibition had no effect on TG concentration50. Thus, repeated AEA treatment may limit TG synthesis and thus the progression of fatty liver in early-lactation.
In addition, we observed higher plasma TG concentration on d 21 p.p. in the AEA group. In mice, activation of the ECS resulted in higher plasma TG and cholesterol levels by impairing apolipoprotein E-mediated clearance51. In contrast to the finding in mice, we did not observe increased plasma cholesterol levels. In general, the increase in plasma TG can be induced either by increased synthesis of the liver or by decreased utilization in the mammary gland. Due to the comparable milk yield and milk fat content between the cow groups in week 3 p.p., a change in TG utilization for milk fat synthesis is rather unlikely. The synthesis and secretion of TG in cows occurs primarily in the liver through the re-esterification of NEFA and subsequent export as VLDL. A previous study described negative correlations between plasma TG and NEFA concentrations and between plasma and hepatic TG levels52. This finding is supported by Van den Top et al., who reported an association between fatty liver and decreased plasma TG concentrations53. Overall, this suggests that lipolysis and plasma TG level are inversely related52. Thus, the elevated plasma TG concentrations in the present study may indicate less lipid accumulation in liver. However, the increase in plasma TG might also be due to an increased VLDL synthesis and secretion. Yet, we observed no difference in the mRNA expression of APOB100 and MTTP, both genes involved in VLDL assembly54, but we cannot exclude that other hepatic genes related to VLDL export account for the different plasma TG concentrations.
Effect of AEA on energy balance, hepatic and mammary gene expressions in week 4 and 5 postpartum
Unexpectedly, AEA treated cows stopped increasing feed intake in week 4 p.p, while feed intake, as expected, further increased in the CON group. The reduced feed intake in the AEA group was also reflected by the lower milk protein percentage as previous studies showed a decrease in milk protein content during restricted feeding55. Moreover, the insufficient energy intake of the AEA group was also accompanied by a higher proportion of the lipid droplet area of the liver, indicating a higher lipid accumulation in the AEA group.
In rodents and cows, AEA administrations either increased or did not affect feed intake14,16,30,56, but there are no studies reporting a decrease in feed intake in mammals, suggesting that the observed reduction in feed intake in the AEA group was presumably not a direct result of the experimental treatment. A potential explanation may be the higher leptin concentration in AEA cows in week 4 p.p. In rodents, humans and ruminants, leptin decreases feed intake and controls energy balance11–13,57−59. Because plasma leptin concentrations are directly related to the amount of stored fat60, a negative energy balance and fat mobilisation results in a decrease in plasma leptin concentration61. However, in our study we found that the AEA group had numerically more adipose tissue and the greater BCS throughout the experimental period, which cannot explain the abrupt increase in leptin concentration in week 4 p.p. Furthermore, the loss of body mass in the AEA group was numerically greater than in the CON group, while the plasma NEFA concentrations were comparable between groups. These facts indicate that plasma leptin concentrations in week 4 p.p. do not correspond to adipose tissue mobilisation.
There is evidence that leptin can modulate the AEA level in non-ruminants10,62. Hence, it is conceivable that the increase in plasma leptin concentration in week 4 p.p. could be a counter-regulatory response to the chronic AEA administrations. However, no study has proved this hypothesis and thus further studies are needed to elucidate the interaction between the AEA tone and leptin release.
In the liver, the FAAH mRNA abundance was higher in the AEA group than in the control group on d 30 p.p. FAAH encodes the enzyme responsible for the degradation of AEA63 and thus, upregulation of FAHH implies increased degradation of AEA. Unfortunately, we were not able to measure the AEA concentration on d 30 p.p. to support this assumption. Previous studies reported increased FAAH mRNA abundance after AEA administration64. However, another reason for the change in FAAH mRNA abundance could be the higher leptin concentration in the AEA group. In rodents, it has been shown that i.p. administration of leptin increased FAAH activity and thus AEA hydrolysis in the hypothalamus, however, FAAH gene expression was unchanged and the authors proposed a post-translational mechanism increasing FAAH activity62. From these findings, we conclude that upregulation of the FAAH mRNA abundance was either triggered directly by repeated administration of AEA or indirectly via leptin.
The analysis of mRNA of genes related to lipid metabolism revealed a tendency to a lower MTTP mRNA abundance in the AEA group on d 30 p.p. Bremmer et al. showed that NEFA administration to hepatocytes reduced MTTP mRNA expression in vitro65, and reported a negative relationship between MTTP mRNA expression and liver TG concentration on d 35 p.p66. Similarly, cows with fatty liver showed downregulation of MTTP mRNA expression relative to controls50. In the present study, MTTP downregulation was also accompanied by higher lipid accumulation, as reflected by the trend to greater lipid droplet area in the liver of AEA cows. However, the biological significance remains elusive, because a change in MTTP mRNA abundance is not necessarily accompanied by a change in MTTP activity65.
As mentioned above, activation of the ECS regulates lipid metabolism in the liver and adipose tissue of rodents and ruminants, and involves among others upregulation of ACC1 in lipogenic tissues20. However, little is known about the influence of endocannabinoids on the metabolism of the mammary gland. In the present study, AEA cows have a lower mammary gland ACC1 mRNA abundance, which is involved in de novo milk fatty acid synthesis67. Surprisingly, downregulation of ACC1 was not accompanied by a reduction in milk fat content, suggesting that the inhibited de novo milk fat synthesis was compensated by an increased NEFA uptake from the circulation. However, the effect of AEA on milk fatty acid composition needs to be evaluated in future studies. Downregulation of ACC1 could also be due to higher leptin concentrations in AEA cows on d 28 p.p. Leptin inhibits ACC1 by activating AMP-activated protein kinase68,69, however, whether this pathway is also activated in the mammary gland of AEA cows requires further investigations. Furthermore, the decreased PLTP activity may be a result of greater leptin concentrations in AEA cows on d 28 p.p. The PLTP mediates the transfer of phospholipids to high-density lipoprotein cholesterol 70, however, high-density lipoprotein cholesterol plasma concentration was unchanged in our study. Nonetheless, our result corresponds to the finding in heterozygous PLTP+/– mice, which had reduced PLTP activity abut no change in high-density lipoprotein cholesterol level compared to the wildtype71.
Comparative analysis of the AEA effect in different stages of lactation
Myers et al. proposed that the sensitivity to endocannabinoids varies in the adipose tissue due to the physiological status39. However, a lactation stage-dependent sensitivity to ECS activation could also be present in other tissues. In mid- and late-lactating cows, AEA administration increased short-term feed intake (1 to 10 h) but had no effect on total daily feed intake15,27, probably due to the short half-life of AEA. In early-lactation, AEA administration also showed no effect on total daily feed intake, but, this may be due to the dominating role of leptin. Nevertheless, endocannabinoid concentrations were found to directly correlate with an increase27 or decrease73 in feed intake during early-lactation, suggesting their involvement in the regulation of feed intake when not disturbed by leptin. When cows in late lactation are treated with AEA, they respond with a reduction in plasma NEFA concentration and thus lower lipomobilisation15. In contrast, AEA administration did not reduce lipomobilisation in early-lactation. Consistent with in vitro studies, CNR1 activation did not alter lipolysis rate in adipose tissue explanted from periparturient cows, whereas the lipolysis rate was reduced in adipose tissue collected from non-lactating and non-gestating cows39.
In the liver, we observed downregulation of the mRNA of genes related to TG synthesis, which may lead to less lipid accumulation in this organ. In contrast, in late-lactating dairy cows, AEA administration did not affect the mRNA abundance of genes involved in fat metabolism15. These results suggest a tissue-specific sensitivity to ECS activation depending on the physiological status of the cows, as proposed by Myers et al. 39. However, further research is needed to elucidate the underlying mechanisms.
In conclusion, the present study shows that repeated AEA administration in the first three weeks p.p. did not affect feed intake, energy balance, milk yield or milk composition. Furthermore, repeated AEA administration did not alter lipomobilisation. However, a three-week AEA treatment affected TG synthesis in the liver, underscoring a tissue-specific AEA insensitivity in early-lactation. Chronic elevation of the AEA level after 4 weeks of administration may resulted in a counter regulatory leptin increase, which coincided with a reduction in feed intake and consequently a higher hepatic lipid accumulation, increased whole-body fat oxidation and lower whole-body carbohydrate oxidation. Further investigations are needed to understand the interaction between leptin and AEA in early-lactation.