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Obesity and Metabolism Laboratory, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, the Hebrew University of Jerusalem, Jerusalem, Israel 9112001
Amplified adipose tissue (AT) lipolysis and suppressed lipogenesis characterize the periparturient period of dairy cows. The intensity of lipolysis recedes with the progression of lactation; however, when lipolysis is excessive and prolonged, disease risk is exacerbated and productivity compromised. Interventions that minimize lipolysis while maintaining adequate supply of energy and enhancing lipogenesis may improve periparturient cows' health and lactation performance. Cannabinoid-1 receptor (CB1R) activation in rodent AT enhances the lipogenic and adipogenic capacity of adipocytes, yet the effects in dairy cow AT remain unknown. Using a synthetic CB1R agonist and an antagonist, we determined the effects of CB1R stimulation on lipolysis, lipogenesis, and adipogenesis in the AT of dairy cows. Adipose tissue explants were collected from healthy, nonlactating and nongestating (NLNG; n = 6) or periparturient (n = 12) cows at 1 wk before parturition and at 2 and 3 wk postpartum (PP1 and PP2, respectively). Explants were treated with the β-adrenergic agonist isoproterenol (1 μM) in the presence of the CB1R agonist arachidonyl-2′-chloroethylamide (ACEA) ± the CB1R antagonist rimonabant (RIM). Lipolysis was quantified based on glycerol release. We found that ACEA reduced lipolysis in NLNG cows; however, it did not exhibit a direct effect on AT lipolysis in periparturient cows. Inhibition of CB1R with RIM in postpartum cow AT did not alter lipolysis. To evaluate adipogenesis and lipogenesis, preadipocytes isolated from NLNG cows' AT were induced to differentiate in the presence or absence of ACEA ± RIM for 4 and 12 d. Live cell imaging, lipid accumulation, and expressions of key adipogenic and lipogenic markers were assessed. Preadipocytes treated with ACEA had higher adipogenesis, whereas ACEA+RIM reduced it. Adipocytes treated with ACEA and RIM for 12 d exhibited enhanced lipogenesis compared with untreated cells (control). Lipid content was reduced in ACEA+RIM but not with RIM alone. Collectively, our results support that lipolysis may be reduced by CB1R stimulation in NLNG cows but not in periparturient cows. In addition, our findings demonstrate that adipogenesis and lipogenesis are enhanced by activation of CB1R in the AT of NLNG dairy cows. In summary, we provide initial evidence which supports that the sensitivity of the AT endocannabinoid system to endocannabinoids, and its ability to modulate AT lipolysis, adipogenesis, and lipogenesis, vary based on dairy cows' lactation stage.
Rapid fetal growth, parturition, and the transition into lactation dramatically increase energy requirements in periparturient dairy cows. Adipose tissues (AT) minimize the nutrient gap between dietary intake and energy requirements by releasing fatty acids (FA) stored in their lipid droplets through lipolysis (
), and the risk of metabolic and infectious disease is exacerbated.
In times of excess dietary nutrient intake, AT energy stores are replenished through 2 primary mechanisms: adipogenesis and lipogenesis. Adipogenesis is defined by the determination and differentiation of new adipocytes from adipocyte progenitors. When pre-existing adipocytes exceed their maximum volume (i.e., hypertrophy) during positive energy balance, the formation of new adipocytes enhances the capacity of AT to continue storing lipids. Lipogenesis is the formation of new lipid molecules that are stored in the fat droplets within adipocytes. Synthesis of lipids involves the assembly of glycerol and FA derived from de novo lipogenesis or existent FA released during intracellular or peripheral lipolysis.
Studies in rodents and humans demonstrate that the endocannabinoid system (ECS) has the capacity to modulate adipogenesis and lipid mobilization (i.e., lipogenesis and lipolysis;
). The eCB are products of FA, the 2 most prominent being the n-6 FA arachidonic acid-derived N-arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (
Although eCB bind and interact with many receptor types including members of the cannabinoid (CB), cytochrome P450, vallinoid, and G protein-coupled families, the interactions of eCB with the metabolically active CB receptor 1 (CB1R) are particularly well documented in AT from rodents and humans (
). In these species, eCB enhance adipogenesis and lipogenesis by activating gene and protein networks that accelerate differentiation of adipocyte progenitors and accumulation of lipids in adipocytes (
). Having the potential to reduce the lipolytic drive observed in periparturient cows, regulating the activity of the ECS by modifying CB1R activation may have the potential to improve metabolic function in these animals. Although previous studies have revealed that eCB concentrations in AT are altered based on physiologic status (
), the effects of CB1R activation on lipid mobilization and adipogenesis in dairy cows are currently unknown. The objective of this study was to determine the effect of CB1R activity on lipolysis, adipogenesis, and lipogenesis in dairy cow AT. We determined that CB1R activation enhances the adipogenic and lipogenic activities of AT and reduces lipolysis in nonlactating, nongestating cows, but, in periparturient cows, does not alter intensity of lipolysis. In addition, these results demonstrate that the lactation stage of cows may influence the ability of AT ECS to modulate lipid mobilization and adipogenesis and its sensitivity to activation by eCB.
MATERIALS AND METHODS
Ethics Statement
All animal use described here followed guidelines established by the Institutional Animal Care and Use Committee of Michigan State University (IACUC; no. 11-16-188-00).
Adipose Tissue Collections and Processing
A total of 6 healthy, nongestating, nonlactating (NLNG) Holstein cows were selected at a local abattoir and assigned a body condition score before slaughter by a trained veterinarian. Only animals with a BCS of 3.0 to 4.0 were selected for the study. Animals were euthanized by captive bolt, and internal organs were evaluated to exclude any cows with evidence of intra-abdominal, thoracic, or gastrointestinal disorders. Then, following skin removal, subcutaneous AT samples were collected from the right paralumbar fossa and further processed for lipolysis assays.
A total of 12 healthy periparturient multiparous Holstein cows from the Michigan State University Dairy Teaching and Research Center (Lansing, MI) were used for this study. At the moment of selection, cows were nonlactating and pregnant (210–240 d of gestation). Cows were blocked by BCS (0.5-point BCS scale), assessed weekly by 3 trained investigators (
). Only animals with a BCS of 3.0 to 3.75 were selected, along with previous-lactation 305-d mature-equivalent yield of 14,500 kg (within 3,150 kg), and parity (up to 1 lactation difference). The values (mean ± SD) for BCS, mature-equivalent yield, and parity were 3.51 ± 0.03, 14,520 ± 119.3 kg, and 3.27 ± 0.19, respectively. Cows were housed in tiestalls bedded with sawdust. All animals received close-up (−21 d relative to parturition) and fresh (1–24 d into lactation) diets that were formulated to meet or exceed nutritional requirements defined by the
. Subcutaneous AT samples were obtained from the right paralumbar fossa at 13 ± 5.1 d before the expected calving date (PreP), and at 14.4 ± 1.9 d and 21 ± 1.9 d postpartum (PP1 and PP2, respectively) using the surgical procedure described by
. A total of 5 g of AT were collected from each cow at PreP, PP1, and PP2, and the biopsy incision was closed using a continuous interlocking suture with Braunamid (USP1, Aesculap). Sutures were removed 12 to 14 d after each procedure.
Ex Vivo Lipolysis Assays
Immediately after collection, 25 g of AT was placed in Krebs-Ringer bicarbonate HEPES buffer (pH 7.4) and transported to the laboratory at 37°C. The AT was minced, and ∼100 mg of tissue fragments was transferred into each well of a 24-well plate containing 1 mL of Krebs-Ringer bicarbonate HEPES buffer supplemented with 3% FA-free BSA (cat. no. A8806-5G, Millipore-Sigma). Explant weight was determined precisely using a high-precision scale (Mettler Toledo). Culture plates with AT explants were pre-incubated at 37°C for 20 min on a shaker. Next, reagents were prepared fresh and added to the culture plates as will be described.
In all lipolysis experiments, basal lipolysis was established without the addition of any reagent. Explants exposed for 3 h to the β-adrenergic agonist isoproterenol (ISO, cat. no. I6504, Millipore-Sigma, 1 μM) were used as a positive control for lipolysis. Glycerol release into the culture medium using free glycerol reagent (Millipore-Sigma, cat. no. F6428) was used to quantify lipolysis. Medium samples (67 µL), blank (Krebs-Ringer bicarbonate HEPES buffer + 3% BSA), and standards (glycerol standard solution, cat. no. G7793, Sigma-Aldrich) were loaded in duplicate into a 96-well plate, and 200 µL of free glycerol reagent was added. After 5 min of incubation at 37°C, optical density was measured at 540 nm. Glycerol release was normalized by the weight of each AT explant (nmol per mg of AT).
The effects of the CB1R agonist arachidonyl-2′-chloroethylamide (ACEA, cat. no. 91054 Cayman Chemical) on the AT lipolytic response were first determined in AT explants from NLNG cows to establish an experimental dose range. The analog ACEA was chosen over its natural counterpart, anandamide, due to its comparatively greater stability and specificity for the CB1R (
Determination of the endocannabinoids anandamide and 2-arachidonoyl glycerol with gas chromatography-mass spectrometry: Analytical and preanalytical challenges and pitfalls.
). The AT explants from NLNG cows were treated with 0, 0.035, 0.09, 0.18, 0.35, and 0.7 µg of ACEA/mg of AT under basal and ISO-stimulated conditions. We added ACEA 1 h before ISO. Results are expressed as nmol glycerol per milligram of AT.
Next, we determined the effect of CB1R inhibition on lipolysis using the selective CB1R antagonist rimonabant (RIM, cat. no. 9000484, Cayman Chemical; dissolved in dimethyl sulfoxide). The AT explants from NLNG cows were incubated for 3 h with basal, ISO, ACEA+ISO, RIM+ISO, and RIM+ACEA+ISO. We added RIM 1 h before ACEA, to ensure enough time was allotted to block the CB1R. After 3 h of incubation, AT explants and medium samples were collected for glycerol quantification or snap-frozen in liquid nitrogen (N2) and stored at −80°C until further analysis. Based on the results in NLNG cow AT (Figures 1A and 1B), 0.7 μg of ACEA/mg of AT and 27 µM RIM were selected for use in the remaining explant experiments.
Figure 1Cannabinoid-1 receptor (CB1R) activation alters adipose tissue (AT) lipolysis in nonlactating, nongestating (NLNG) but not in periparturient cows. (A) Glycerol released by subcutaneous AT explants from NLNG cows exposed to no reagents (Basal), isoproterenol (ISO; 1 µM), in the presence of arachidonyl-2′-chloroethylamide (ACEA; 0, 0.035, 0.18, 0.35, 0.7 µM) for 3 h; n = 6. (B) Basal and ISO glycerol release from NLNG cow AT explants in the presence of ACEA (0.7 µg/mg AT), rimonabant (RIM; 0, 0.27, 2.7, 27 µM), and ACEA+RIM. (C) Basal glycerol release from AT collected from periparturient cows after 3 h of basal or ISO stimulation; n = 12. PreP = prepartum; PP1 = 2 wk postpartum; PP2 = 3 wk postpartum. (D) Glycerol released from PreP, PP1, and PP2 AT samples exposed to basal and ISO for 3 h in the presence of ACEA (0.7 µg/mg AT), RIM (27 µM), and ACEA+RIM; n = 12. (E) Glycerol released from AT from periparturient cows exposed to basal and ISO for 3 h in the presence of ACEA (0.7 µg/mg AT), RIM (27 µM), and ACEA+RIM; n = 12. Values are LSM of glycerol nmol/mg of AT. Error bars represent the SEM. Bars with * or different lowercase letters (a–d) vary significantly (P < 0.05).
In periparturient cows, we compared ISO-induced lipolysis between PreP, PP1, and PP2 AT explants to determine the effects of sample collection times relative to calving on lipid mobilization. Following, lipolysis was quantified in periparturient AT samples exposed to basal and ISO with or without ACEA ± RIM.
Cell Isolation and Culture
Preadipocytes were harvested from AT obtained from the right paralumbar fossa region of multiparous NLNG Holstein cows (n = 5) as described previously (
). Tissue samples were placed in 30 mL of filtered Krebs-Ringer bicarbonate buffer containing NaCl 135 mM; KCl 5 mM; MgSO4 1 mM; KH2PO4 0.4 mM; glucose 5.5 mM; HEPES 20 mM (pH 7.4; Teknova, cat. no. H1030) supplemented with 100 units/mL of penicillin; 100 µg/mL of streptomycin, 0.25 µg/mL of amphotericin B, and 50 µg/mL of gentamicin. Upon arrival to the laboratory, approximately 50 mg of AT were finely minced using sterile surgical scissors into 1- to 2-mm3 pieces. Tissue fragments were then transferred into 24-well plates, and 5 mm were left between each piece. After 10 min of incubation, 200 µL of MesenPro RS Complete Medium (Thermo Fisher Scientific, cat. no. 12746012) was carefully added, and tissue fragments were incubated at 37°C. Medium was changed every 48 h until preadipocytes migrated from within the tissue and reached confluency on the plate surface, at which point they were passed into flasks for expansion. Upon passage to flasks and as described by
, preadipocyte medium containing 10% fetal bovine serum, Dulbecco's modified Eagle's medium/F12, 44.05 mM sodium bicarbonate (Corning, cat. no. 61-065-RO), 100 µM ascorbic acid (Sigma-Aldrich, cat. no. A4544-100G), 33 µM biotin (Sigma-Aldrich, B4501-1G), 17 µM pantothenate (Sigma-Aldrich, P5155-100G), 1% l-glutamine (Gibco, cat. no. 25030-081), 1 µg/mL amphotericin (Sigma-Aldrich, cat. no. A-2942), 10 µg/mL ampicillin (Sigma-Aldrich, cat. no. A0166-5G), and 20 mM HEPES was used for outgrowth of preadipocytes, with replacement every 2 d.
In Vitro Adipogenesis and Lipogenesis Experiments
Following 2 to 3 serial passages, preadipocytes were seeded in 6-, 12-, and 96-well plates at a density of 20,000 cells/cm2. Upon confluency, preadipocyte medium was removed and replaced with induction (adipocyte) medium with treatments. The adipocyte medium contained 10 mg/mL insulin, 1 µM octanoate, 10 mM acetate, 10 µg/mL transferrin, 5 µM troglitazone, 1 µM 3-Isobutyl-1-methylxanthine, and 0.5 µM dexamethasone and was supplemented with 300 µM of a 60% palmitic acid:40% oleic acid mixture prepared as indicated in (
). Treated cells were pre-incubated for 1 h at 37°C in adipocyte medium with 0.1 µM RIM or vehicle. Following pre-treatment, ACEA in adipocyte medium was added to achieve well concentrations of 10 µM, or an equivalent volume of adipocyte medium ± 0.1 µM RIM was added. After 48 h, treatments and medium were replaced at 60% of the well volume with fresh 0.1 µM RIM ± 10 µM ACEA in maintenance medium and incubated for an additional 48 h. Cell viability after 48 h and at every collection time point was evaluated using a cell viability/cytotoxicity kit according to the manufacturer's protocol (cat. no. 30002, Biotium;
). Adipocyte viability was unaffected by treatments and vehicles (Figure 2C).
Figure 2Cannabinoid-1 receptor (CB1R) activation enhances adipogenesis in dairy cow adipocytes. (A) Experimental timeline. (B) Representative images of adipocytes (n = 5) cultured for 4 d in the presence of adipocyte medium, arachidonyl-2′-chloroethylamide (ACEA; 10 µM), rimonabant (RIM; 0.1 µM), and ACEA+RIM. Lipid droplets were stained with Bodipy (Thermo Fisher; green) and nuclei with NucRed (Biotium; red). Scale bar = 400 µm; n = 5. (C) Cell viability of preadipocytes treated with adipocyte medium, vehicle controls [methyl acetate, dimethyl sulfoxide (DMSO)], ACEA, ACEA+RIM, and RIM for 96 h. Values are percent of cells viable, as measured with the Calcein AM viability assay (Biotium); n = 5. Dots represent individual data points. Error bars indicate SEM. Significant differences are denoted by * (P < 0.05). (D) Adipogenic efficiency as calculated by the IncuCyte imager (Sartorius; number of cells with 1 or more lipid droplets over total number of cells per well); n = 5. Dots indicate individual data points. Error bars indicate SEM. Significant differences are denoted by a, b, c (P < 0.05). (E) Relative gene expression of PPARG, SCD1, FASN, FAAH, CNR1, and CNR2. Values are relative mRNA abundance after normalization with the reference genes EIF3K and RPS9. Dots represent individual data points. Significant differences are indicated by lowercase letters (a–c); n = 5; P < 0.05. (F) PPARγ abundance in adipocytes treated 496 with adipocyte medium, ACEA, ACEA+RIM, and RIM, as measured by capillary electrophoresis. Dots represent individual data points. Bars are the LSM of PPARγ expression relative to adipocyte values following normalization with vinculin. Significant differences are indicated by lowercase letters (a, b); n = 5; P < 0.05.
After 96 h of induction, adipogenesis was evaluated using the live cell imaging IncuCyte S3 system (Sartorius). Cells were stained with Bodipy 493/503, a neutral lipid stain (10 µM; cat. no. D3922, Thermo Fisher Scientific) and the nuclear stain NucSpot Live 650 (1×; Biotium, cat. no. 40082). Images were analyzed using the IncuCyte ZOOM software (version 2018A). Adipogenesis is reported as Bodipy fluorescence intensity/nuclei count.
For lipogenesis experiments, cells were cultured in maintenance medium for 48 h following induction. Next, medium was replaced at 60% of the well volume with fresh, treated maintenance medium to reach well concentrations of 0.1 µM RIM ± 10 µM ACEA. Cells were incubated for a total of 14 d post-induction, with 60% medium changes every 48 h. Fresh ACEA and RIM treatments were included in all medium changes. Lipogenesis was quantified in 96-well plates using the AdipoRed assay (cat. no. PT-7009, Lonza) and a Synergy H1 Microplate Reader (Biotek) as described by
On the day of collection, culture medium was removed, and cells were rinsed twice with ice-cold 1× PBS before the addition of 100 μL of RNA lysis buffer (cat. no. R1060-1-100, Zymo Research) to each well. Wells containing cells treated in duplicate were combined into 0.5-mL microfuge tubes, flash-frozen using liquid N2, and stored at −80°C until further analysis. Just before extractions, samples were thawed on ice. RNA was extracted using the Promega simplyRNA Cells Kit (cat. no. AS1390) in the Maxwell RSC Instrument (Promega) with some modifications to the protocol previously described (
). A total of 200 μL of each sample was transferred into a microfuge tube along with 200 μL of 1-thioglycerol/homogenization solution. Next, homogenate was vortexed and was added to Maxwell RSC cartridges previously loaded with 10 μL of DNase I. RNA was eluted by the addition of 50 μL of nuclease-free water. RNA purity and concentration were determined using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). All samples had a 260/280 absorbance ratio >2. Conversion to cDNA was performed using the qScript cDNA SuperMix (cat. no. 95048-025, Quantabio). The protocol began by combining 4 μL of the qScript cDNA SuperMix, ∼500 ng of suspended template RNA, and variable volumes of RNase- and DNase-free water to reach a volume of 20 μL in microfuge tubes. Next, tubes were sealed, vortexed, and centrifuged at 300 × g for 10 min at 21°C. Samples were then incubated for 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C, and held at 4°C. Resulting cDNA was then diluted to a concentration of 5 ng/μL in RT-PCR-grade water. Transcriptional studies were performed using the SmartChip real-time PCR system as described in
. SYBR gene expression primers for quantitative PCR assays were used and were either commercially available or designed from bovine sequences and synthesized by Sigma-Aldrich or Integrated DNA Technologies (Coralville, IA; Supplemental Table S1; https://doi.org/10.6084/m9.figshare.21587370.v1;
). Samples were assayed in duplicate. Each 100-nL PCR reaction contained 1× LightCycler 480 SYBR Green Master Mix (Roche), 200 nM primer assays, and 5 ng/μL of sample cDNA. A non-template control and non-reverse transcriptase control were used to monitor contamination and primer-dimer formation that could produce false-positive results and confirm the absence of genomic DNA. The following cycling conditions were used on the Wafergen SmartChip real-time PCR system: initial enzyme activation at 95°C for 10 min, 45 cycles of denaturation at 95°C for 10 sec, and annealing at 60°C for 53 sec.
Gene expression data of 3 endogenous control genes (EIF3K, RPL9, and RPS9) were analyzed using qBase+ analysis software (version 3.1, Biogazelle), which calculates the stability of endogenous control genes (M-value). Following qBase+ analysis of gene expression data, the endogenous control genes EIF3K and RPS9 were ranked best. The quantification cycle values of the target genes (ABHD5, AGPAT2, CNR1, CNR2, FAAH, FASN, LIPE, LPIN1, LPIN3, PPARG, and SCD1; Supplemental Table S1) were converted to normalized relative gene expression as described previously (
To harvest protein from cultured cells, medium was flicked off, wells were rinsed twice with ice-cold 1× PBS, and 200 μL of protein extraction buffer containing radio immunoprecipitation assay buffer (Teknova, cat. no. R3792) supplemented with cOmplete mini EDTA-free protease inhibitor (Roche Diagnostics, cat. no. 11873580001) and phosphatase inhibitors (phosphatase inhibitor cocktail II, cat. no. J61022, Thermo Fisher Scientific) was added to each well. Adherent cells were lifted using cell scrapers, and the resulting suspensions were pipetted into microfuge tubes; cells treated in duplicate were combined. Samples were flash-frozen using liquid N2 and stored at −80°C until further analysis. Protein concentrations were quantified using the Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, cat. no. 23225). Optimal protein concentrations varied by antibody as established in the 12- to 230-kDa Wes Separation Module capillary cartridges of the Simple Protein Wes system (cat. no. SM-W004, ProteinSimple;
): 0.5 mg/mL for perilipin-1 and 0.75 mg/mL for PPARγ and CB1R antibodies. Antibodies and dilutions were as follows: rabbit polyclonal anti-perilipin-1 (1:50; cat. no. PA5-72921, Thermo Fisher Scientific), anti-PPARγ (1:25; cat. no. 2435, Cell Signaling Technology), anti-CB1R (1:25; cat. no. ab23703, Abcam), mouse monoclonal anti-vinculin (1:100; cat. no. sc-73614, Santa Cruz Biotechnology). The anti-rabbit module for the Wes kit (cat. no. DM-001, ProteinSimple) that included luminol-S, peroxide, antibody diluent 2, streptavidin-HRP, and anti-rabbit secondary antibody was used for detection of perilipin-1, PPARγ, and CB1R. For vinculin detection, the anti-mouse detection module was used (cat. no. DM-002, Bio-Techne). Microcapillary electrophoresis was used to separate sample proteins, and chemiluminescence signal peaks were generated for analysis. Once transformed into digital images depicting bands, the signal peaks were analyzed using Compass software (version 4.1.0; ProteinSimple). The peak areas of CB1R, PPARγ, and perilipin-1 proteins were estimated and normalized against the reporter protein vinculin. As further described by
, the peak areas correspond directly to the target protein quantities. Normalized CB1R, PPARγ, and perilipin-1 data are expressed as relative increase versus adipocyte or maintenance for adipogenesis and lipogenesis, respectively.
Statistical Analysis
Statistical analyses were performed using the Proc Mixed program from SAS version 9.4 (SAS Institute Inc.). The normality of the variables was checked using the Shapiro-Wilk test (P < 0.05). Residuals of the models were checked and found to be normally distributed. Nonsignificant (P > 0.05) 2-way interactions were removed from the model. Tukey's post-hoc adjustment test was used for pairwise comparisons. For AT explant experiments, time relative to parturition (PreP, PP1, and PP2) was included as a repeated measure using a mixed model with the first-order autoregressive covariance structure. The random effects included cows and time. The effect of time of periparturient sampling was significant in the model. For in vitro experiments, one-way ANOVA pairwise comparisons were performed using Tukey's post-hoc test. Mean differences were considered significant when P ≤ 0.05 and tendencies when P < 0.10. Average values throughout the text and figures are shown as mean ± standard error of the mean.
RESULTS
CB1R Activation by ACEA Alters AT Lipolysis in Dairy Cows
We treated AT explants from NLNG cows exposed to basal and ISO with 0.035, 0.09, 0.18, 0.35, and 0.7 µg ACEA/mg AT; in these animals, ACEA at 0.7 µg/mg AT reduced ISO-induced lipolysis but not at lower doses (Figure 1A; P = 0.03). The CB1R agonist did not affect basal lipolysis (Figure 1A; P = 0.82). Based on these results, we selected 0.7 µg/mg AT ACEA as our working dose for the remaining AT explant experiments. Next, we treated the AT explants with the selective CB1R antagonist RIM (0.27, 2.7, 27 µM) under basal and ISO conditions with or without ACEA. We found that NLNG cow AT lipolysis was highest when exposed to ISO and 27 µM RIM (Figure 1B; P < 0.05); based on these results, 27 µM RIM was selected for the periparturient cow AT experiments. During ISO stimulation, ACEA failed to reduce lipolysis below that of RIM-treated explants (Figure 1B; P = 0.88). The combination of ACEA and RIM, however, reduced ISO-induced lipolysis, and this effect was greatest with 27 µM RIM (Figure 1B; P < 0.05).
In the AT of periparturient cows, ISO-induced lipolysis was higher at PreP compared with PP1 and PP2 (Figure 1C; P < 0.0001). Among the periparturient AT samples, and as expected, basal glycerol release was lower than ISO (Figures 1D and 1E; P < 0.05). We found that CB1R stimulation with ACEA in periparturient cow AT did not reduce ISO-induced lipolysis (Figures 1D and 1E; P = 0.99). Inhibition of CB1R signaling with RIM did not affect the intensity of ISO-induced lipolysis at any periparturient sampling point (Figures 1D and 1E; P = 0.89).
CB1R Activation by ACEA Enhances Adipogenesis in Dairy Cow Adipocytes
Preadipocytes from 5 NLNG cows were induced to differentiate into adipocytes (adipogenesis) in the presence of ACEA (10 µM) and RIM (0.1 µM), and cells were collected at d 4 post-induction (Figure 2A). Exposure to vehicles, ACEA, and RIM for 96 h did not affect the viability of preadipocytes and adipocytes (Figure 2C; P < 0.05). Treatment with ACEA enhanced adipogenesis compared with adipocyte medium alone (Figure 2B and 2D; P < 0.05). Both RIM and ACEA+RIM reduced adipogenic efficiency in adipocytes to levels comparable to those observed in cells exposed to adipocyte medium alone (Figure 2D; P < 0.05). Cells treated with adipocyte medium increased the transcription of key adipogenic genes including PPARG (peroxisome proliferator-activated receptor γ), SCD1 (stearoyl-coenzyme A desaturase 1), and FASN (fatty acid synthase) in comparison to those cultured in preadipocyte medium (Figure 2E; P < 0.05). We further found that ACEA enhanced the transcription of PPARG compared with adipocyte medium alone. Both ACEA+RIM and RIM reduced PPARG expression to levels similar to those of adipocyte medium (Figure 2E; P < 0.05). The transcription of SCD1 in cells treated with ACEA increased compared with those in adipocyte medium, RIM, and ACEA+RIM (Figure 2E; P < 0.05). Compared with RIM, expression of FASN was reduced in cells treated with adipocyte medium or ACEA (Figure 2E; P < 0.05). At d 4, the transcription of FAAH (fatty acid amide hydrolase), an eCB-degrading enzyme, was reduced in adipocyte medium and ACEA compared with preadipocyte medium and RIM (Figure 2E; P < 0.05). Expressions of the genes encoding CB1R (CNR1) and CB2R (CNR2) were not affected by any of the treatments (Figure 2E; P > 0.1). Additionally, ACEA increased the relative abundance of PPARγ compared with all other treatments (Figure 2F; P < 0.05).
CB1R Activation Enhances Lipogenesis in Dairy Cow Adipocytes
To evaluate the lipogenic capacity of cells under continuous CB1R activation, adipocytes and preadipocytes from 5 NLNG cows were exposed to ACEA and RIM for 12 d following the first 48 h of induction. We found that ACEA increased the triglyceride (TG) content compared with ACEA+RIM, RIM, and maintenance medium (Figure 3A and B; P < 0.05). Lipid contents in ACEA+RIM and RIM were greater than in those cells cultured in maintenance medium (Figure 3A and B; P < 0.05). Expression of lipogenesis-related genes revealed that ACEA enhanced the transcription of AGPAT2 compared with ACEA+RIM, RIM, and maintenance medium (Figure 3C; P < 0.05). We observed no treatment effect on the expression of SCD1, lipin 1 (LPIN1) and 3 (LPIN3), or FASN (Figure 3C; P < 0.05). Continuous stimulation of CB1R with ACEA or RIM did not affect the expression of FAAH. Similarly, we detected no treatment effect on the transcription of the key lipolytic genes LIPE (hormone-sensitive lipase) and ABHD5 (abhydrolase domain-containing 5, lysophosphatidic acid acyltransferase; Figure 3C; P < 0.05). Reflecting greater TG accumulation, the content of perilipin-1 increased in ACEA compared with maintenance (Figure 3D; P < 0.05). We found that RIM reduced perilipin-1 content compared with maintenance, ACEA, and ACEA+RIM (Figure 3D; P < 0.05). Likewise, CB1R abundance was reduced in RIM in contrast to all other treatments (Figure 3D; P < 0.05). The expression of CB1R was elevated in ACEA compared with maintenance, but the levels did not differ significantly from the combination of ACEA+RIM (Figure 3D; P < 0.05).
Figure 3Cannabinoid-1 receptor (CB1R) activation enhances lipogenesis in dairy cow adipocytes. (A) Representative images of adipocytes cultured for 12 d in the presence of maintenance medium, arachidonyl-2′-chloroethylamide (ACEA; 10 µM), rimonabant (RIM; 0.1 µM), or ACEA+RIM. Lipid droplets were stained with Bodipy (Thermo Fisher; green) and nuclei with NucRed (Biotium; red). Scale bar = 400 µm; n = 5. (B) Lipogenic efficiency of adipocytes treated with maintenance medium, vehicle controls [methyl acetate, dimethyl sulfoxide (DMSO)], or CB1R ligands as calculated using the Adipored assay [Lonza; reported as relative fluorescence units (RFU) per ng dsDNA; n = 5]. Dots represent individual data values. Bars indicate mean adipogenic efficiency ± SEM. Lowercase letters (a–c) indicate significant differences (P < 0.05). (C) Expression of AGPAT2, SCD1, LPIN1, LPIN3, FASN, FAAH, LIPE, and ABHD5. Dots represent individual data points. Bars are relative mRNA abundance after normalization with the reference genes EIF3K and RPS9 (P < 0.05). (D) Perilipin-1 abundance in adipocytes treated with maintenance, ACEA, ACEA+RIM, and RIM for 12 d as measured by capillary electrophoresis. Dots represent individual data points. Bars are the LSM of protein expression relative to maintenance values ± CI. Bars with different lowercase letters (a–c) are different (n = 5; P < 0.05).
Endocannabinoid signaling through CB1R modulates lipid metabolism by reducing lipolysis and enhancing the accumulation of lipids in AT of rodents and humans. In this study we demonstrate that, in dairy cows, CB1R activation with ACEA modulates AT lipolysis in a physiologic state-dependent manner. In NLNG cows, ACEA reduced lipolysis; however, in peripartum dairy cows, no anti-lipolytic response was observed. Similarly, stimulation of CB1R activity in NLNG dairy cows' preadipocytes and adipocytes with ACEA increases adipogenesis and lipogenesis.
Lipolytic responses in AT are sensitive to changes in dairy cows' metabolic status and to the quantity of TG stored in the lipid droplets of adipocytes (
). Basal and stimulated lipolysis per adipocyte is higher during nonlactating stages and, in the majority of cows, is reduced after parturition and tapers gradually until cows are in positive energy balance (
Changes in lipid metabolism and β-adrenergic response of adipose tissues of periparturient dairy cows affected by an energy-dense diet and nicotinic acid supplementation.
). Our results coincide with these studies and show that intensity of lipolysis is higher in AT from PreP cows and is reduced as lactation progresses (PP1 and PP2).
Data from this study demonstrate that the AT lipolytic response is altered by CB1R activation in NLNG dairy cows, as ACEA reduced ISO-induced lipolysis. These results correspond with observations in AT from humans and rodents, in which CB1R activation reduced the production of cAMP (
). By reducing intracellular cAMP, the activation of hormone-sensitive lipase, the enzyme responsible for limiting the rate of lipolysis, is inhibited, and therefore lipolysis is reduced (
). In contrast to our findings in NLNG cows, we did not observe ACEA's anticipated anti-lipolytic effects in AT from periparturient cows. This lack of response to CB1R stimulation may be related to the reductions in circulating insulin concentrations and signaling impairment within adipocytes of periparturient cows—important homeorhetic mechanisms that promote the release of stored energy from AT and are necessary to meet periparturient energy demands (
). Combined with the results of our present study, these findings suggest that the anti-lipolytic effects of eCB, which are dependent on insulin signaling through IRS/Akt, may be limited around the time of calving.
The AT of peripartum cows may be more resistant to effects associated with ECS activation than that of NLNG cows. The notion of ECS (CB1R) insensitivity in peripartum cow AT is evidenced by our present experimental results, in which CB1R failed to modulate lipolysis, and further supported by results reported by
Effects of omega-3 supplementation on components of the endocannabinoid system and metabolic and inflammatory responses in adipose and liver of peripartum dairy cows.
in vivo. In their study, suppression of AT ECS activity in postpartum cows by supplementation with α-linolenic acid, a FA that lowers arachidonic acid content in fat and plasma, did not reduce lipolysis intensity in AT. Additionally, limited ECS activity in cows supplemented with α-linolenic acid did not affect body weight loss, plasma nonesterified FA, or energy balance peripartum. This apparent resistance to AT ECS activation in periparturient cows may result from variations in the sensitivity of CB1R to eCB, receptor expression and sensitivity, enzymatic activity and profile, or degree of local AT and systemic inflammation, although these areas are yet to be explored in detail.
Enhancing adipogenesis in AT may reduce dyslipidemia in dairy cows—characterized by high plasma free fatty acids. The mechanism would involve a higher capacity to store free fatty acids by increasing the number of adipocytes in AT (
). Our results demonstrate that activating CB1R with ACEA enhances the efficiency of adipogenesis and the transcription of the key adipogenic genes encoding PPARγ and SCD1. These results coincide with studies in monogastrics that show a strong activation of the master regulator of adipogenesis, PPARγ, upon activation of CB1R with eCB (i.e., anandamide) and synthetic CB such as ACEA (
). Enhanced activity of SCD1 upon CB1R activation with eCB or synthetic CB (i.e., ACEA) is a well-characterized response in monogastric hepatocytes and adipocytes (
). Products of SCD1 reduce the transcription and activity of FAAH and therefore amplify the adipogenic effect of CB1R activation by reducing the breakdown of eCB (
). To our knowledge, this is the first study to evaluate the role of the ECS on lipolysis, adipogenesis, and lipogenesis in bovine adipocytes. Results from this project indicate that the same effect is observed in ruminants as ACEA increases TG accumulation in differentiated NLNG dairy cows' adipocytes. The response is driven by stimulation of PPARγ, which activates the machinery for FA synthesis and TG assembly. We determined that the expression of AGPAT2 is increased upon CB1R activation. AGPAT2 is one of the rate-limiting enzymes for the assembly of TG, and its activation promotes strong lipogenic responses in bovine adipocytes (
). AGPAT2 catalyzes the acylation of lysophosphatidic acid with acyl-CoA FA. The resulting products, phosphatidic acids, are then metabolized into diglycerides in the endoplasmic reticulum (
). Our results show that the expression of CB1R on adipocytes is upregulated by ACEA and downregulated by RIM during lipogenesis but not during adipogenesis. These findings suggest autoregulatory capabilities of CB1R; however, the abundance of receptors appears to vary based on the stimulation provided and the developmental stage of the adipocyte. The mechanisms behind the differential expression and autoregulation of CB1R during adipogenesis and lipogenesis remain to be revealed.
Limitations exist for this set of experiments. First, ACEA, although at considerably lower affinities and efficacies, is capable of binding and activating other ECS receptors (
The anti-inflammatory mediator palmitoylethanolamide enhances the levels of 2-arachidonoyl-glycerol and potentiates its actions at TRPV1 cation channels.
). Second, the half-lives of ACEA and RIM are presumably longer than those of natural eCB (i.e., anandamide, 2-arachidonoylglycerol) and related compounds, but, with this in mind, the dynamics of eCB synthesis and degradation cannot be mimicked under in vitro or ex vivo conditions as they occur in vivo (
). In the same way, eCB and synthetic analog concentrations used for our experiments and those measured, administered, and reported in previous studies likely vary from true physiologic concentrations based on tissue, handling conditions, species, and assays used (
). In addition to these considerations, CB1R stimulation may exert various AT depot-specific effects, including alterations in lipid mobilization and adipogenesis (
Cannabinoid type 1 receptor mediates depot-specific effects on differentiation, inflammation and oxidative metabolism in inguinal and epididymal white adipocytes.
). To better characterize these interactions as they pertain to the AT ECS in peripartum dairy cows, further studies are required.
CONCLUSIONS
Stimulation of the CB1R enhances energy conservation in NLNG dairy cows by enhancing adipogenesis and lipogenesis in vitro and reducing lipolysis ex vivo in AT. In periparturient cows, however, AT lipolysis is not altered by CB1R activation or inhibition. Based on our study's findings, the capacity of CB1R to modulate lipid mobilization in AT varies between periparturient and NLNG cows. Furthermore, these results suggest physiologic state-dependent differences in the sensitivity of the AT ECS to activation by eCB, which may shift transiently to compensate for disparities between nutrient intake and energy expenditure throughout the productive cycle of dairy cows. Additional research is required to reveal the complex interactions between the AT ECS, AT lipid mobilization, and the physiologic status of cows, and to define targetable pathways that exhibit potential to improve cow health, reproductive and milking efficiency, and overall sustainability of the dairy industry.
ACKNOWLEDGMENTS
This project was funded by the US-Israel Binational Agricultural Research and Development Fund (Rishon LeZion, Israel) Grant IS-5167-19, Michigan Alliance for Animal Agriculture (East Lansing, MI) project AA18-006, and the United States Department of Agriculture National Institute for Food and Agriculture (Washington, DC) Competitive Project 2019-67015-29443. Madison Myers was supported in part by NIH Grant 5T35OD016477-19 to Michigan State University (East Lansing, MI), the United States Department of Agriculture National Institute for Food and Agriculture's Agriculture and Food Research Initiative Project 2021-67037-34657, and the Office of the Associate Dean for Research and Graduate Studies of the Michigan State University College of Veterinary Medicine. The authors have not stated any conflicts of interest.
REFERENCES
Buch C.
Muller T.
Leemput J.
Passilly-Degrace P.
Ortega-Deballon P.
Pais de Barros J.-P.
Vergès B.
Jourdan T.
Demizieux L.
Degrace P.
Endocannabinoids produced by white adipose tissue modulate lipolysis in lean but not in obese rodent and human.
Changes in lipid metabolism and β-adrenergic response of adipose tissues of periparturient dairy cows affected by an energy-dense diet and nicotinic acid supplementation.
Effects of omega-3 supplementation on components of the endocannabinoid system and metabolic and inflammatory responses in adipose and liver of peripartum dairy cows.
Determination of the endocannabinoids anandamide and 2-arachidonoyl glycerol with gas chromatography-mass spectrometry: Analytical and preanalytical challenges and pitfalls.
The anti-inflammatory mediator palmitoylethanolamide enhances the levels of 2-arachidonoyl-glycerol and potentiates its actions at TRPV1 cation channels.
Cannabinoid type 1 receptor mediates depot-specific effects on differentiation, inflammation and oxidative metabolism in inguinal and epididymal white adipocytes.
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