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State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin Province, 130062, China
Activated autophagy-lysosomal pathway (ALP) can degrade virtually all kinds of cellular components, including intracellular lipid droplets, especially during catabolic conditions. Sustained lipolysis and increased plasma fatty acids concentrations are characteristic of dairy cows with hyperketonemia. However, the status of ALP in adipose tissue during this physiological condition is not well known. The present study aimed to ascertain whether lipolysis is associated with activation of ALP in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes. In vivo, blood and subcutaneous adipose tissue (SAT) biopsies were collected from nonhyperketonemic (nonHYK) cows [blood β-hydroxybutyrate (BHB) concentration <1.2 mM, n = 10] and hyperketonemic (HYK) cows (blood BHB concentration 1.2–3.0 mM, n = 10) with similar days in milk (range: 3–9) and parity (range: 2–4). In vitro, calf adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30–40 kg) were differentiated and used for (1) treatment with lipolysis inducer isoproterenol (ISO, 10 µM, 3 h) or mammalian target of rapamycin inhibitor Torin1 (250 nM, 3 h), and (2) pretreatment with or without the ALP inhibitor leupeptin (10 μg/mL, 4 h) followed by ISO (10 µM, 3 h) treatment. Compared with nonHYK cows, serum concentration of free fatty acids was greater and serum glucose concentration, DMI, and milk yield were lower in HYK cows. In SAT of HYK cows, ratio of phosphorylated hormone-sensitive lipase to hormone-sensitive lipase, and protein abundance of adipose triacylglycerol lipase were greater, but protein abundance of perilipin 1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC) was lower. In addition, mRNA abundance of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), protein abundance of lysosome-associated membrane protein 1, and cathepsin D, and activity of β-N-acetylglucosaminidase were greater, whereas protein abundance of sequestosome-1 (p62) was lower in SAT of HYK cows. In calf adipocytes, treatment with ISO or Torin1 decreased protein abundance of PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes, but increased glycerol content in the supernatant of calf adipocytes. Moreover, the mRNA abundance of ATG5, ATG7, and MAP1LC3B was upregulated, the protein abundance of lysosome-associated membrane protein 1, cathepsin D, and activity of β-N-acetylglucosaminidase were increased, whereas the protein abundance of p62 was decreased in calf adipocytes treated with ISO or Torin1 compared with control group. Compared with treatment with ISO alone, the protein abundance of p62, PLIN1, and CIDEC, and triacylglycerol content in calf adipocytes were higher, but the glycerol content in the supernatant of calf adipocytes was lower in ISO and leupeptin co-treated group. Overall, these data indicated that activated ALP is associated with increased lipolysis in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes.
During the transition period, high-yielding dairy cows often experience a state of negative energy balance (NEB) induced by the decline in DMI (approximately 30%;
). In mammals, periods of excess supply of energy allow adipose tissue to serve as a major site of energy storage in the form of triacylglycerols (TG). In contrast, when there is a shortfall in the supply of dietary energy, the TG are hydrolyzed to provide fatty acids, which can be oxidized by peripheral organs (
). Up to 25% of circulating fatty acids are removed by the liver and subsequently oxidized, re-esterified, or metabolized into ketone bodies in hepatocytes (
), which can have an economic effect on the herd, decreasing milk production and increasing the risk of other postpartum disorders such as fatty liver, metritis, and displaced abomasum (
Lysosomes are acidic cytosolic vesicles that can sense intracellular nutrient availability and hydrolyze macromolecules to contribute to energy homeostasis during periods of energy shortfall (
). Remarkably, the rate-limiting enzymes adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL) accounted for more than 90% of TG hydrolase activity in white adipose tissues of mice (
Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis.
Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis.
). Thus, neutral lipases-mediated degradation of lipid droplet plays an essential role in the adipose tissues. In recent years, an alternative pathway of lipid droplet degradation through ALP has been described and termed lipophagy (
). This process, initially implicated in hepatocyte, have now been complemented by study on adipose tissue in autophagy-related 7 (ATG7) knockout mice (
). In addition, the fact that blockage of autophagy lessened isoproterenol (ISO) or starvation-induced lipolysis confirmed the importance of ALP in lipolysis of adipocytes (
Inhibition of cell death inducing DNA fragmentation factor-α-like effector c (CIDEC) by tumor necrosis factor-α induces lipolysis and inflammation in calf adipocytes.
). As evidenced by greater numbers of autolysosomes and increased autophagosome-lysosome fusion, a shortfall in supply of nutrients leads to activation of ALP in bovine aortic endothelial cells, mouse embryonic stem cells and Hela cells (
). Thus, because of the similarity in physiological adaptations that characterize undernutrition, we hypothesized that increased lipolysis in postpartum dairy cows with hyperketonemia may be associated with enhanced autophagy-lysosomal activity in adipose tissue. The specific objectives were to investigate the association between ALP activity and lipolysis in adipose tissues of dairy cows with hyperketonemia and in calf adipocytes.
MATERIALS AND METHODS
Ethics
The animal use protocol covering cow and calf was authorized by the Animals Care and Ethics Committee at Jilin University (Changchun, China, SY202012009). The animals received humane care according to the principles and specific guidelines presented in Guidelines for the Care and Use of Agricultural Animals in Research and Teaching (
To evaluate differences in the relative abundance of cathepsin D (CTSD), one of the major lysosomal proteases, in the adipose tissue of nonhyperketonemic (nonHYK) versus hyperketonemic (HYK) cows, we assumed that the standard deviation of relative CTSD abundance was 0.5, and our goal was to see a 2-fold difference in relative CTSD abundance between nonHYK and HYK cows. Then, to ensure that the study had a 95% power to detect this difference as significant (P = 0.01), 9 cows per group were required as calculated by the IACUC sample size calculations online tool (https://www.bu.edu/researchsupport/compliance/animal-care/working-with-animals/research/sample-size-calculations-iacuc/). An additional cow was added to each group in case of any sample lose. Thus, 10 cows in each group were needed.
Sixteen dairy cows were categorized as suspected nonHYK and 25 dairy cows were categorized as suspected HYK based on nitroprusside tests for ketone bodies in milk (
). Subsequently, the selected cows were housed in a tiestall barn. Blood concentrations of BHB were measured for 3 consecutive days to select nonHYK and HYK cows. Lastly, 15 dairy cows with serum 1.2 mM < BHB <3.0 mM for all 3 d were classified as HYK cows, and 12 cows with serum BHB <1.2 mM for all 3 d were classified as nonHYK. Then, 10 cows from each group were randomly chosen by using Excel randomization tool (Excel 2019; Microsoft) for further analyses. The basic description of the cows used in the study is reported in Supplemental Table S2 (https://figshare.com/articles/figure/Activated_autophagy-lysosomal_pathway_in_dairy_cows_with_hyperketonemia_is_associated_with_lipolysis_of_adipose_tissues/19524622).
Sample Collection
Before feeding, blood samples were collected without anticoagulant between 0730 and 0830 h by jugular venipuncture for 3 consecutive days. Subsequently, blood samples were centrifuged at 3,500 × g for 15 min at 4°C and serum was harvested and stored at −80°C until further analysis. During collection of blood samples, milk yield was recorded at 0530 and 1500 h for 3 consecutive days.
On the last day of blood sample collection, subcutaneous adipose tissue (SAT; approximately 3 g) biopsies from all cows in each group were harvested as described previously (
Inhibition of cell death inducing DNA fragmentation factor-α-like effector c (CIDEC) by tumor necrosis factor-α induces lipolysis and inflammation in calf adipocytes.
). Briefly, before biopsy, a 5 × 5 cm area of shaved skin at the tailhead was disinfected with iodine scrub and 75% alcohol, and then anesthetized with 2% lidocaine HCl (Sigma-Aldrich Co.). A 1.5- to 2.5-cm incision was made with a scalpel blade, and SAT removed from the incision site by blunt dissection. Samples were then clamped with tweezers, snipped with scissors, and snap-frozen in liquid nitrogen before storage at −80°C until analysis.
Determination of Blood Parameters
Serum concentrations of glucose, BHB, and free fatty acids were measured using a Hitachi 3110 autoanalyzer (Hitachi) with commercially-available kits (glucose: cat. no. GL3815; BHB: cat. no. RB1008; free fatty acids: cat. no. FA115; Randox Laboratories). The limits of quantification of glucose, BHB, and free fatty acids were 0.335 to 34.1 mM, 0.1 to 5.75 mM, and 0.072 to 2.24 mM, respectively.
Isolation of Primary Adipocytes
Calf adipocytes were isolated from 5 clinically healthy Holstein female newborn calves (1 d old, 30–40 kg) following published protocols with minor modifications (
). Adipose tissue from the peritoneal omentum and mesentery were obtained surgically under sterile conditions. After harvesting adipose tissue, calves underwent a meticulous postoperative recovery period involving delivery of flunixin meglumine (Schering-Plough Sante Animale; 1.1 mg/kg i.v. every 12 h) for 5 d. Veterinarians monitored wound healing, behavior, appetite, urination, and defecation of calves twice daily. Calves returned to the herd until suture removal 10 to 14 d following surgery.
Blood was removed from the surface of adipose tissue by rinsing with sterile PBS containing penicillin (2500 U/mL) and streptomycin (2500 μg/mL). Then, the fascia and visible blood vessels in the tissue were peeled away, and adipose tissue cut into small pieces of approximately 1 mm. The resulting adipose tissue was digested with 50 mL of Dulbecco's Modified Eagle Medium (DMEM)-F12 (SH30023.01; HyClone) digestion solution containing collagenase type I (1 mg/mL; C0130; Sigma-Aldrich) and incubated in a shaking water bath at 37°C for 1.5 h. The mixture was filtered sequentially through 80- and 40-µm cell strainers and the filtrate separated from adipocytes and medium by centrifugation at 175 × g for 10 min at room temperature. The resulting cell pellet was incubated with ammonium-chloride-potassium (ACK) lysis buffer (C3702; Beyotime Institute of Biotechnology) to remove residual erythrocytes and centrifuged at 175 × g for 10 min at room temperature. The suspension was discarded, and resulting cells resuspended in basal culture medium (BCM) composed of DMEM-F12 with 10% fetal bovine serum (SH30084.03; HyClone) and 1% penicillin-streptomycin (Sv30010; HyClone). After cell counting, the cell suspension was adjusted to a concentration of 1 × 104 cells/mL and inoculated in a cell culture flask. Adipocytes were then incubated at 37°C in a humidified atmosphere with 5% CO2 in a cell incubator with BCM replacement every 48 h.
Cell Culture
Adipocytes were inoculated in 6-well tissue culture plates (Corning Costar Corp.). After cells were approximately 70% confluent, the BCM was replaced with freshly prepared differentiation culture medium (DCM)1 to differentiate preadipocytes; BCM was with 0.5 mM 3-Isobutyl-1-methylxanthine (I-7018; Sigma-Aldrich), 1 μM dexamethasone (D-4902; Sigma-Aldrich), and 1 μg/mL insulin (I-5500; Sigma-Aldrich). After 48 h, DCM1 was discarded and replaced by DCM2. The DCM2 had a final concentration of 1 μg/mL insulin in BCM. The DCM2 was replaced every other day for 10 d until visible lipid droplets appeared in the cells, a sign that adipocytes were differentiated. After differentiation, the number of mature adipocytes was set to 4.0 × 105 per 6-well plate.
Cell Treatment
Experiments with calf in vitro differentiated adipocytes were divided into the following 2 sections: (1) mature adipocytes were incubated with DMEM-F12 containing 10 μM ISO (S2566; Selleck Chemicals) for 3 h (
) followed by treatment with ISO for additional 3 h.
Quantitative Reverse-Transcription PCR
Total RNA from SAT and adipocytes was isolated with the RNAiso Plus kit (9109; TaKaRa Biotechnology Co. Ltd.) according to the manufacturer's instructions. The RNA concentration and quality were measured using a Nanophotometer N50 Touch (Implen GmbH) and by electrophoresis (1% agarose gels). In our study, the optical density 260/280 ratio of RNA samples, used to evaluate the RNA quality, ranged from 1.9 to 1.98 and met purity requirements specified in the MIQE guidelines (
). The concentration of purified RNA was determined by UV spectrum at 260 nm. Then, cDNA was synthesized from total RNA using a reverse-transcription kit (RR047A; TaKaRa Biotechnology Co. Ltd.) according to the manufacturer's instructions. Subsequently, the SYBR Green plus reagent kit (RR420A; TaKaRa Biotechnology Co. Ltd.) was used to conduct quantitative reverse-transcription PCR on a 7500 Real-Time PCR System (Applied Biosystems Inc.). The reaction conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Each sample was run in triplicate. Relative expression of each target gene was normalized against the geometric mean of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) that were stably expressed in different groups (
; Supplemental Figure S1). The relative gene expression was calculated with the 2-ΔΔCt method. In this study, the optimal number and choice of housekeeping genes were not performed. This is a limitation of this study. The primers of target genes were designed by Primer Express software 3.0 (ThermoFisher Scientific) according to bovine reference sequences from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) and verified by BLAST searching at NCBI for primer specificity. The quality of the primers was evaluated by agarose gel electrophoresis (a single band of correct size) and melt curve (a single peak). The PCR efficiency (E, 95% < E <105%) and the correlation coefficient (r, higher than 0.99) were also used to assess the primers (
Total protein from SAT and adipocytes was extracted using a commercial protein extraction kit (C510003; Sangon Biotech Co. Ltd.) according to manufacturer's instructions. The bicinchoninic acid Protein Assay Kit was used to determine protein concentration (P1511; Applygen Technologies). Protein samples (20 μg per lane) were separated by 12% SDS-polyacrylamide gels and electrophoretically transferred onto 0.45-µm polyvinylidene fluoride membranes. Following transfer, membranes were blocked in Tris-buffered saline solution with 0.01% Tween-20 containing 3% bovine serum albumin at room temperature for 4 h. Blocked membranes were incubated with specific antibodies against β-actin (ab8226; Abcam; 1:2,000), lysosome-associated membrane protein 1 (LAMP1; ab24170; Abcam; 1:1,000), CTSD (21327–1-AP; Proteintech Group; 1:1,000), sequestosome-1 (p62; ab101266; Abcam; 1:2,000), phosphorylated HSL (p-HSL; 4139; Cell Signaling Technology Inc.; 1:1,000), HSL (4107; Cell Signaling Technology; 1:1,000), ATGL (ab99532; Abcam; 1:1,000), perilipin-1 (PLIN1; ab3526; Abcam; 1:1,000), and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC; ab198204; Abcam; 1:1,000) at 4°C overnight. Subsequently, membranes were washed 3 times with Tris-buffered saline solution with 0.01% Tween-20 and incubated with horseradish peroxidase-conjugated antimouse or antirabbit antibody (Boster Biological Technology Co. Ltd.) for 45 min at room temperature. Immunoreactive bands were visualized via a protein imager (ProteinSimple) using an enhanced chemiluminescence solution (WBKLS0500; Millipore). All protein bands were quantified with Image-Pro Plus 6.0 (Media Cybernetics Inc.). Individual samples were run in triplicate. In this study, β-actin was used as a reference protein, the phosphorylation level of HSL was calculated as p-HSL/total HSL. To ensure that the measurement is done in the linear range, the following pre-experiments were performed: (1) a standard curve was generated for signal intensity and sample amount to ensure that the sample amount used in the experiment was in the linear range; (2) under the condition of certain sample amount, different time of protein electrophoretically transferred onto polyvinylidene fluoride (PVDF) membrane was tested to observe whether the membrane capacity was saturated, and the shortest time for forming linear signal was selected; and (3) image acquisition time was optimized to ensure that all signals were acquired (i.e., weak signals were collected, whereas strong signal proteins were not overexposed). It was determined that (1) the sample amount was 20 μg, (2) CIDEC and β-actin were electrophoretically transferred onto PVDF membrane at 80 V for 45 min; CTSD, ATGL, p62, PLIN1, p-HSL, and HSL were electrophoretically transferred onto PVDF membrane at 80 V for 60 min; and LAMP1 was electrophoretically transferred onto PVDF membrane at 80 V for 75 min, and (3) the image acquisition time of CIDEC, β-actin, and CTSD was 1 s, that of p-HSL, HSL, PLIN1, p62, and ATGL was 5 s, and that of LAMP1 was 10 s.
Determination of Glycerol Content
Glycerol (GC) content in the supernatant of adipocytes was determined using a commercial assay kit (E1002; Applygen Technologies Inc.) as described by the manufacturer. The cell-free supernatants were collected from each group. A 50-μL sample of cell-free supernatant was mixed with 150 μL of chromogenic liquid before heating in an incubator (37°C) for 10 min to measure GC. Absorbance at 550 nm was proportional to the concentration of GC.
Determination of Triacylglycerol Content
Triacylglycerol content in adipocytes was determined using a commercial kit (E1013; Applygen Technologies Inc.) according to the manufacturer's protocols. Approximately 1 × 106 cells were mixed with 0.1 mL of lysis buffer, and kept at room temperature for 10 min. Total protein concentration of supernatant was detected by bicinchoninic acid assay (P1511, Applygen Technologies Inc.). Fifty microliters of supernatant were heated in a 70°C water bath for 10 min and centrifuged at 2,000 × g for 5 min at room temperature. A total of 10 μL of supernatant was mixed with 190 μL of chromogenic liquid and incubated at 37°C for 15 min. The mixture absorbance at 550 nm was proportional to the concentration of TG in each sample.
Determination of β-N-acetylglucosaminidase Activity
β-N-acetylglucosaminidase (NAG) activity in SAT and adipocytes was determined using a commercial assay kit (ab204705; Abcam). In this assay, NAG uses a synthetic p-nitrophenol derivative as a NAG substrate and releases p-nitrophenol, which can be measured at absorbance (optical density = 400 nm). First, cells were lysed in radio immunoprecipitation assay (RIPA) buffer, and then 10 µg of protein from each sample were normalized to an equal volume and measured for NAG activity following the protocol provided by the supplier. Absorbance at 400 nm was proportional to the activity of NAG.
Statistical Analysis
All data were analyzed using SPSS 23.0 software (IBM Corp.). All data were tested for normality and homoscedasticity using the Shapiro-Wilk and Levene test, respectively. For data with normal distribution, unpaired t-tests or 2-way mixed-ANOVA with subsequent Bonferroni correction was performed for data analysis. For data with nonnormal distribution, nonparametric statistical analysis was performed using the Wilcoxon test. P < 0.05 was considered significant and P < 0.01 was marked significant. Data throughout the text and figures are presented as means ± SEM.
RESULTS
Performance and Biochemical Parameters
Compared with nonHYK cows, milk yield, and DMI were lower (P < 0.01) and BCS greater (P < 0.05) in HYK cows (Supplemental Table S2). No significant difference in BW was observed between control and HYK cows (Supplemental Table S2). Serum concentrations of free fatty acids and BHB were greater (P < 0.01) in HYK cows (Supplemental Table S2). In contrast, the concentration of glucose was lower (P < 0.01) in HYK compared with nonHYK cows (Supplemental Table S2).
Lipolysis in Adipose Tissues
Ratio of p-HSL/HSL and protein abundance of ATGL were greater (P < 0.01, Figure 1A–C), whereas protein abundance of PLIN1 and CIDEC were lower (P < 0.01, Figure 1A, D, and E) in adipose tissue of HYK compared with nonHYK cows.
Figure 1Lipolysis status in adipose tissue. (A) Representative blots of phosphorylated hormone-sensitive lipase (p-HSL), hormone-sensitive lipase (HSL), adipose triacylglycerol lipase (ATGL), perilipin1 (PLIN1), and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC). (B–E) Quantification of protein levels of p-HSL and HSL, ATGL, PLIN1, and CIDEC in nonhyperketonemic (nonHYK; n = 10) and hyperketonemic cows (HYK; n = 10). Data were analyzed using unpaired t-tests and expressed as means ± SEM.
The mRNA abundance of autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) was greater (P < 0.01, Figure 2A–C) in adipose tissues of SCK cows. Protein abundance of LAMP1 and CTSD was greater (P < 0.01, Figure 2D–F), whereas abundance of p62 was lower (P < 0.01, Figure 2D and G) in adipose tissue of SCK compared with nonHYK cows. Compared with nonHYK cows, NAG activity was greater (P < 0.01, Figure 2H) in adipose tissue of HYK cows.
Figure 2Status of autophagy-lysosomal pathway in adipose tissue. (A–C) Relative mRNA abundance of ATG5, ATG7, and MAP1LC3B in adipose tissues of nonhyperketonemic cows (nonHYK; n = 10) and hyperketonemic cows (HYK; n = 10). (D) Representative blots of lysosome-associated membrane protein 1 (LAMP1), cathepsin D (CTSD), and sequestosome-1 (p62). (E–G) Quantification of protein levels of LAMP1, CTSD, and p62 in nonHYK cows (n = 10) and HYK cows (n = 10). (H) Relative activity of β-N-acetylglucosaminidase (NAG) in adipose tissues of nonHYK cows (n = 10) and HYK cows (n = 10). Data were analyzed using unpaired t-tests and expressed as means ± SEM.
Effects of ISO and Torin1 on Lipolysis in Calf Adipocytes
Compared with the control group, treatment with lipolysis inducer ISO or mTOR inhibitor Torin1 decreased protein abundance of PLIN1 and CIDEC (P < 0.01, Figure 3A, B and C) and TG content (P < 0.01, Figure 3D) in calf adipocytes. Conversely, the GC content in supernatant of calf adipocytes treated with ISO or Torin1 was higher than control group (P < 0.05, Figure 3E).
Figure 3Effects of isoproterenol (ISO) and Torin1 on lipolysis in calf adipocytes. Calf adipocytes were treated with 10 µM ISO for 3 h or 250 nM Torin1 for 3 h. (A) Representative blots of perilipin1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC). (B–C) Quantification of protein levels of PLIN1 and CIDEC. (D) The triacylglycerol (TG) content in calf adipocytes. (E) The content of glycerol (GC) in the supernatant of calf adipocytes. Data were analyzed using 2-way mixed-ANOVA with subsequent Bonferroni correction and expressed as means ± SEM.
Effects of ISO and Torin1 on ALP in Calf Adipocytes
Compared with the control group, treatment with ISO or Torin1 upregulated mRNA abundance of ATG5, ATG7 and MAP1LC3B (P < 0.01, Figure 4A–C) in calf adipocytes. Protein abundance of LAMP1 and CTSD was greater (P < 0.01, Figure 4D–F), whereas p62 was lower (P < 0.01, Figure 4D and G) in calf adipocytes treated with ISO or Torin1 than control group. In addition, compared with the control group, treatment with ISO or Torin1 increased NAG activity in calf adipocytes (P < 0.05, Figure 4H).
Figure 4Effects of isoproterenol (ISO) and Torin1 on autophagy-lysosomal pathway in calf adipocytes. Calf adipocytes were treated with 10 µM ISO for 3 h or 250 nM Torin1 for 3 h. (A–C) Relative mRNA abundance of ATG5, ATG7, and MAP1LC3B in calf adipocytes. (D) Representative blots of lysosome-associated membrane protein 1 (LAMP1), cathepsin D (CTSD), and sequestosome-1 (p62). (E–G) Quantification of protein levels of LAMP1, CTSD, and p62. (H) Relative activity of β-N-acetylglucosaminidase (NAG). Data were analyzed using 2-way mixed-ANOVA with subsequent Bonferroni correction and expressed as means ± SEM.
Effects of Leupeptin on Lipolysis in ISO-Treated Calf Adipocytes
Compared with ISO-treated calf adipocytes, protein abundance of p62, PLIN1. and CIDEC was higher in ISO plus leupeptin-treated calf adipocytes (P < 0.01, Figure 5A–D). The TG content in calf adipocytes was higher, whereas the GC content in supernatant of calf adipocytes was lower in response to co-treatment with ISO and leupeptin (P < 0.05, Figure 5E and F).
Figure 5Effects of leupeptin on lipolysis in isoproterenol (ISO)-treated calf adipocytes. Calf adipocytes were pretreated with or without 10 μg/mL leupeptin for 4 h and then treated with 10 µM ISO for another 3 h. (A) Representative blots of sequestosome-1 (p62), perilipin1 (PLIN1), and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC). (B–D) Quantification of protein levels of p62, PLIN1, and CIDEC. (E) The triacylglycerol (TG) content in calf adipocytes. (F) The content of glycerol (GC) in the supernatant of calf adipocytes. Data were analyzed using two-way mixed-ANOVA with subsequent Bonferroni correction and expressed as means ± SEM.
During early lactation, adipose mobilization is necessary to offset the NEB experienced by dairy cows, but a sustained lipolytic state increases the risk of ketosis (
), the potential role of ALP is not well understood. In this study, activated ALP along with lipolysis in SAT of dairy cows with HYK suggested a role of the former in the tissue response to a catabolic state. Data generated from adipocytes revealed that pharmacologic activation of ALP induced lipolysis, whereas blocking of ALP weakened ISO-induced lipolysis (Figure 6). Thus, the data suggested that ALP is a potential therapeutic target for limiting excessive fat mobilization of dairy cows during the early postpartum period.
Figure 6The increased lipolytic response is associated with activated autophagy-lysosomal pathway (ALP) in dairy cows. Lipid droplet (LD) surface proteins perilipin 1 (PLIN1) and cell death-inducing DNA fragmentation factor-α-like effector c (CIDEC) are degraded by autophagy. Then, LD are taken by autophagosomes and delivered to lysosomes for degradation and subsequent release of free fatty acids (FFA). HSL, hormone-sensitive lipase; ATGL, adipose triacylglycerol lipase; p62, sequestosome-1; LC3-II, microtubule-associated protein 1 light chain 3-II. Figure created with BioRender.com.
Hormone-sensitive lipase protein expression and extent of phosphorylation in subcutaneous and retroperitoneal adipose tissues in the periparturient dairy cow.
), the greater protein abundance of ATGL and activity of HSL in SAT of HYK cows underscored the intense lipolytic state of those cows. Lipid droplets are surrounded by lipid droplet-associated proteins including PLIN1 and CIDEC (
). A reduction in the expression of PLIN1 and CIDEC leads to increased lipolysis, attributed to the greater interaction of HSL and ATGL with lipid droplets (
). Thus, the upregulation of autophagosome formation genes, ATG5, ATG7, and MAP1LC3B in the present study are suggestive of activated ALP during ketosis. Lysosomes are major organelles carrying out degradative functions in cells. A greater mass of lysosomes was observed in SAT of HYK cows, as evidenced by greater protein abundance of LAMP1, which is a lysosomal marker (
). In addition, NAG is a high molecular-weight (∼140 kDa) hydrolytic lysosomal enzyme and is necessary for degradation and disposal of various cellular fractions (
). Given the essential role of CTSD and NAG in lysosomal function, the greater protein abundance of CTSD and activity of NAG in HYK cows were suggestive of enhanced lysosomal activity. The protein p62 serves as a crucial adaptor and interacts with autophagosomes and ubiquitin-conjugated substrates, recruiting both for lysosomal degradation (
). Thus, lower protein abundance of p62 in SAT confirmed the elevated degree of autophagy-lysosomal activity in HYK cows. Taken together, the current data indicated that SAT of HYK cows experiences an activation in ALP.
Recent work has identified lipophagy as a branch of the ALP, which can recruit the autophagosome to lipid droplets for engulfment and delivery to the lysosome (
demonstrated that autophagosome formation genes were upregulated in adipose tissue of HYK cows, implying that lipophagy might have been more active due to enhanced lysosomal activity and further contribute to catabolism of adipose tissue. It should be noted that chaperone-mediated autophagy (CMA) selectively degrades lipid droplet proteins on their surface, thus facilitating the recruitment of cytosolic lipases and autophagy effector proteins (
). Thus, increased lysosomal function may result in CMA to degrade lipid droplet proteins and subsequent lipolysis. The lower protein abundance of PLIN1 and CIDEC observed in adipose tissue of SCK cows and Torin1-treated adipocytes supports this speculation. Therefore, our data and that of previous studies indicate that the greater autophagy-lysosomal activity may promote lipolysis of adipose tissue via multiple ways.
Hypoglycemia induced by NEB is a characteristic of dairy cows with ketosis (
). Compared with nonHYK cows, HYK cows experienced more severe NEB, as evidenced by higher concentrations of free fatty acids and BHB in the present and previous studies (
reported that autophagy-lysosomal degradation activity was activated under condition of nutritional deprivation in mouse embryonic fibroblasts. Consistent with this study, the greater expression of autophagosomes formation and lysosomal function-regulated genes in SAT of HYK cows suggested activated ALP, thus, energy deficiency may contribute to enhance autophagy-lysosomal activity in adipose tissues of HYK cows. The pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) was reported to upregulate autophagy-related genes in murine 3T3-L1 adipocytes (
). The fact that several studies, including ours, detected increased plasma and serum concentrations of TNF-α in HYK cows suggest the inflammatory state associated with ALP (
) and activated ALP in adipose tissue from HYK cows suggested the occurrence of this process. Together, multiple factors could potentially alter status of ALP in adipose tissue of dairy cows.
There is a limitation of the present study. Although Torin1 was used as autophagy activator in previous studies (
). Thus, activation of autophagy by overexpression of autophagy-regulated genes, such as ATG5 and MAP1LC3B, might be a better approach to investigate the function of autophagy during lipolysis.
CONCLUSIONS
Combined data from the present in vivo and in vitro studies underscored the positive association between lipolysis and autophagy-lysosomal activity in adipose tissue during periods of NEB. These findings provide new insights regarding the role of ALP during lipolysis in dairy cows with ketosis.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (Beijing, China; grant no. 32172927) and the Science Foundation of Education Department of Jilin Province (Changchun, China; grant no. JJKH20221038KJ). The authors have not stated any conflicts of interest.
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