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. 2018 May 9;96(7):2646–2658. doi: 10.1093/jas/sky162

Downregulated angiopoietin-like protein 8 production at calving related to changes in lipid metabolism in dairy cows

Misato Nakano 1,#, Yutaka Suzuki 2,#, Satoshi Haga 3, Eri Yamauchi 1, Dahye Kim 1, Koki Nishihara 1, Keiichi Nakajima 4, Takafumi Gotoh 5, Seungju Park 6, Myunggi Baik 6, Kazuo Katoh 1, Sanggun Roh 1,✉
PMCID: PMC6095384  PMID: 29746655

Abstract

Acute physiological adaptation of lipid metabolism during the postpartum transition period of cows facilitates peripheral metabolic regulation. Hepatokines, which are hormones secreted from hepatocytes, are presumed to play a critical role in systemic metabolic regulation. Angiopoietin-like protein 8 (ANGPTL8) has been identified as a novel hepatokine associated with circulating triglyceride concentrations in mice and humans. However, regulation of ANGPTL8 and its physiological effects is still unknown in cattle. The present study aimed to reveal changes in ANGPTL8 expression and secretion during the periparturient period, and to investigate its regulatory effect on adipocytes and mammary epithelial cells. In the peripartum period, liver ANGPTL8 mRNA expression was lesser on the day of parturition and 1 wk postpartum than it was 1 wk before parturition (P < 0.05). Moreover, plasma ANGPTL8 concentrations decreased on the day of parturition as compared with that 1 wk before parturition (P < 0.05). In addition, ANGPTL8 expression in cultured bovine hepatocytes was downregulated after oleate and palmitate treatment but upregulated after insulin treatment (P < 0.05). ANGPTL8 decreased hormone-sensitive lipase (HSL) expression in differentiated adipocytes and cluster of differentiation 36 (CD36), fatty acid synthase (FAS), acetyl-coa carboxylase (ACC), and stearoyl-coa desaturase (SCD) in cultured bovine mammary epithelial cells (P < 0.05). These data suggest that hepatic ANGPTL8 production was downregulated postpartum when the cows experienced a negative energy balance. This downregulation was associated with increased concentrations of NEFA and decreased concentrations of insulin in lactating cows, and it facilitated lipid mobilization from adipose tissue to the mammary glands. We speculate that ANGPTL8 might have beneficial effects in reverting or improving the physiological adaptation and pathological processes of lipid metabolism during the peripartum period.

Keywords: angiopoietin-like protein 8, dairy cows, hepatokine, lipid metabolism, negative energy balance

INTRODUCTION

Angiopoietin-like protein 8 (ANGPTL8) has been identified as a novel adipokine/hepatokine that is significantly associated with the metabolism of circulating triglyceride (TG), and the loss of its function has been shown to selectively reduce adipose tissue mass; moreover, this effect increases with age (Wang et al., 2013). The physiological role of ANGPTL8 has been attributed to the inhibition of lipoprotein lipase (LPL) activity (Zhang, 2012; Zhang and Abou-Samra, 2014). Mice deficient in the ANGPTL8 gene (Gm6484) display decreased serum triacylglycerol concentrations in response to feeding after starvation (Wang et al., 2013). Plasma ANGPTL8 concentrations have been found to be associated with obesity and diabetes. ANGPTL8 concentrations are elevated in db/db and ob/ob mice and subjects with type 1 diabetes (T1D) and type 2 diabetes (T2D) (Espes et al., 2014; Fenzl et al., 2014; Fu et al., 2014). In addition, genome-wide association studies in humans have revealed that SNPs in ANGPTL8 are associated with lipid metabolism. A maximum of 15% lower plasma high-density lipoprotein cholesterol (HDL-C) was reported for c.194C>T, rs2278426, and 15% lower plasma TG for c.361C>T, rs145464906, and c.2136A>G, rs737337 (Teslovich et al., 2010; Quagliarini et al., 2012; Peloso et al., 2014).

Drastic changes in the lipid metabolic process in parturient cows interactively cause hormonal regulation of various tissues, including liver, to adapt to these metabolic transition periods (Donkin and Armentano, 1995; Baldwin et al., 2004; Roh et al., 2016). Previously, we had reported that chemerin, an endocrine factor secreted mainly from hepatocytes, regulates insulin secretion and lipid metabolism in ruminants (Suzuki et al., 2012b, 2015, 2016). Other studies indicated a potential role of fibroblast growth factor 21 (FGF21) in lipid mobilization at the onset of lactation (Schoenberg et al. 2011; Akbar et al. 2015). Thus, ANGPTL8 as a hepatokine may perform important functions in periparturient dairy cows via regulation of peripheral lipid metabolism. In addition, we hypothesized that insulin and NEFA changed around parturition would be key factors for hepatic ANGPTL8 production. The aims of the present study were: 1) to reveal changes in the expression pattern of ANGPTL8 in the liver tissue of cattle during parturition and lactation, 2) to identify the factors regulating ANGPTL8 in cultured hepatocytes, and 3) to understand the function of ANGPTL8 in adipocyte and mammary epithelial cells, for the understanding of the role of this hormone in periparturient cows.

MATERIALS AND METHODS

Animals and Tissue Sampling

Experiments were performed with Holstein, Japanese Black cattle, and Korean Native cattle at 5 institutions: Tohoku University; Division of Grassland Farming, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization (NARO); Kyushu University; Division of Dairy Production Research, Hokkaido Agricultural Research Center, NARO; and Seoul National University. All the experiments were conducted in accordance with the Institute Guide for the Care and Use of Experimental Animals at each facility.

In the first experiment, we aimed to understand the expression pattern of ANGPTL8 in liver, subcutaneous adipose tissue (ScAT), mesenteric adipose tissue (MesAT), perirenal adipose tissue (PeriAT), epididymal adipose tissue (EpiAT), skeletal muscle, and rumen epithelium, which were sampled from 5 male Japanese Black cattle at the age of 11 wk. All the tissues were rapidly separated from cattle euthanized by administering an overdose of sodium pentobarbital via the jugular vein followed by exsanguination. Tissues were trimmed and immediately frozen in liquid nitrogen. The samples were stored at −80 °C until RNA and protein extraction.

The second experiment was conducted to assess the relationships between hepatic ANGPTL8 expression, plasma ANGPTL8, and various metabolic indicators. Liver tissue was biopsied from each cow at −4, −1, 0, 1, and 4 wk from parturition. The procedure for biopsy was described previously (Miura et al., 1987). Briefly, the cows were administered 2% lidocaine (Bayer, Leverkusen, Germany) for sedation and 5% tranexamic acid (Meiji-Seika Pharma, Tokyo, Japan) for hemostasis. The biopsy site was selected at the intersection of the right 11th to 12th intercostal space and the line from the right point of the acromion to tuber coxa. The biopsy site was anesthetized by an application of lidocaine. A 0.5-cm-long stab incision was made in the skin and the biopsy was performed using a Bard Magnum Biopsy Gun system with 12G, 10 cm Bard Magnum needles (Bard Biopsy Systems, Tempe, AZ, USA). After biopsy, the spot was disinfected with 10% isodine gel, and a dose of suspension procaine penicillin G injection NZ (ZENOAQ, Tokyo, Japan) was administered. All the tissues were rapidly separated, immediately frozen in liquid nitrogen, and stored at −80 °C until RNA extraction.

The third experiment was conducted to analyze the gene expression of ANGPTL8 in the liver during lactation and dry-off in Holstein dairy cows. Liver tissue was rapidly separated from cows slaughtered at 1 wk (i.e., during the early lactation stage; n = 3), 5 mo (i.e., at the mid lactation stage; n = 4), 9 mo (i.e., at the late lactation stage; n = 3), and 15 mo (i.e., during the at dry-off period; n = 3) post parturition. The cows used in this experiment were multiparous, expect for 2 primiparous cows at mid lactation stage. All the cows at late lactation were pregnant, whereas all other cows were nonpregnant. The feeding strategy was designed to meet the Japanese Feeding Standard for Dairy Cattle (NARO, 2008). Tissue samples were immediately frozen in liquid nitrogen; and stored at −80 °C until RNA extraction.

The fourth experiment investigated the effects of nutrition on hepatic ANGPTL8 gene expression in male Japanese Black cattle. Six Japanese Black calves were fed with milk replacers containing 26% CP, 10% crude fat (CF), and 99% TDN at a rate of a maximum of 1,800 g/d until 2.5 mo of age followed by weaning via a gradual reduction in volume by 200 g each day for 10 d until 3 mo. The quantity of milk administered was based on the Japanese feeding standard for beef cattle (NARO, 2008). Dry calf starter (72% TDN, 18% CP, and 2% ether extract) was provided until 3.5 mo of age, after which the calves were randomly assigned to 2 groups [concentrate-fed group (Con): n = 3; hay-fed group (Hay): n = 3]. Calves in the Hay group were fed only with hay (9.4% CP, 37.6% crude fiber, and 55.3% TDN) ad libitum, whereas those in Con group were fed only with concentrate (12% CP, 6.5% crude fiber, and 73% TDN) at 2.5% of BW until 10 mo. Liver tissue was collected from the calves at 10 mo by biopsy and used for protein and gene expression analyses.

The fifth experiment was conducted to analyze the gene expression of ANGPTL8 in the liver, abdominal adipose tissue (ABF), and longissimus dorsi muscle (LM) used tissue samples from 10 bulls and 10 steers of male Korean Native cattle using a previously described feeding method (Bong et al., 2012). The animals were fed concentrate diets that consisted of 15% CP and 71% TDN until 14 mo of age, 13% CP and 72% TDN until 20 mo of age, and 11% CP and 73% TDN from 21 mo of age. Hay was offered ad libitum, and the animals had free access to fresh water. Animals were slaughtered at 20 mo for bulls and 28 mo for steers with mean carcass weights of 347 and 398 kg, respectively. Carcass characteristics are described in more detail in our previous paper (Bong et al., 2012). Briefly, steer LM had 3.7-fold greater intramuscular fat (IMF) content (11%) than bull LM did (3.0%, P < 0.001). All tissues were collected immediately after slaughter as previously described (Bong et al., 2012). ABF samples were dissected from the abdomen. Five tissue samples each from 10 bulls and 10 steers were randomly chosen for the analysis of ANGPTL8 mRNA.

Hormone and Metabolite Analysis

Plasma concentrations of GH, insulin, NEFA, and TG were analyzed according to the methods described in our previous report (Suzuki et al., 2016).

Hepatocyte Culture

The caudate lobes of the liver were sampled from male Japanese Black cattle at approximately 4–5 mo of age. Collagenase solution was recirculated through the lobes to collect cells in the medium. Cells were seeded in 12-well plates (5 × 105 cells per well) and cultured for 48 h, and then they were washed 3 times with fresh medium and cultured for an additional 6 h in serum-free medium. Thereafter, the medium was exchanged with fresh serum-free medium in the absence or presence of insulin (1, 10, and 100 nM), palmitate (50 and 250 μM), and oleate (50 and 250 μM) followed by incubation for 24 h. The treated cells were harvested for analysis of mRNA expression.

Effects of ANGPTL8 on Differentiated Bovine Adipocytes

To assess the physiological effects of ANGPTL8 on lipid metabolism, cultured bovine adipocytes were incubated with the protein. Stromal vascular (SV) cells containing an abundant population of preadipocytes were isolated from ScAT of male Holstein cattle by collagenase digestion as previously reported (Song et al., 2010). The SV cells were plated on 6-well plates at 2.5 × 104 cells per cm2 and incubated at 37 °C in a humidified 5% CO2 atmosphere. The culture medium was changed 24 h after cell seeding and subsequently once every 2 d. After the cells reached confluence, the medium was replaced with differentiation-induction medium consisting of Dulbecco’s Modified Eagle’s medium (DMEM):Nutrient Mixture F-12 Ham (Ham’s F-12) (1:1) supplemented with the antibiotic mix solution, octanoic acid (5.0 × 10−3 M), sodium acetate (10−6 M), lipid mixture (L0288, Sigma-Aldrich, Inc., St. Louis, MO, USA) (100-fold dilution), T3 (2.0 × 10−9 M), dexamethasone (10−8 M), insulin (10−7 M), and troglitazone (10−7 M).

Differentiated adipocytes were incubated with DMEM/HAMF12, 0.5% (wt/vol) BSA, and 1% (wt/vol) penicillin for 3 h before treatment with ANGPTL8. The culture medium was subsequently refreshed with the same incubation medium containing mouse ANGPTL8 (0, 1, and 10 ng/mL) for 3 h, and the treated cells were harvested for gene expression analysis.

Effects of ANGPTL8 on Bovine Mammary Epithelial Cells

To assess the physiological effects of ANGPTL8 on lipid metabolism, immortalized bovine mammary epithelial cells (MAC-T cells) were cultured according to the methods described in our previous study (Suzuki et al., 2015). MAC-T cells were differentiated in DMEM containing 10% (wt/vol) fetal bovine serum, 1% (wt/vol) penicillin-streptomycin mixture, bovine insulin, human GH, bovine prolactin, dexamethasone, and lipid mixture for 6 d. After that, the differentiated MAC-T cells underwent serum starvation with DMEM, 0.5% (wt/vol) BSA, 1% (wt/vol) penicillin/streptomycin, and lipid mixture (100-fold dilution) for 3 h. Following serum starvation, the culture medium was refreshed with the same incubation medium containing mouse ANGPTL8 (0, 1, and 10 ng/mL) for 3 h. Treated cells were harvested for mRNA expression analysis.

RNA Extraction and Quantitative Real-Time PCR

Total RNA was extracted from bovine tissues and cultured cells, according to the instructions supplied with the RNAiso Plus Kit (Takara Bio Inc., Otsu, Japan). The purity and integrity of the RNA were checked by spectrophotometry and agarose gel electrophoresis, respectively, and semiquantitative reverse transcription-PCR was performed as previously described (Song et al., 2010; Suzuki et al., 2012a; Kato et al., 2016). Five hundred nanograms of RNA was reverse-transcribed to cDNA with the PrimeScript RT reagent Kit (Takara Bio Inc.). Quantitative real-time PCR (qRT–PCR) was performed using SYBR Premix Ex Taq II (Takara Bio Inc.), and the thermal cycling parameters were as follows: 95 °C for 15 min followed by 40 cycles at 94 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. The 2−∆∆CT method was used to determine relative fold changes. Expressions of the target genes were normalized with the geometric mean of β-actin, cyclin G-associated kinase (GAK), and vacuolar protein sorting 4 homolog A (VPS4A) as internal standards, and expressions of each gene shown in Figs. 5–8 were normalized with the 18S rRNA gene as an internal standard. Primer information is shown in Supplementary Table S1.

Figure 5.

Figure 5.

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Expression of ANGPTL8 in liver and adipose tissues between Korean Native bulls and castrated steers. (A) Abdominal adipose tissue (ABF) was sectioned to investigate its morphology and lipid content by hematoxylin and eosin staining. (B) ANGPTL8 expression in the liver, ABF, and LM were compared by qRT–PCR between bulls and steers (n = 5). Data are shown as fold changes relative to the expression in the bulls.

Figure 6.

Figure 6.

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Fatty acid and insulin regulation of ANGPTL8 expression in bovine hepatocytes. Hepatocytes prepared from cattle were cultured and proliferated to approximately 80% confluence. Cultured primary bovine hepatocytes were treated with palmitate (A), oleate (B), and insulin (C) at the concentrations indicated for 24 h. After treatment, total RNA was extracted from the cells and ANGPTL8 expression was analyzed using qRT–PCR. Data are shown as fold changes in expression relative to untreated control cells. Different letters indicate significant differences between groups (P < 0.05).

Figure 7.

Figure 7.

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ANGPTL8 regulation of hormone-sensitive lipase (HSL) expression in cultured bovine differentiated adipocytes. Preadipocytes from bovine connective tissue were cultured, and differentiation was induced in vitro with adipogenic hormones for 12 d. Differentiated adipocytes underwent serum starvation for 3 h and then were subsequently treated with medium containing mouse ANGPTL8 (0, 1, and 10 ng/mL) for 3 h. Gene expressions of lipoprotein lipase (LPL), cluster of differentiation 36 (CD36), fatty acid synthase (FAS), and HSL were measured by qRT–PCR. Data show fold changes relative to expression in untreated control cells. Different letters indicate significant differences between groups (P < 0.05).

Figure 8.

Figure 8.

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ANGPTL8 regulation of expression of cluster of differentiation 36 (CD36), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD) in cultured bovine mammary epithelial cells. Immortalized bovine mammary epithelial cells (MAC-T cells) were cultured, and differentiation was induced in vitro with lactogenic hormones for 6 d. MAC-T cells underwent serum starvation for 3 h, and subsequent treatment with mouse ANGPTL8 (0, 1, and 10 ng/mL) for 3 h. Gene expressions of lipoprotein lipase (LPL), CD36, FAS, ACC, SCD, glycerol-3-phosphate acyltransferase 1 (GPAT), and liver X receptor α (LXRA) were quantified by qRT–PCR. Data show fold changes relative to expression in untreated control cells. Different letters indicate significant differences between groups (P < 0.05).

Western Blot Analysis

The liver, adipose, muscle, and rumen tissues were lysed in cell lysis buffer (Cell Signaling Technology, Boston, MA, USA) with a 1% protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). Plasma samples were diluted 5-fold with PBS, and 10 µg of protein from tissue samples and 5 µL of diluted plasma samples were subjected to SDS–PAGE in 15% polyacrylamide gels. Electrophoresis gels were transferred to a polyvinylidene difluoride (PVDF) membrane (ATTO, Tokyo, Japan), which were blocked with 3% skimmed milk for 1 h and washed several times in Tris-buffered saline (TBS) with 0.5% Tween-20. Blots were incubated with primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase (HRP). Primary antibodies used were anti-ANGPTL8 rabbit polyclonal IgG (#7619; Prosci, CA, USA) at a dilution of 1:10,000 and anti-β-actin (sc-47778; Santa Cruz Biotechnology, TX, USA) at 1:5,000 dilution. The secondary antibodies used were anti-rabbit IgG HRP-conjugated (W4011; Promega, WI, USA) at 1:5,000 dilution and anti-mouse IgG HRP-conjugated (W4021; Promega) at a dilution of 1:5,000. Protein bands were detected by an ECL Prime Western Blotting Detection System (GE Healthcare, UK) and an ImageQuant LAS 4000 (GE Healthcare).

Hematoxylin and Eosin Staining

Fresh adipose tissues from calves were fixed with 4% formaldehyde/PBS and embedded in paraffin. Tissue sections were deparaffinized in xylene and dehydrated with a dilution series of ethanol. Thereafter, hematoxylin and eosin (HE) staining was performed on sections of fat tissues.

Statistical Analysis

All data are expressed as means ± SEM. In Figs. 1–3, differences in mRNA expression and protein abundance were analyzed using a 1-way ANOVA followed by Tukey’s multiple range test. The data in Fig. 4 were analyzed using a Student’s t-test. In Fig. 5, differences among various tissues were determined using Dunnett’s modified Tukey–Kramer pairwise multiple comparison test (DTK) in R. Additionally, statistical significance of the differences between bulls and steers was determined with a t-test in R. Values in Figs. 6–8 are representative of at least 2 separate series of cultures (5 to 6 replicates were performed for each treatment).

Figure 1.

Figure 1.

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mRNA expression and protein expression of ANGPTL8 in various tissues of Japanese Black cattle. (A) ANGPTL8 expression was quantified in liver, adipose, and muscle tissues of 5 cattle by qRT–PCR. Data are shown as fold changes relative to expression in the liver tissue. ND, not detected. (B) ANGPTL8 protein expression was evaluated in the liver, perirenal adipose tissue (PeriAT), skeletal muscle, and rumen of cattle by immunoblotting. β-Actin was used as an internal standard. (C) ANGPTL8 protein was detected in the plasma of cattle by immunoblotting. Liver lysate served as a positive control.

Figure 2.

Figure 2.

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Production of hepatic ANGPTL8 at parturition in dairy cows. (A and B) Plasma was sampled from multiparous Holstein dairy cows around parturition (−4, −1, 0, 1, and 4 wk from parturition; n = 6 each time), and metabolite plasma concentrations (NEFA and TG) and hormones (insulin and GH) were measured. (C) Biopsied liver tissues from the same cows were subjected to qRT–PCR analysis to determine ANGPTL8 expression. Data are shown as fold change relative to ANGPTL8 expression at −4 wk. (D) Plasma from periparturient cows was analyzed by immunoblotting. Abundance of ANGPTL8 was calculated and the data are shown as arbitrary levels at each week relative to average ANGPTL8 level during the whole experimental period. Different letters indicate significant differences between groups (P < 0.05).

Figure 3.

Figure 3.

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ANGPTL8 expression in dairy cows during different lactation periods and the dry-off period. Liver tissue samples were collected from slaughtered dairy cows at early, mid, and late lactation periods (i.e., 1, 5, and 9 mo postpartum, respectively) and from the dry-off period (i.e., 15 mo postpartum) to examine expression of ANGPTL8 and genes related to hepatic lipid metabolism. ANGPTL8, glycerol-3-phosphate acyltransferase 1 (GPAT1), low-density lipoprotein receptor (LDLR), and fatty acid synthase (FAS) were analyzed by qRT–PCR. The data are shown as fold changes relative to gene expression during early lactation.

Figure 4.

Figure 4.

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Effects of a long-term nutritional treatment on the expression of ANGPTL8. Young beef cattle were fed concentrate diet (C) or hay (H) from 3–10 mo of age. Liver tissue samples were biopsied when the cattle were at an age of 10 mo. Gene expressions of ANGPTL8, fatty acid synthase (FAS), pyruvate carboxylase (PC), and phosphoenolpyruvate carboxykinase 1 (PCK1) were analyzed by qRT–PCR. The data are shown as fold changes relative to gene expression of cattle on the hay diet. *P < 0.05 vs. H.

RESULTS

Sequences and Comparisons of Bovine ANGPTL8

We identified bovine ANGPTL8 gene in the opposite strand of the Dedicator of cytokinesis 6 (DOCK6) gene (NCBI Reference Sequence: NM_001192166.2) (Supplementary Fig. S1). Bovine ANGPTL8 had 198 AA, and similarities with mouse and human ANGPTL8 AA sequences were 67% and 69%, respectively (Supplementary Fig. S2). Further, the Specific epitope 1 (SE1) region, the binding domain of LPL, had 100% homology among these species.

Gene and Protein Expression of ANGPTL8 in Bovine Tissues

Relative expression of ANGPTL8 revealed that the greatest ANGPTL8 expression occurred in the liver (Fig. 1A). In addition, it was detected in ScAT, MesAT, and PeriAT, and western blot analysis showed that it was expressed in liver and adipose tissue, but not in skeletal muscles or rumen papillary tissues (Fig. 1B). Moreover, circulating ANGPTL8 was detected in bovine plasma (Fig. 1D).

Hepatic ANGPTL8 Expression and Plasma ANGPTL8 Levels During the Transition from Pregnancy to Lactation

A negative energy balance was associated with increased plasma NEFA (the last 4 wk of pregnancy vs. 1 wk of lactation: 0.14 vs. 0.94 mEq/L; P < 0.001) and decreased plasma TG (13.8 vs. 3.3 mg/dL, P < 0.01) as shown in Fig. 2A. Simultaneously, plasma GH tended to be greater at parturition and in the first week of lactation, whereas insulin decreased during parturition and did not recover in the first 4 wk of lactation. Cows re-established their energy balance by 4 wk of lactation and had NEFA plasma concentrations of 0.47 mEq/L that were no longer different from those during pregnancy.

ANGPTL8 expression in the liver measured in the same cows was increased at 4 wk and 1 wk before parturition and decreased rapidly during parturition and in the 1 wk following it (Fig. 2C). Gene expression recovered 4 wk after parturition. Western blot analysis showed that relative abundance of plasma ANGPTL8 was decreased at parturition as compared with that at −1 wk (Fig. 2D). No significant changes were observed in the expression of ANGPTL8 or lipid metabolism-related genes [glycerol-3-phosphate acyltransferase 1 (GPAT1), low-density lipoprotein receptor (LDLR), and fatty acid synthase (FAS)] in the liver from the early lactation, mid lactation, late lactation, and dry-off periods (Fig. 3).

ANGPTL8 Gene Expression in Liver Biopsied from Young Cattle Fed Different Diets

Catte fed concentrate (Con) with high nutrient content showed 61% increase in BW and 12% increase in height, as compared with cattle fed with hay, which has low nutrient content. A comparison of ANGPTL8 gene expression in the liver of cattle revealed that ANGPTL8 expression did not differ between the 2 groups. There were no significant differences in expression of the FAS or phosphoenolpyruvate carboxykinase 1 (PCK1) genes (Fig. 4); however, significant differences were observed in pyruvate carboxylase (PC) gene expression.

Expression of ANGPTL8 in Liver and Adipose Tissue of Bulls and Steers

Hematoxylin and eosin staining showed that the size of adipocytes in adipose tissue was larger in steers than in bulls (Fig. 5A) indicating that castration might induce adipose hypertrophy. Expression of the ANGPTL8 gene was not different between bulls and steers in the liver, ABF, or LM (Fig. 5B).

Factors Regulating ANGPTL8 Expression in Cultured Bovine Hepatocytes

Figure 6 shows the effect of fatty acids (palmitate and oleate) and insulin, which might be possible regulating factors, on ANGPTL8 gene expression in cultured bovine hepatocytes. Palmitate and oleate treatments of 250 µM decreased the expression of ANGPTL8 (Fig. 6A and B). Moreover, treatment with insulin (10 and 100 nM) significantly increased ANGPTL8 expression (Fig. 6C).

Effects of ANGPTL8 on Cultured Differentiated Adipocytes and Mammary Epithelial Cells

Figure 7 shows the effects of ANGPTL8 on genes associated with lipid metabolism in adipose tissues. Expression of LPL, cluster of differentiation 36 (CD36), and FAS were not changed by ANGPTL8 treatment in cultured bovine adipocytes. However, expression of the hormone-sensitive lipase (HSL) gene was downregulated by the treatment.

Figure 8 shows the effect of ANGPTL8 on genes associated with lipid metabolism in MAC-T cells. The expression of CD36, FAS, acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD) were downregulated by ANGPTL8 treatment, but the expression of LPL was not altered by the treatment.

DISCUSSION

Dramatic endocrine changes resulted in NEFA mobilization from adipose tissues in most dairy cows, beginning a few weeks prepartum (Bell, 1995). This adaptive mechanism was presumed to be favorable for providing energy and substrates for lactogenesis, whereas a disturbance in the underlying process would lead to pathological situations and improper hormonal control. The present study was conducted to understand the role of ANGPTL8 in lipid metabolism in cows, including the change in its plasma concentration, the regulation of its production, and the physiological effect on cell types involving lactogenesis. The results demonstrated that ANGPTL8 was produced most abundantly in the liver and that the gene expression and ANGPTL8 in blood declined during parturition, which was the beginning of the negative energy balance period, with implications for lipid metabolism at the onset of lactation. Decreased ANGPTL8 expression in the liver at parturition in dairy cattle is slightly different from that observed in humans. The latter exhibit a decline in serum ANGPTL8 during late pregnancy, whereas the plasma insulin concentration is elevated (Zielińska et al., 2016). Therefore, we aimed to identify the factors regulating hepatic ANGPTL8 expression in cows. Some studies of ANGPTL8 expression regulatory factors and other related hormones have been conducted. The concentrations of ANGPTL8 in mice derived from brown adipocytes have been found to be significantly decreased postpartum as compared with those during pregnancy (Martinez-Perez et al., 2016). The hepatic ANGPTL8 expression in mice is upregulated by a high-fat diet or administration of an insulin receptor antagonist associated with elevated insulin concentration (Zhang, 2012; Zhang and Abou-Samra, 2014), and another study revealed that treatment with insulin induces ANGPTL8 in 3T3-L1 cells (Ren et al., 2012). In dairy cows, it is well known that plasma insulin concentrations decline around parturition despite elevated peripheral insulin resistance, which is associated with increased plasma NEFA (Bell, 1995). Indeed, insulin treatment for 24 h increased ANGPTL8 gene expression in cultured bovine hepatocytes, whereas palmitate and oleate, which are representative NEFA, downregulated it. Lactating cows that are supposed to have insulin-resistant peripheral tissues had decreased insulin (0.15-fold decrease at 1 wk vs. −4 wk) and increased NEFA concentrations (6.8-fold increase at 1 wk vs. −4 wk) than nonlactating cows. Thus, declined insulin and elevated NEFA concentration around the time of parturition should directly downregulate hepatic ANGPTL8 gene expression in cows. This raises the possibility that the expression of ANGPTL8 and its release from the liver declined after parturition leading to the adaptation of lipid metabolism during negative energy balance via an endocrine pathway in postpartum lactating cows.

The results of ANGPTL8 downregulation immediately after parturition led us to consider whether this endocrine alteration was associated with acute metabolic adaptation during parturition. The lack of significant changes in hepatic ANGPTL8 expression among cows during the early, mid, and late lactation, and dry-off periods indicated that drastic alterations in plasma metabolites and hormones acutely downregulated ANGPTL8 expression. To support this idea, the effects of castration and a long-term nutrition treatment were investigated in beef cattle. Castration in calves leads to increased hepatic and plasma lipid content owing to the diminished effect of testosterone and altered energy metabolism (Baik et al., 2015). In the present study, ANGPTL8 expression was not different between bulls and steers, although hepatic lipid content was greater in steers than in bulls. In addition, there was no difference in hepatic ANGPTL8 expression between young cattle fed with concentrate diet and those fed with hay from the age of 3 to 10 mo. The plane of nutrition from 3 to 10 mo after weaning in Japanese Black cattle altered both the cows’ endocrine profiles and metabolites. Insulin concentrations increased in the high-nutrition group as compared with that in the roughage group at 5 and 10 mo of age with no significant differences in glucose concentration (Ebara et al., 2013). Elevated insulin owing to high nutrition over the long term did not change the expression of ANGPTL8 in the liver. These results suggested that hepatic ANGPTL8 expression declined acutely during parturition, but were not altered in cattle with sufficiently long adaptation periods to counteract the effects of different diets or lack of testosterone over a long period. However, further studies on dairy cows fed with feed containing different levels of dietary energy or subjected to long-term insulin treatment are required to validate this idea.

In the present study, adipose tissue and mammary glands were assumed to be the main peripheral targets of ANGPTL8. Several studies have reported that this protein has regulatory effects on lipid metabolism owing to its regulation of TG and cholesterol concentrations in plasma through the inhibition of LPL with the cooperation of ANGPTL3 and ANGPTL4 (Ren et al., 2012; Zhang, 2012; Wang et al., 2013). This mechanism encourages the utilization of circulating TG in metabolically active tissue, such as skeletal muscle and adipose tissue (for storage), depending on systemic energy levels (e.g., fed or fasted state) (Zhang, 2016). LPL expression in mammary glands is considerably upregulated in early than mid or late lactation or in the dry-off period (Zhao et al., 2014) indicating that LPL may facilitate the uptake of lipids from circulating TG for lactogenesis in mammary glands. The present study showed that the AA sequence of the functional domain called the SE1 region, which is thought to bind to LPL to inhibit its activity (Siddiqa et al., 2016), was completely conserved between cattle, humans, and mice. This result suggests the mode of action of ANGPTL8 in cattle is similar among these species. In this study, however, ANGPTL8 did not directly act on LPL gene expression in bovine mammary epithelial cells or adipocytes; however, we did not investigate whether bovine ANGPTL8 inhibits peripheral LPL activity. Meanwhile, HSL expression in adipocytes and CD36, FAS, ACC, and SCD expression in mammary epithelial cells were downregulated by ANGPTL8. These observations suggested that the reduction of ANGPTL8 secretion induced by parturition acted on adipocytes and mammary epithelial cells. In adipocytes, decreased ANGPTL8 is thought to upregulate HSL, which facilitates the release of FFA in blood thereby supplying substrate to the mammary glands for lactation. In addition, lipogenesis in mammary epithelial cells is facilitated by reduced serum ANGPTL8, which might lead to the upregulation of genes involved with NEFA uptake or lipogenesis as discussed above. A reduction of ANGPTL8 secretion and production during a negative energy balance suggests a causal relationship between ANGPTL8 production in liver tissue and whole-body lipid homeostasis including lipid mobilization from adipose tissues and lactogenesis in mammary glands (as illustrated in Fig. 9). However, further experiments using bovine ANGPTL8 instead of mouse ANGPTL8 are necessary to support these hypotheses.

Figure 9.

Figure 9.

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Proposed model for the regulation and physiological role of ANGPTL8 released from hepatocytes on adipocytes and mammary epithelial cells under normal and postpartum conditions in cattle. Postpartum, the reduction of ANGPTL8 expression in hepatocytes, which is induced by a negative energy balance, upregulates HSL in adipocytes and consequently facilitates the release of FFA in blood thereby supplying the substrate for lactation to the glands (left). Under normal conditions, the recovery of ANGPTL8 secretion induces an increase of lipogenesis in adipocytes and mammary epithelial cells (right).

In conclusion, expression and production of ANGPTL8 were altered by metabolic adaptation during the transition periods in cows, possibly by the direct effect of insulin and NEFA on hepatocytes. This alteration appeared to be beneficial for lipid mobilization at the onset of lactation, which was associated with a negative energy balance in postpartum cows, through the regulation of genes associated with lipid metabolism in adipose tissue and mammary glands. Thus, ANGPTL8 appears to be a novel key factor for metabolic transition and the pathological processes related to lipid metabolism in lactating cows, which would provide beneficial effects of reverting or improving the condition of negative energy balance.

SUPPLEMENTARY DATA

Supplementary data are available at Journal of Animal Science online.

Supplementary Material
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Supplementary Figures
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ACKNOWLEDGMENTS

This work was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (18H02325). We would like to thank the farm staff at the NARO Institute, Kyushu University, and Seoul National University for managing the animals and for their cooperation in sample collection.

Conflict of interest statement. None declared.

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