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Journal of Animal Science - Animal Physiology

Postweaning changes in the expression of chemerin and its receptors in calves are associated with the modification of glucose metabolism1

 

This article in JAS

  1. Vol. 94 No. 11, p. 4600-4610
     
    Received: May 27, 2016
    Accepted: Sept 02, 2016
    Published: October 27, 2016


    2 Corresponding author(s): sanggun.roh@tohoku.ac.jp
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doi:10.2527/jas.2016-0677
  1. Y. Suzuki*,
  2. S. Haga*†,
  3. M. Nakano,
  4. H. Ishizaki,
  5. M. Nakano*,
  6. S. Song,
  7. K. Katoh* and
  8. S. Roh 2*
  1. * Laboratory of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 9818555, Japan
     Division of Grassland Utilization, Institute of Livestock and Grassland Science, NARO, Nasushiobara, Tochigi 3292793, Japan
     Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 6908504, Japan

Abstract

Chemerin, originally known as a chemoattractant derived from adipose tissue and the liver, has been reported to have regulatory functions in gluconeogenesis, peripheral insulin sensitivity, and insulin secretion. This study was conducted to assess the postweaning changes in expression of this cytokine and its physiological role in the modification of glucose metabolism associated with weaning. Eighteen tissue samples were collected from Holstein calves (90 d of age; n = 4) to investigate the tissue distributions of chemerin and its receptors genes. Chemerin was highly expressed in the liver, and secreted chemerin protein was found in the plasma. Among the receptors of chemerin, chemokine-like receptor 1 and C-C chemokine receptor-like 2 were ubiquitously expressed whereas G protein-coupled receptor 1 was predominantly expressed in the liver. The changes in glucose metabolism and expression of these genes after weaning were assessed by comparing suckling calves (n = 6) and weaned calves (n = 8) of Japanese Black cattle. No significant difference was observed in plasma glucose levels between suckling and weaned calves (P = 0.22), whereas the plasma level of total ketone bodies was significantly higher in weaned calves (P < 0.01). Plasma levels of insulin and cortisol did not differ between suckling and weaned calves. The mRNA levels of certain key enzymes involved in hepatic gluconeogenesis were also altered; for instance, pyruvate carboxylase level was lower in postweaning calves (P < 0.05) and phosphoenolpyruvate carboxykinase 2 (PCK2) level tended to be higher after weaning (P = 0.08). However, PCK1 was not altered after weaning. The plasma levels of hepatic stress indicators were also changed, with aspartate transaminase, alanine transaminase, and lactate dehydrogenase being significantly elevated in postweaning calves (P < 0.05). Chemerin protein in liver tissue was less abundant in weaned calves (P < 0.05), although there were no changes in its transcript levels. The abundance of plasma chemerin protein did not change after weaning (P = 0.95). In summary, these data indicate that as a consequence of weaning, which causes physiological stress and alters hepatic metabolism, chemerin protein expression within the liver is downregulated, indicating that chemerin plays a role in the upregulation of hepatic PCK2 expression via its inhibitory effect on hepatic gluconeogenesis.



INTRODUCTION

Calves experience marked alteration in nutrient metabolism following weaning, which is defined by the onset of utilization of VFA produced by ruminal fermentation for hepatic gluconeogenesis (Donkin and Hammon, 2005; Kato et al., 2016). The role of hormonal regulation in the metabolic development of weaning calves has been intensively investigated, and the major hormones involved in this process have been identified. For example, glucagon, glucocorticoids, and catecholamine act as inducers of hepatic gluconeogenesis, whereas insulin has an antagonizing effect (Donkin and Armentano, 1994; Hocquette, 2010; Hammon et al., 2012; Jacometo et al., 2016).

Recent studies have revealed the emerging roles of hepatokines, hormone-like endocrine factors derived from the liver, in the energy metabolism of nonruminant animals. Of these endocrine factors, chemerin was originally identified as a novel chemoattractant for antigen-presenting cells (Wittamer et al., 2003). Chemerin was later found to be highly expressed in adipose tissue and less expressed in the liver and to play regulatory roles in energy metabolism (Ernst and Sinal, 2010; Roh et al., 2016). Chemerin induces insulin resistance in skeletal muscle and liver tissues (Sell et al., 2009; Ernst et al., 2010). Notably, chemerin knockout mice displayed impaired basal and glucose-stimulated insulin secretion and upregulated gene expression of hepatic gluconeogenic enzymes (Takahashi et al., 2011). Limited information exists for the potential roles for chemerin in calves, although observations in other species suggest chemerin is a novel regulator of glucose metabolism. This study aimed to 1) identify the origin and target tissues of chemerin protein in calves, 2) investigate changes in the expression of chemerin and its receptors (chemokine-like receptor 1 [CMKLR1], C-C chemokine receptor-like 2 [CCRL2], and G protein-coupled receptor 1 [GPR1]) after weaning, and 3) determine its regulatory role in glucose metabolism during weaning.


MATERIALS AND METHODS

Management of Animals and Collection of Samples

All animals were bred at the National Agriculture and Food Research Organization (NARO) Institute of Livestock and Grassland Science, Tochigi, Japan. The study was conducted in accordance with the “Guideline for the NARO Institute of Livestock and Grassland Science” (NARO, 2011) and was approved by the Animal Care Committees of the NARO Institute of Livestock and Grassland Science.

To investigate the gene expression of chemerin and its receptors in calf tissues, 4 Holstein calves (3 females and 1 male) were used, all of which were born between February and March 2011. These calves were individually housed and raised on commercial milk replacer and grower feed (Zenrakuren, Tokyo, Japan) before weaning at 56 d of age (the guaranteed ingredient compositions of the milk replacer and the grower feed for Holstein calves are shown in Supplemental Table S1; see the online version of the article at http://journalofanimalscience.org). After weaning, they were limited to grower feed and timothy hay (Phleum pratense) fed twice per day. The calves had ad libitum access to water throughout the experimental period. At 90 d of age, the calves were euthanized by overdose administration of sodium pentobarbital via the jugular vein and exsanguination, and subsequently, plasma and the following tissues were collected: liver, perirenal adipose, skeletal muscle, cardiac muscle, adrenal gland, renal cortex, lung, thymus, spleen, rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, rectum, and colon.

To examine changes in the expression of chemerin and its receptors after weaning, 14 male Japanese Black calves born between February 2013 and April 2014 were randomly assigned to 2 groups: suckling (n = 6) and weaned (n = 8). All calves were raised by natural suckling and were allowed free access to the dams’ feed before weaning. The calves in the suckling group were euthanized at 38 ± 6 d of age. The calves in the weaned group were separated from their dams for weaning at 88 ± 3 d of age. Thereafter, they were raised by group feeding until being slaughtered at 107 ± 6 d of age. They were fed with commercial grower feed for Japanese Black calves (ZEN-NOH, Tokyo, Japan) and timothy hay at 0900 and 1600 h each day to achieve an ADG of 0.6 kg, which is in accordance with the Japanese Feeding Standard for Beef Cattle (Minister of Agriculture, Forestry and Fisheries (MAFF), 2000). The guaranteed ingredient composition of the grower feed for Japanese Black calves is shown in Supplemental Table S1 (see the online version of the article at http://journalofanimalscience.org). Calves were also allowed ad libitum access to water and commercial mineral blocks containing Fe, Cu, Co, Zn, Mn, I, Ca, Se, Cl, and Na (Zenoaq, Fukushima, Japan). They were denied access to feed on the morning of the sampling day. Jugular blood was sampled before euthanasia. Euthanasia was performed by overdose administration of sodium pentobarbital via the jugular vein followed by exsanguination. Tissue samples were collected from euthanized calves, including the liver, skeletal muscle (longissimus lumborum), and adipose tissues (subcutaneous adipose tissue, mesenteric adipose tissue [MesAT], epididymal adipose tissue, and perirenal adipose tissue).

Plasma was isolated from heparinized jugular blood samples by centrifugation at 1,000 × g for 20 min at 4°C and stored in microtubes at −80°C. Tissue samples were snap frozen with liquid nitrogen and stored at −80°C until further analyses. Sampling was conducted at approximately 1200 h each day.

Plasma Hormone, Enzyme, and Metabolite Analysis

The plasma cortisol concentration was measured by competitive ELISA provided by Enzo Life Sciences, Inc. (Farmingdale, NY) following the manufacturer’s protocol. The concentrations of the following plasma metabolites and enzymes were measured using a blood analyzer at Obihiro Clinical Laboratory Inc. (Hokkaido, Japan): glucose, total cholesterol, triglyceride, phospholipid, NEFA, acetoacetate, β-hydroxyl butyrate, total ketone bodies, blood urea nitrogen, creatinine, sodium, potassium, calcium, magnesium, chloride ion, inorganic phosphorus, creatine phosphokinase, albumin, total protein, aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH).

Quantitative Reverse Transcription PCR

Total RNA was extracted using RNAiso Plus (TaKaRa Bio Inc., Shiga, Japan). The concentration of isolated total RNA was determined by optical density measurement at 260 nm and its purity was checked by the ratios of optical densities of 260:280 nm and 260:230 nm, using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) . The Ratios of 260:280 nm and 260:230 nm were greater than 1.8 in all samples. The integrity of RNA was checked by agarose gel electrophoresis. First-strand cDNA was synthesized from 500 ng of total RNA using the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa Bio Inc.). Gene expression analyses were performed via quantitative reverse transcription PCR using SYBR Premix Ex Taq II (Tli RNaseH Plus; TaKaRa Bio Inc.) with a DNA Engine Opticon 2 Continuous Fluorescence Detector (Bio-Rad Laboratories, Hercules, CA). Primer sequences and performances are described in Supplemental Table S2 (see the online version of the article at http://journalofanimalscience.org). The PCR efficiencies were checked by constructing standard curves of serial dilutions of pooled cDNA. β-Actin, cyclin G-associated kinase (GAK), and vacuolar protein sorting 4 homolog A (VPS4A) were used as reference genes. Transcript expression relative to that in the suckling group was calculated using the method described by Vandesompele et al. (2002).

Western Blotting

Frozen animal tissues were homogenized in radioimmunoprecipitation assay buffer supplemented with 1 mM Na3VO4 and a protease inhibitor cocktail (Nacalai Tesque Inc., Kyoto, Japan). Plasma samples were diluted with PBS by a factor of 10. A total of 10 μg of extracted protein or 10 μL of diluted plasma samples were separated by 15% SDS-PAGE and electroblotted onto a Clear Blot-P Plus membrane (ATTO Corporation, Tokyo, Japan). The membranes were blocked using 3% BSA in Tris-buffered saline and Tween 20 (TBST) for 30 min. Membranes were incubated with primary antibodies that were diluted in antibody dilution buffer (TBST containing 0.5% BSA, 0.1% polyvinylpyrrolidone 360, and 0.5% polyethylene glycol 6000) overnight at 4°C followed by incubation with secondary antibodies for 1 h at room temperature. The antibodies used for western blotting were as follows: anti-bovine chemerin (custom-made rabbit polyclonal IgG raised against antigen peptide; CVTSVDNAADTLFPAGQF, purified by antigen affinity chromatography), anti β-actin (sc47778; Santa Cruz Biotechnology, Dallas, TX), anti-rabbit IgG horseradish peroxidase (HRP)-conjugated (W4011; Promega Corporation, Madison, WI), and anti-mouse IgG HRP-conjugated (W4021; Promega Corporation). Recombinant human chemerin corresponding to Glu21-Ser157 (R&D Systems, Inc., Minneapolis, MN) was used as a positive control. Detection of the probed band was achieved using an ECL Prime Western Blotting Detection System and ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK).

Immunohistochemistry

Fresh liver tissues from Japanese Black calves were fixed with 10% formaldehyde/PBS and were then embedded in paraffin. Tissue sections were deparaffinized in xylene and were rehydrated with a dilution series of ethanol followed by antigen retrieval via autoclave treatment in citrate buffer (pH 6.0). Sections were then incubated in 1% peroxide diluted in methanol to inactivate endogenous peroxidase and were blocked with blocking buffer (2% normal goat serum diluted in phosphate buffered saline containing 0.05% tween-20. [PBST]). Sections were incubated with anti-bovine chemerin rabbit polyclonal IgG (described above), anti-human CMKLR1 rabbit polyclonal IgG (ab82773; Abcam, Cambridge, UK), or anti-human CCRL2 rabbit polyclonal IgG (LS-A1094; LifeSpan BioSciences Inc., Seattle, WA) diluted with Can Get Signal Immunostain Immunoreaction Enhance Solution (Toyobo Co., Ltd., Osaka, Japan) at 4°C overnight. After washing in PBST, tissue sections were incubated with HRP-labeled goat anti-rabbit IgG (424141; Nichirei Bioscience, Tokyo, Japan) at room temperature for 1 h. Sections were washed in PBST, and the immunocomplex was visualized using diaminobenzidine substrate (Nichirei Bioscience). To evaluate the specificity of the anti-bovine chemerin antibody, polyclonal IgG was absorbed by an antigen-immobilized Sepharose column (GE Healthcare, Little, Chalfont, UK), and column flow through was used as a negative control.

Statistical Analysis

Overall data from quantitative reverse transcription PCR, western blotting, and plasma hormones, metabolites, and enzymes are presented as least squares means ± SEM. The mRNA levels of chemerin and its receptors among the 18 tissues from Holstein calves were statistically analyzed using 1-way ANOVA. If a significant difference was detected between tissues (P < 0.05), Tukey’s honest significant difference test was used to analyze the difference in mean values of mRNA levels between the tissues. Differences between the data from suckling and weaned Japanese Black calves were analyzed using the Mann–Whitney U test, because of the non-normal distribution of the data. Differences between suckling and weaned calves were considered significant when P < 0.05, and P < 0.1 was deemed to indicate tendencies. All statistical analyses were performed using the statistical package R (version 2.8.1; available as a free download from https://www.r-project.org [accessed 30 Nov. 2011]).


RESULTS AND DISCUSSION

Tissue Distributions of Chemerin and Its Receptors mRNA and Protein

Although our previous study reported mRNA expression of chemerin in bovine adipose and liver tissues, the relative mRNA levels in the tissues were unclear (Song et al., 2010). In weaned Holstein calves, chemerin mRNA was highly expressed in the liver, and lower levels of mRNA were detected in other tissues, including adipose tissue (Fig. 1A). Chemerin protein was detected in both the liver (Fig. 1E and 2A) and plasma (Fig. 1F). CMKLR1 mRNA was highly expressed in the lung, and lower amounts of mRNA were detected in the remainder of the tissues investigated, including the spleen, liver, and adrenal tissues (Fig. 1B). CCRL2 mRNA was highly expressed in lung and colon tissues, with lower expression in all other tissues (Fig. 1C). In contrast, GPR1 mRNA was expressed almost predominantly in the liver but was also detected in the forestomach at lower levels (the rumen, reticulum, and omasum; Fig. 1D). Expression of CMKLR1 and CCRL2 proteins was detected in the liver tissue (Fig. 2B and 2C).

Figure 1.
Figure 1.

Messenger RNA and protein expression of chemerin and its receptors in a range of bovine tissues and plasma. Relative mRNA levels of chemerin (A), chemokine-like receptor 1 (CMKLR1; B), C-C chemokine receptor-like 2 (CCRL2; C), and G protein-coupled receptor 1 (GPR1; D) in 18 tissues from 4 Holstein calves were quantified using quantitative reverse transcription PCR. The mRNA levels are represented as least squares means ± SEM, relative to values detected in the liver. a,bSignificant differences (P < 0.05, as determined by Tukey’s honest significant difference test) among the values for each gene in the tissues. Chemerin protein was detected in the liver tissues (E) and plasma (F) of Japanese Black calves via western blotting. Commercial recombinant human chemerin was used as a positive control. AT = adipose tissue; Actb = beta-actin.

 
Figure 2.
Figure 2.

Protein expression of chemerin and its receptors in calf liver tissues. Protein expression and the localization of chemerin (A), chemokine-like receptor 1 (CMKLR1; B), and C-C chemokine receptor-like 2 (CCRL2; C) in liver slices from Japanese Black calves were visualized using immunohistochemistry. Cells expressing chemerin, CMKLR1, or CCRL2 were visualized using diaminobenzidine substrate (brown); scale bar = 20 μm. The antibody specificity of chemerin was assessed using an absorption treatment with an antigen peptide-immobilized column (D). Nonspecific staining was checked using immunohistochemistry with anti-rabbit IgG (E) only.

 

The relative expressional levels of chemerin in tissues varies among animal species (Bozaoglu et al., 2007; Goralski et al., 2007; Song et al., 2010; Takahashi et al., 2011); however, the consensus indicates that chemerin is highly expressed in the liver and adipose tissues, which may contribute to the circulating chemerin. Our data showed that the highest expression levels of chemerin mRNA in calves were detected in the liver and that these levels were 17-fold higher than those detected in the perirenal adipose tissues. Protein expression of chemerin was also observed in the liver, and secreted chemerin was detected in the plasma. These results indicate that in cattle, chemerin is a hepatokine localized to the liver rather than an adipokine. Furthermore, the expression patterns of chemerin receptors (CMKLR1, CCRL2, and GPR1) in cattle tissues differed from those observed in mice. For example, CMKLR1 was highly expressed in the lung, spleen, liver, and adrenal tissues of cattle, whereas it was highly expressed in the white adipose tissues, cardiac muscle, and testis of mice (Takahashi et al., 2011). In addition, CCRL2 was highly expressed in the lung, ileum, cecum, and colon tissues of cattle but was highly expressed in the cardiac muscle and pancreas tissues of mice (Zabel et al., 2008; Takahashi et al., 2011). GPR1 was highly expressed in the liver, rumen, reticulum, and omasum tissues of cattle, whereas it was highly expressed in the white adipose tissue, brown adipose tissue, and skeletal muscle tissues of mice (Rourke et al., 2014). These results indicate that in cattle, CMKLR1 is ubiquitously expressed, CCRL2 is expressed mainly in tissues containing epithelia (namely lung and intestinal), and GPR1 is expressed in the liver and forestomach, which are prominent sites of VFA metabolism. Therefore, chemerin is suggested to be a novel hepatokine that exhibits both endocrine (systemic effect in the body) and paracrine (local effect within the liver) characteristics in cattle

Plasma Metabolite Concentrations in Suckling and Weaned Calves

Before investigating changes in the expression of chemerin and its receptors in suckling and weaned Japanese Black calves, the metabolic conditions of these animals were monitored. The BW of suckling and weaned calves at slaughter was 48 ± 1 and 131 ± 8 kg, respectively. As shown in Table 1, there were no significant differences in the plasma concentrations of glucose (P = 0.22). Regarding lipid metabolism, concentrations of total cholesterol and triglycerides were not altered following weaning (P = 0.13 and P = 0.17, respectively). However, the plasma concentrations of phospholipids tended to be lower in postweaning calves (P = 0.09). Although concentrations of NEFA were not significantly different between suckling and weaned calves (P = 0.22), the plasma concentrations of acetoacetate, β-hydroxyl butyrate, and total ketone bodies were elevated 2- to 3-fold in postweaning calves (P < 0.05, P < 0.01, and P < 0.01, respectively). There were no significant changes in the plasma levels of albumin, total protein, blood urea nitrogen, and creatinine, which are indicators of protein metabolism. Plasma inorganic phosphorus levels were lower (P < 0.05) and chloride ion tended to be higher (P = 0.08) in weaned calves.


View Full Table | Close Full ViewTable 1.

Plasma levels of metabolites and minerals in suckling and weaned calves

 
Item Suckling calves Weaned calves
Glucose, mg/dL 98.2 ± 7.3 83.6 ± 1.3
Total cholesterol, mg/dL 104.2 ± 14.2 78.3 ± 4.8
Triglyceride, mg/dL 17.8 ± 3.3 12.6 ± 1.3
Phospholipid, mg/dL 128.5 ± 16.0 95.3 ± 5.0†
NEFA, mEq/L 0.285 ± 0.04 0.360 ± 0.05
Acetoacetate, μmol/L 3.00 ± 0.37 7.86 ± 1.91*
Beta-hydroxybutyrate, μmol/L 106.0 ± 21.7 303.0 ± 54.5**
Total ketone bodies, μmol/L 109.0 ± 21.9 310.9 ± 56.3**
Albumin, g/dL 3.70 ± 0.12 3.93 ± 0.06
Total protein, g/dL 6.28 ± 0.25 6.81 ± 0.11†
BUN,1 mg/dL 7.32 ± 0.79 8.20 ± 1.10
Creatinine, mg/dL 1.02 ± 0.04 0.94 ± 0.05
Na, mEq/L 142.8 ± 1.0 143.0 ± 0.7
K, mEq/L 4.63 ± 0.14 4.54 ± 0.14
Ca, mg/dL 10.07 ± 0.29 9.80 ± 0.14
Mg, mg/dL 1.85 ± 0.11 2.04 ± 0.06
Cl, mEq/L 100.8 ± 0.6 102.9 ± 0.9†
iP,2 mg/dL 8.15 ± 0.53 6.77 ± 0.35*
1BUN = blood urea nitrogen.
2iP = inorganic phosphorus.
P < 0.1; *P < 0.05; **P < 0.01; suckling vs. weaned.

The abrupt weaning method is a common strategy of weaning in meat cattle. This method is considered to induce more acute changes in the metabolism of weaning calves compared with the gradual weaning method that is mainly applied to dairy calves (Enríquez et al., 2011). Because we intended to assess the effect of weaning on the gene expression of chemerin and its receptors more clearly, we used abrupt weaning in beef cattle calves in the present study. The results showed plasma levels of phospholipids tended to be lower in weaned calves. The plasma level of NEFA appeared to be slightly greater and triglyceride was lower in weaned calves, although the differences were not statistically significant. However, there were significant increases in the levels of plasma ketone bodies. These alterations could be attributed to the following 2 metabolic changes associated with weaning: the development of ruminal and hepatic functions for metabolism of VFA produced during ruminal fermentation and the slight energy deficiency caused by the abrupt weaning method. However, there was no change in plasma glucose level in weaned calves. Furthermore, neither suckling nor weaned calves showed any abnormalities in the markers for protein and mineral metabolism. Therefore, the management strategy and weaning method for calves was sufficiently appropriate to maintain a healthy condition and did not appear to induce severe energy deficiency in weaned calves.

Levels of Genes and Hormones Related to Gluconeogenesis

Pyruvate carboxylase (PC) mRNA levels in the liver were lower in weaned calves (P < 0.05), whereas those of hepatic phosphoenolpyruvate carboxykinase 2 (PCK2) tended to be greater (P = 0.08; Fig. 3A). PCK1 mRNA levels did not differ between suckling and weaned calves (P = 0.59; Fig. 3A). Plasma levels of insulin and cortisol, which are potent hormones that inhibit and stimulate hepatic gluconeogenesis, respectively, did not differ between suckling and weaned calves (P = 0.18 and P = 0.29, respectively; Fig. 3B and 3C). Furthermore, mRNA expression levels of atrogin1 and muscle RING-finger protein-1 (MuRF1), which are regulators of muscular autophagy, were not altered in the skeletal muscle of weaned calves (P = 0.23 and P = 0.95, respectively; Fig. 3D).

Figure 3.
Figure 3.

Modification of glucose metabolism in suckling and weaned calves. The mRNA levels of hepatic gluconeogenic enzymes (A) and autophagy-related factors in skeletal muscle (D) of suckling and weaned Japanese Black calves (n = 6 and n = 8, respectively) were quantified using quantitative reverse transcription PCR. Plasma insulin (B) and cortisol (C) levels in the same calves were measured using ELISA. The data are represented as the mean ± SEM, relative to values detected in suckling calves. *P < 0.05 between suckling and weaned calves (Mann–Whitney U test). PC = pyruvate carboxylase; PCK1 = phosphoenolpyruvate carboxykinase 1; PCK2 = phosphoenolpyruvate carboxykinase 2; MuRF1 = muscle RING-finger protein-1.

 

The adaptation of hepatic gluconeogenesis in weaned calves is attributed to alteration in the amount and ratio of glycogenic precursors, hepatic gluconeogenic enzymatic activity, and hormonal regulation. The contribution of propionate, derived from ruminal fermentation, to hepatic gluconeogenesis in weaned calves is almost predominant, whereas gluconeogenesis in suckling calves utilizes lactate and AA (Baldwin et al., 2004; Donkin and Hammon, 2005; Hammon et al., 2012). In response to this alteration in substrates, the activity and expressional levels of hepatic gluconeogenic enzymes are also regulated. PC, an enzyme essential for glucose production from lactate and some of glycogenic AA, is gradually downregulated in the liver of weaned ruminants. In contrast, the expression of PCK fluctuates during the weaning process (Haga et al., 2008; Jacometo et al., 2016). Our data showing decreased PC mRNA, increased PCK2 mRNA, and unchanged PCK1 mRNA in weaned calves is considered consistent with previous studies, even in beef calves that have undergone abrupt weaning, and indicates an alteration in hepatic gluconeogenesis. Hormonal regulation plays an important role in the regulation of glucose metabolism at the time of weaning. Insulin strongly inhibits the activity of gluconeogenesis in the liver. Cortisol, in contrast, induces the expression of gluconeogenic enzymes and the activity of the gluconeogenic pathway, together with glucagon under conditions of fasting and energy shortage. However, the basal plasma levels of these hormones in weaned calves have been shown to vary in previous studies (increased, unchanged, or decreased compared with suckling calves; Hickey et al., 2003; Katoh et al., 2004; Haga et al., 2008; Laarman et al., 2012). These differences may reflect the differences in weaning method and/or the nutritional status of calves. Our results indicated that the plasma concentrations of insulin and cortisol were not altered in weaned calves. Additionally, the expression levels of atrogin1 and MuRF1 in skeletal muscle, which are induced in cases of severe energy deficiency to facilitate autophagy, did not differ between suckling and weaned calves (Kandarian and Jackman, 2006). These results indicate that the abrupt weaning method used in this study did not cause any energy deficiency that was sufficiently severe to induce acute alteration in hormone secretion or to upregulate muscle autophagy to supply AA for hepatic gluconeogenesis.

Physiological States of the Liver in Suckling and Weaned Calves

The plasma levels of AST, ALT, and LDH, which are the major enzymes released when hepatocytes are degraded, were elevated in weaned calves compared with suckling calves (P < 0.05, P < 0.01, and P < 0.01; Fig. 4A, 4B, and 4C, respectively). In contrast, hepatic mRNA levels of protein translation-related proteins, namely eukaryotic translation initiation factor 4E (EIF4E) and eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), did not differ between suckling and weaned calves (Fig. 4D).

Figure 4.
Figure 4.

Plasma levels of the enzymes used to indicate hepatic condition and mRNA levels of protein translation initiation factors in the livers of suckling and weaned calves. Plasma concentrations of aspartate transaminase (AST; A), alanine transaminase (ALT; B), and lactate dehydrogenase (LDH; C) and mRNA levels of translation initiation factors in liver tissues were quantified in suckling and weaned Japanese Black calves (n = 6 and n = 8, respectively). The data are represented as the mean ± SEM, relative to values detected in suckling calves. *P < 0.05 and **P < 0.01 between suckling and weaned calves (Mann–Whitney U test). EIF4E = eukaryotic translation initiation factor 4E; EIF4EBP1 = eukaryotic translation initiation factor 4E binding protein 1.

 

The abrupt weaning method used in beef cattle often causes physiological stress in calves, resulting in decreased food intake and daily gain (Enríquez et al., 2011). Weaning stress can be assessed by plasma concentrations of enzymes indicating hepatic function, in addition to monitoring the behavioral responses, such as frequencies of vocalization and walking. The weaning process used in this study induced physiological stress in the liver, as indicated by increased plasma AST, ALT, and LDH levels, although these increases were not excessively severe and were similar to previously reported responses (Pavlík et al., 2009; Jacometo et al., 2016). We further investigated the activity of protein translation from mRNA, because this process is known to be altered by the types of physiological stress associated with elevated AST and ALT levels, such as hepatic steatosis (Soronen et al., 2016). However, although we measured the mRNA levels of EIF4E and EIF4EBP1, which are prominent factors of the protein translation system (Kindler et al., 2005), no significant changes were observed in the mRNA levels of these genes, indicating that the calves were not experiencing intense physiological stress during the postweaning period.

Changes in Expression Levels of Chemerin and Its Receptors

The mRNA levels of chemerin and its receptors were investigated in the liver, adipose tissue, and longissimus muscle of suckling and weaned calves, to assess the expressional regulation of these genes at the time of weaning. The mRNA expression of chemerin in MesAT was greater in weaned calves (P < 0.05; Fig. 5A) but not in the other tissues investigated. The levels of CMKLR1 mRNA tended to be greater in the subcutaneous adipose tissue of weaned calves (P = 0.09; Fig. 5B). Messenger RNA expression of hepatic CCRL2 and GPR1 was also greater in weaned calves (P < 0.05 and P < 0.01; Fig. 5C and 5D). Regarding protein expression and secretion of chemerin, hepatic chemerin protein was less abundant in weaned calves (P < 0.05; Fig. 6A), whereas the plasma levels of chemerin did not differ between suckling and weaned calves (Fig. 6B). Furthermore, the protein content of chemerin in MesAT did not differ between suckling and weaned calves (Fig. 6A).

Figure 5.
Figure 5.

Changes in mRNA levels of chemerin and its receptors in suckling and weaned calves. The mRNA levels of chemerin (A), chemokine-like receptor 1 (CMKLR1; B), C-C chemokine receptor-like 2 (CCRL2; C), and G protein-coupled receptor 1 (GPR1; D) in the liver, skeletal muscle (Muscle), subcutaneous adipose tissue (SubAT), mesenteric adipose tissue (MesAT), perirenal adipose tissue (PeriAT), and epididymal adipose tissues (EpiAT) of suckling and weaned Japanese Black calves (n = 6 and n = 8, respectively) were quantified using quantitative reverse transcription PCR. The mRNA levels are represented as the mean ± SEM, relative to values detected in the livers of suckling calves. *P < 0.05 and **P < 0.01 between suckling and weaned calves (Mann–Whitney U test).

 
Figure 6.
Figure 6.

Protein levels of chemerin in the liver, adipose tissues, and plasma of suckling and weaned calves. Relative protein contents of chemerin in the liver and adipose tissues (subcutaneous adipose tissue [SubAT], mesenteric adipose tissue [MesAT], perirenal adipose tissue [PeriAT], and epididymal adipose tissue [EpiAT]; A) and plasma (B) of suckling and weaned Japanese Black calves (n = 6 and n = 8, respectively) were quantified using western blotting. The protein content data are represented as the mean ± SEM, relative to the values detected in suckling calves. *P < 0.05 between suckling and weaned calves (Mann–Whitney U test).

 

Several studies have investigated the expressional regulation of hepatic chemerin, and the results identified various regulators of chemerin and its receptors, including a high-fat diet (Roh et al., 2007), fatty liver (Krautbauer et al., 2013), insulin signal (Krautbauer et al., 2013), palmitate (Bauer et al., 2011), farnesoid X receptor agonist (Deng et al., 2013), adipokines (Suzuki et al., 2012a), and cytokines (Parlee et al., 2010; Monnier et al., 2012). Moreover, the results indicated a close relationship between chemerin and both energy metabolism and immune response (Buechler, 2014). The present study determined that the expression of hepatic chemerin receptors in calves was altered as a result of weaning and that the hepatic chemerin protein content decreased in postweaning calves without alteration in transcript levels. However, most of the previously discussed factors associated with chemerin expression do not appear to be relevant to the observed postweaning decrease in hepatic chemerin protein. We initially considered that insulin is the regulator of hepatic chemerin, because of its significant role in postweaning metabolism in the liver. However, this possibility was excluded because basal plasma insulin levels did not differ between suckling and weaned calves. Our unpublished data show that insulin downregulates chemerin mRNA in cultured hepatocytes in suckling calves but that this effect was diminished in weaned calves. We also treated hepatocytes with palmitate, but no potent effect was observed on hepatic chemerin expression (unpublished data). Therefore, we consider that another factor is involved in the regulation of hepatic chemerin production during weaning, with weaning stress being regarded as a potential factor. Deng et al. (2013) reported that physiological stress in the liver, such as dietary-induced lipid accumulation and inflammation, decreases hepatic chemerin expression. Our results showed decreased plasma phospholipid levels and elevated plasma levels of AST and ALT, implicating physiological stress in the altered hepatic function of weaned calves. We also found no alteration in gene expression of protein translation–related factors, indicating there is another mechanism regulating hepatic chemerin content, such as chemerin-degrading enzymes. Although the detailed mechanism should be investigated in future studies, the present study demonstrated that hepatic chemerin cellular protein decreased during the postweaning period (2 wk after weaning) in calves, possibly due to stress in the liver caused by the abrupt weaning method.

Hepatokines, which are recently identified endocrine factors derived from the liver (Misu et al., 2010; Stefan and Häring, 2013), play significant roles in the regulation of systemic glucose homeostasis. The expression and secretion of hepatokines is closely related to energy balance in the body, as demonstrated by studies investigating the effects of high-fat diet feeding, fasting, and feeding (Iroz et al., 2015). This led us to determine the role of chemerin as a hepatokine in the glucose metabolism of weaning calves, because chemerin might act as a signal from the liver to the rest of the body in response to an alteration in dietary nutrition. According to previous studies, chemerin is considered to have 2 potent functions in glucose metabolism: the regulation of insulin signaling and hepatic gluconeogenesis (Takahashi et al., 2011; Ferland and Watts, 2015). Because our previous study showed that the administration of a chemerin peptide analog sharply induced insulin secretion in sheep (Suzuki et al., 2012b), we investigated the basal plasma levels of insulin in suckling and weaned calves. In this study, no change in plasma insulin and glucose levels was observed after weaning. Similarly, there was no significant difference in plasma chemerin protein levels, even though hepatic chemerin protein levels decreased. Therefore, this raised the following 2 possibilities: chemerin stabilizes insulin levels in weaned calves by maintaining its circulating level or it does not play a significant role in the regulation of insulin secretion in weaned calves. These possibilities will be investigated in more detail in future studies

Chemerin knockout mice have been shown to exhibit increased clamp hepatic glucose production and elevated expression of the gluconeogenic enzymes glucose 6-phosphatase and PCK (Takahashi et al., 2011), indicating that chemerin could have an inhibitory effect on hepatic gluconeogenesis. Indeed, our results showed a potential relationship between hepatic chemerin and gluconeogenic genes in cattle. In weaned calves, hepatic PKC2, an isozyme of PCK present in mitochondria, tended to increase as hepatic chemerin protein levels decreased. Although this isozyme is considered to be essential for gluconeogenesis from lactate, one study has revealed its significant role in hepatic gluconeogenesis from propionate (Mendez-Lucas et al., 2013). Because chemerin and its receptors are highly expressed in the liver, the decreased PCK2 expression in weaned calves recorded in the present study could be attributed to the paracrine activity of chemerin within the liver. The results showing that plasma insulin and cortisol levels did not differ between suckling and weaned calves support the idea that chemerin could be a regulator of hepatic PCK expression, because these hormones are well known to regulate PCK activity and expression (Granner et al., 1983; Donkin and Armentano, 1995; Scheuer et al., 2006). Therefore, the modification of glucose metabolism in weaned calves is believed to be associated with the alteration of hepatic metabolism, which is regulated, in part, by chemerin.

Conclusion

In this study, we made the following novel observations regarding chemerin in calves around weaning period. Chemerin was highly expressed in the liver of calves and was secreted into the circulatory system, indicating that chemerin functions as a novel hepatokine, acting in both endocrine and paracrine manners. Hepatic chemerin protein levels decreased during the postweaning period, which was associated with moderate energy deficiency and physiological stress in the liver. Decreased hepatic chemerin protein expression in weaned calves is believed to contribute to metabolic modification during the postweaning period via the regulation of gluconeogenesis. Our results reveal a new aspect of metabolic regulation in calves at the time of weaning, wherein the liver acts as an endocrine and paracrine organ, partly via chemerin signaling.

 

References

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