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.

 


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.

 


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.

 


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).