Fig 1.
(A) alignment of chemerin protein sequences from 9 species. (B) dendrogram showing an evolutionary tree of those species. (C) human and mouse chemerin C-terminal peptides. Chemerin sequences (RARRES2) were extracted from the Ensembl database and compared using Clustal omega.
Table 1.
Properties of chemerin forms.
Fig 2.
Biological activities of mouse chemerin forms.
(A) the indicated concentrations of mchem161T(blue), mchem157R(red), mchem156S(green), mchem155F(purple), and mchem154A (brown) were assayed for their chemotactic activity on mCMKLR1/L1.2 cells. (B) Calcium flux in mCMKLR1/L1.2 cells in response to these proteins.
Fig 3.
Characterization of specific antibodies against chemerin forms.
(A) purified antibodies specific for different chemerin forms were characterized by western blot analysis of mchem161T (lane 1), mchem157R (lane 2), mchem156S (lane 3), mchem155F (lane 4), and mchem154A (lane 5) with anti-mchem162K, anti-mchem157R, anti-mchem156S, anti-mchem156F, and anti-mchem154A. Molecular mass markers are shown on the left of the panels. (B) mchem161T (blue), mchem157R (red), mchem156S (green), mchem155F (purple) and mchem154A (brown) were detected with specific ELISAs using anti-mchem162K, anti-mchem157R, anti-mchem156S, anti-mchem155F, and anti-mchem154A respectively. Bottom right panel displays the results of mchem161T (blue), mchem157R (red), mchem156S (green), mchem155F (purple) mchem154A (brown) and R&D standard (black) in the R&D total mouse chemerin ELISA.
Fig 4.
Weight and body fat % of mice fed with a low-fat or high-fat diet.
Weight (A) and body fat % (B) of 12 weeks old mice fed with either a LF (designated with a blue circle) or HF (designated with a red square) diet, and 26 weeks old mice fed with either a LF (designated with a blue triangle) or HF (designated with a red triangle) diet were determined by DEXA scanner. Horizontal lines show the mean ± SEM. (n = 10–14). Multigroup comparisons were by ANOVA followed by Kruskal-Wallis analysis. *: p<0.05; **: p<0.01; *** p<0.001; ****: p<0.0001. (C) relationship between mouse body fat % and weight (n = 49), The Pearson correlation coefficient was calculated with a two-tailed p value.
Table 2.
Chemerin forms in plasma of mice fed with low-fat or high-fat diet.
Fig 5.
Chemerin levels in plasma of mice fed a low-fat or high-fat diet.
Total chemerin (A), mchem162K (B), mchem157R (C), mchem156S (D), mchem155F (E), and mchem154A (F) levels in plasma of 12 weeks old mice fed with either a low-fat (LF) (designated with a blue circle) or high-fat (HF) (designated with a red square) diet, and 26 weeks old mice fed with either a LF (designated with a blue triangle) or HF (designated with a red triangle) diet were determined using total chemerin ELISA and specific ELISAs. Horizontal lines show the mean ± SEM. (n = 10–14). (G) chemerin forms in plasma presented as a percentage of the sum of the five specific ELISAs (mchem162K + mchem157R + mchem156S + mchem155F + mchem154A). Comparisons were by ANOVA followed by post-hoc Kruskal-Wallis analysis with two-tailed p value. *: p<0.05; **: p<0.01; ****: p<0.0001. Relationship between mouse plasma total chemerin and weight (H), plasma total chemerin and body fat % (I), plasma mchem162K and weight (J), and plasma mchem162K and body fat % (K) (n = 47). The Pearson correlation coefficient was calculated with a two-tailed p value.
Fig 6.
Chemerin levels in adipose tissue extracts of mice fed a low-fat or high-fat diet.
Total chemerin in brown fat extracts (A) and epididymal fat extracts (B), mchem162K, mchem155F and mchem154A in epididymal fat extracts (C, D, and E) of 12 weeks old mice fed with either a LF (designated with a blue circle) or HF (designated with a red square) diet, and 26 weeks old mice fed with either a LF (designated with a blue triangle) or HF (designated with a red triangle) diet were determined by ELISA. Horizontal lines show the mean ± SEM. (n = 10–14). There was no mchem157R and mchem156S detected in epididymal fat extracts. (F) chemerin forms in epididymal fat extracts presented as a percentage of the sum of the five specific ELISAs. Relationship between epididymal fat extracts total chemerin and weight (G), body fat % (H), or plasma total chemerin (I), brown fat extracts total chemerin and weight (J), body fat % (K), or plasma total chemerin (L) (n = 47), multigroup comparisons were by ANOVA followed by Kruskal-Wallis analysis. **: p<0.01; *** p<0.001 ****: p<0.0001. The Pearson correlation coefficient was calculated with a two-tailed p value.
Table 3.
Total chemerin and adiponectin in mice fed with low-fat or high-fat diet.
Fig 7.
Adiponectin levels in plasma and adipose tissue extracts of mice fed a low-fat or high-fat diet.
Adiponectin in plasma (A), epididymal fat extracts (B), and brown fat extracts (C) of 12 weeks old mice fed with either a LF (designated with a blue circle) or HF (designated with a red square) diet, and 26 weeks old mice fed with either a LF (designated with a blue triangle) or HF (designated with a red triangle) diet were determined with the adiponectin ELISA. Horizontal lines show the mean ± SEM. (n = 4 for A, n = 5–11 for B, and n = 10–14 for C). Multigroup comparison was by ANOVA followed by Kruskal-Wallis analysis. *: p<0.05; **: p<0.01; *** p<0.001 ****: p<0.0001. (D) relationship between adiponectin and total chemerin in epididymal fat extracts (n = 30). The Pearson correlation coefficient was calculated with a two-tailed p value.
Fig 8.
Summary of chemerin changes with diet induced obesity.
Mice were started on either a high fat diet (HFD) or low fat diet (LFD) at 6 weeks old (wo) and were sacrificed at 12 wo or 26 wo for analysis of chemerin levels in plasma, epididymal (white) and brown adipose tissue (fat). Mice on HFD were heavier and had higher %fat at 26 wo. Increase in levels shown as red arrows and decrease in levels shown as a green arrow.