Subfatin is a novel adipokine and unlike Meteorin in adipose and brain expression

Abstract

Aims: Adipose tissue releases adipokines that play important roles in metabolic and cardio-cerebro-vascular homeostasis. This study was to discover novel adipokines using caloric restriction model.

Methods: Adipokine candidates were captured by gene array and bioinformatics analysis and verified by preparation of recombinant protein and antibody.

Results: We established a potential secreted protein database containing 208 genes and identified a novel adipokine, Subfatin, that was the highest expressed in subcutaneous fat of both rodents and humans among 15 detected tissues. The secreted mammalian Subfatin was a glycosylated protein. Subfatin was located diffusely throughout the adipose tissue except lipid droplets, with comparable expression between adipocytes and stromal cells, but much lower expression in macrophages than adipocytes. Subfatin was downregulated in white adipose tissue of caloric restriction rats, whereas dramatically upregulated during white adipocyte differentiation as well as in white adipose tissue of diet-induced obese mice. Subfatin was annotated as Meteorin-like (Metrnl) in public databases, a similar transcript of Meteorin (Metrn, also known as glial cell differentiation regulator). Meteorin displayed a brain-specific expression and was scarce in various adipose tissues, in contrast to the tissue expression patterns of Subfatin.

Conclusions: Subfatin is a novel adipokine regulated by adipogenesis and obesity, with tissue distribution different from its homologue Meteorin.

Keywords: Adipokines; Brain; Caloric restriction; Meteorin; Subfatin.

Figure 1

Food intake, body weight, and serum parameters in ad libitum (AL) and caloric restriction (CR) rats. (A) Food consumption of AL and CR rats. (B) Body weight curve of AL and CR rats. **P < 0.01 versus AL. (C) Serum glucose, insulin, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol in AL and CR rats. n = 8 in each group.

Figure 2

Screening and identification of Subfatin from adipose tissues. (A) Flowchart screening process of novel adipokines. (B) Graphical output from SignalP Server showing a 45 aa signal peptide in Subfatin. (C) Graphical output from TMHMM Server showing no transmembrane domain in Subfatin. (D) Amino acid alignment of human, rat, and mouse Subfatin sequences. Residues identical in all proteins were marked in black boxes, and similarity was shown in gray boxes. The putative NH2‐terminal signal sequences were indicated by the solid overline, and potential glycosylation site in mouse and rat Subfatin was indicated by red star. (E) The AVG_signal of Subfatin in various adipose tissues under CR. The mean value of global gene expression profiles was indicated by dotted line. AL, ad libitum; CR, caloric restriction; SAT, subcutaneous adipose tissue; MAT, mesenteric adipose tissue; PVAT, perivascular adipose tissue.

Figure 3

Identification of Subfatin as a novel adipokine. (A) Cloning of Subfatin open reading frame. (B) The purification of Subfatin prokaryotic recombinant protein. (C) Verification of Subfatin antibody using Subfatin recombinant proteins with Western blotting. (D) Subfatin was detected in both culture medium and cell lysate of Subfatin‐transfected HEK‐293 and COS‐7 cells. (E) Purification of Subfatin recombinant protein from the serum‐free culture medium of transfected HEK‐293 cells. (F) and (G) Identification of Subfatin mammalian recombinant protein with anti‐Subfatin or anti‐His₆ antibody. (H) Subfatin mammalian recombinant protein was detected with Western blotting after PNGase F digestion. (I) Subfatin in serum‐free incubation medium of adipose tissue was detected with Western blotting. (J) Identification of Subfatin mammalian recombinant proteins with mass spectrometry. CM, culture medium; PNGase F, glycopeptidase F.

Figure 4

The expression of Subfatin in different tissues and cell types. (A) The expression of Subfatin in different tissues of mice (n = 3). (B) The expression of Subfatin in different tissues of human (n = 3). (C) Comparison of Subfatin expression between subcutaneous adipose tissue (SAT) and interscapular adipose tissue (IAT) of mice (n = 3). **P < 0.01 versus IAT. (D) Comparison of Subfatin expression between adipocytes and stromal cells isolated from the adipose tissue of mice (n = 4). (E) Comparison of Subfatin expression between 3T3‐L1 adipocytes and RAW 264.7 macrophage cells. **P < 0.01 versus macrophages. (F) Subfatin in mouse subcutaneous adipose tissue was detected by immunohistochemistry. Bar = 100 μm.

Figure 5

Subfatin expression is regulated by adipogenesis and obesity. (A) Representative phase‐contrast images of 3T3‐L1 preadipocytes and differentiative adipocytes. Bar = 100 μm. (B) Subfatin expression during adipocyte differentiation (n = 3). *P < 0.05, **P < 0.01 versus D0. (C) Body weight of diet‐induced obesity mice (n = 4). **P < 0.01 versus NCD. (D) Subfatin expression in subcutaneous adipose tissue of diet‐induced obesity mice (n = 4). NCD, normal chow diet; HFD, high‐fat diet. **P < 0.01 versus NCD.

Figure 6

Different tissue expression patterns of Subfatin (A) and Meteorin (B) in mice (n = 3).