Identification and validation of novel adipokines released from primary human adipocytes

Abstract

Adipose tissue is a major endocrine organ, releasing signaling and mediator proteins, termed adipokines, via which adipose tissue communicates with other organs. Expansion of adipose tissue in obesity alters adipokine secretion, which may contribute to the development of metabolic diseases. Although recent profiling studies have identified numerous adipokines, the amount of overlap from these studies indicates that the adipokinome is still incompletely characterized. Therefore, we conducted a complementary protein profiling on concentrated conditioned medium derived from primary human adipocytes. SDS-PAGE/liquid chromatography-electrospray ionization tandem MS and two-dimensional SDS-PAGE/matrix-assisted laser desorption ionization/time of flight MS identified 347 proteins, 263 of which were predicted to be secreted. Fourty-four proteins were identified as novel adipokines. Furthermore, we validated the regulation and release of selected adipokines in primary human adipocytes and in serum and adipose tissue biopsies from morbidly obese patients and normal-weight controls. Validation experiments conducted for complement factor H, αB-crystallin, cartilage intermediate-layer protein, and heme oxygenase-1 show that the release and expression of these factors in adipocytes is regulated by differentiation and stimuli, which affect insulin sensitivity, as well as by obesity. Heme oxygenase-1 especially reveals to be a novel adipokine of interest. In vivo, circulating levels and adipose tissue expression of heme oxygenase-1 are significantly increased in obese subjects compared with lean controls. Collectively, our profiling study of the human adipokinome expands the list of adipokines and further highlights the pivotal role of adipokines in the regulation of multiple biological processes within adipose tissue and their potential dysregulation in obesity.

Fig. 1.

Flow chart—work scheme.

Fig. 2.

Representative Western blots of CILP, HO-1, CRYAB, and CFH protein level and release by human primary adipocytes. Adipocytes were differentiated and concentrated CM was generated as described in the text. A, Release of novel adipokines at day 14 of differentiation. 1–5 μl of concentrated CM were analyzed by SDS-PAGE and Western blotting. B, Protein level of novel adipokines during adipocyte differentiation. 10 μg of total lysates were analyzed by SDS-PAGE and Western blotting. Signals were detected by ECL. C, 10 μg of total lysates derived from adipocytes (Ad) and macrophages (MØ) were analyzed by SDS-PAGE and Western blotting, with subsequent signal detection by ECL.

Fig. 3.

Protein level of CFH and CRYAB in adipocytes and relative serum concentrations in lean and obese subjects. Human primary adipocytes were differentiated as described in the text. CFH (A) and CRYAB (D) protein levels during differentiation were analyzed by SDS-PAGE and Western blotting. Data were normalized to the protein level of actin and are expressed relative to day 0. Data are mean values ± S.E., n ≥ 5, *p < 0.05 versus preadipocytes. B, Regulation of CFH protein expression. Differentiated adipocytes were treated with 5 μmol/L troglitazone (Tro), 10 ng TNFα, 50 mmol/L insulin (I), 5 nm adiponectin (A), or incubated under hypoxic conditions (H) for 24 h. CFH protein level was analyzed by SDS-PAGE and Western blotting. Data were normalized to the protein level of actin and are expressed relative to unstimulated control (Con). Data are mean values ± S.E., n = 6, *p < 0.05 versus control. (C, E) Relative serum concentration of CFH and CRYAB were determined in samples obtained from lean (n = 12) and obese (n = 9) subjects participating in study 1. Sera samples were diluted and analyzed by SDS-PAGE and Western blotting. Data are mean values ± S.E., *p < 0.05 versus lean controls.

Fig. 4.

Protein level of CILP in adipocytes and relative CILP serum concentration in lean and obese subjects. A, Human primary adipocytes were differentiated as described in the text, and CILP protein level during differentiation was analyzed by SDS-PAGE and Western blot. Data were normalized to the protein level of actin and are expressed relative to day 0. Data are mean values ± S.E., n ≥ 3, *p < 0.05 versus preadipocytes. B, Differentiated adipocytes were treated as described in Fig. 3 legend. CILP protein level was analyzed by SDS-PAGE and Western blotting. Data were normalized to the protein level of actin and are expressed relative to unstimulated control (Con). Data are mean values ± S.E., n = 5, *p < 0.05 versus control. C, CILP-1 mRNA expression (Ra) in human adipose tissue. Expression of CILP-1 was determined by real-time PCR in subcutaneous (sc) and visceral (visc) adipose tissue biopsies collected during abdominal surgery of normal weight males (n = 12). Data were normalized for RPS18 expression and expressed as mean ± S.E. *p < 0.05 versus subcutaneous adipose tissue. D, Relative serum concentration of CILP was determined in samples obtained from lean (n = 12) and obese (n = 9) subjects participating in study 1. Sera samples were diluted and analyzed by SDS-PAGE and Western blotting. Data are mean values ± S.E., *p < 0.05 versus lean controls.

Fig. 5.

HO-1 protein level in adipocytes and relative HO-1 serum concentration in lean and obese subjects. A, HO-1 protein level during adipocyte differentiation was analyzed by SDS-PAGE and Western blotting. Data were normalized to the protein level of actin and are expressed relative to day 0. Data are mean values ± S.E. n ≥ 5, *p < 0.05 versus pre-adipocytes. B, Secretion of HO-1 during differentiation of adipocytes was analyzed by ELISA. Data are mean values ± S.E., n = 5. C, Differentiated adipocytes were treated for 24 h, as described in Fig. 3B legend. HO-1 secretion was measured by ELISA. Data are mean values ± S.E., n = 6. D, Sera from lean (n = 20) and morbidly obese men (n = 20) participating in study 1 were analyzed for their HO-1 concentration by ELISA. Data are mean values ± S.E., *p < 0.05 versus lean group. E, Linear regression analysis of HO-1 serum concentration and size of subcutaneous adipocytes (p = 0.031; r = 0.40).

Fig. 6.

CRYAB and HO-1 protein expression in subcutaneous and visceral adipose tissue from lean and obese patients. CRYAB (A) and HO-1 (B) level were determined in adipose tissue lysates from paired subcutaneous and visceral adipose tissue of lean (n = 9) and obese (n = 15) patients. 5 μg of tissue lysates were analyzed by SDS-PAGE and Western blotting. Data were normalized to the protein level of GAPDH and are expressed relative to subcutaneous adipose tissue of lean subjects. Data are mean values ± S.E., *p < 0.05 versus lean.