Chemerin is a novel adipocyte-derived factor inducing insulin resistance in primary human skeletal muscle cells

Abstract

Objective: Chemerin is an adipokine that affects adipogenesis and glucose homeostasis in adipocytes and increases with BMI in humans. This study was aimed at investigating the regulation of chemerin release and its effects on glucose metabolism in skeletal muscle cells.

Research design and methods: Human skeletal muscle cells were treated with chemerin to study insulin signaling, glucose uptake, and activation of stress kinases. The release of chemerin was analyzed from in vitro differentiated human adipocytes and adipose tissue explants from 27 lean and 26 obese patients.

Results: Human adipocytes express chemerin and chemokine-like receptor 1 (CMKLR1) differentiation dependently and secrete chemerin (15 ng/ml from 10(6) cells). This process is slightly but significantly increased by tumor necrosis factor-alpha and markedly inhibited by >80% by peroxisome proliferator-activated receptor-gamma activation. Adipose tissue explants from obese patients are characterized by significantly higher chemerin secretion compared with lean control subjects (21 and 8 ng from 10(7) cells, respectively). Chemerin release is correlated with BMI, waist-to-hip ratio, and adipocyte volume. Furthermore, higher chemerin release is associated with insulin resistance at the level of lipogenesis and insulin-induced antilipolysis in adipocytes. Chemerin induces insulin resistance in human skeletal muscle cells at the level of insulin receptor substrate 1, Akt and glycogen synthase kinase 3 phosphorylation, and glucose uptake. Furthermore, chemerin activates p38 mitogen-activated protein kinase, nuclear factor-kappaB, and extracellular signal-regulated kinase (ERK)-1/2. Inhibition of ERK prevents chemerin-induced insulin resistance, pointing to participation of this pathway in chemerin action.

Conclusions: Adipocyte-derived secretion of chemerin may be involved in the negative cross talk between adipose tissue and skeletal muscle contributing to the negative relationship between obesity and insulin sensitivity.

FIG. 1.

Expression of chemerin and CMKLR1 in human skeletal muscle cells and adipocytes. A: Human adipocytes from different donors were differentiated for the indicated time, and total cell lysates were resolved by SDS-PAGE. Western blots for chemerin, adiponectin, and CMKLR1 as well as normalization for actin are shown. Data are the means ± SEM of three to four independent experiments. All data were normalized to the level of actin expression and are expressed relative to the expression level at day 0. *Significantly different from day 0. B: Skeletal muscle cells from different donors were differentiated for the indicated time, and total cell lysates were resolved by SDS-PAGE. Western blots for CMKLR1 and MHC as well as normalization for tubulin are shown. Data are the means ± SEM of three independent experiments. All data were normalized to the level of tubulin expression and are expressed relative to the expression level at day 0. The right panel shows that skeletal muscle cells from two different donors (SkM1 and SkM2) have no expression of chemerin compared with adipocytes harvested at day 0, 1, and 3 of differentiation. *Significantly different from day 0.

FIG. 2.

Regulation of chemerin and CMKLR1 expression and chemerin secretion in human adipocytes. A: Human adipocytes from different donors were differentiated and incubated with either 2.5 nmol/l TNF-α, 5 nmol/l adiponectin, or 5 μmol/l troglitazone overnight. Total cell lysates were resolved by SDS-PAGE. Western blots for chemerin, adiponectin, and CMKLR1 as well as normalization for actin are shown. Data are the means ± SEM of three to four independent experiments. All data were normalized to the level of actin expression and are expressed relative to the unstimulated control. *Significantly different from control. B: Human adipocytes from different donors were differentiated, and conditioned medium was collected after different periods of incubation. The release of chemerin was analyzed using a chemerin ELISA. Data are the means ± SEM of three independent experiments. C: Human adipocytes from different donors were treated with 2.5 nmol/l TNF-α, 5 nmol/l adiponectin, or 5 μmol/l troglitazone overnight, and the conditioned medium was collected for chemerin measurement. Data are the means ± SEM of three independent experiments. All data are expressed relative to the unstimulated control. *Significantly different from control.

FIG. 3.

Secretion of chemerin from adipose tissue explants derived from lean and obese female subjects. Adipose tissue explants were treated as detailed in the research design and methods section, and the release of chemerin was measured by ELISA. Data are the means ± SEM of tissue explants from 53 individuals. *Significantly different from lean control subjects.

FIG. 4.

Correlation of chemerin release from adipose tissue explants with different parameters. A: Adipose tissue explants were treated as detailed in the research design and methods section, and the release of chemerin was measured by ELISA. Chemerin release was correlated with BMI, waist-to-hip ratio, and adipocyte volume. B: Lipogenesis and insulin-stimulated antilipolysis was measured as described in the research design and methods section. Values for antilipolysis are not normally distributed, but the correlation remains significant, using a nonparametric test (Spearman rank test).

FIG. 5.

Effect of chemerin on insulin signaling and glucose uptake in human skeletal muscle cells. A: Myocytes from different donors were cultured with increasing concentrations of chemerin (250 ng/ml and 1 μg/ml) for 24 h. After acute stimulation with insulin, total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific Akt antibody and tubulin antibody. Data are the means ± SEM of five independent experiments. All data were normalized to the level of tubulin expression and are expressed relative to the insulin-stimulated control value. B: Myocytes from different donors were cultured as outlined in A. After acute stimulation with insulin, total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific GSK3 antibody and GSK3 antibody. Data are the means ± SEM of four independent experiments. All data were normalized to the level of tubulin expression and are expressed relative to the insulin-stimulated control value. C and D: Skeletal muscle cells were cultured for 24 h in absence or presence of chemerin (1 μg/ml). IRS-1 phosphorylation and glucose uptake was assessed after acute stimulation with insulin, as outlined in the research design and methods section. Data are the means ± SEM of three independent experiments. *Significantly different from insulin-stimulated control; §significantly different from respective insulin-stimulated control. ■, insulin; □, basal.

FIG. 6.

Additive effect of chemerin and conditioned medium (CM) on insulin signaling in skeletal muscle cells. Skeletal muscle cells from different donors were incubated with chemerin, conditioned medium, or a combination of both overnight. After insulin stimulation, total cell lysates were resolved by SDS-PAGE and immunoblotted with a phosphospecific antibody for Akt and tubulin for loading control. Representative blots are shown. Data are the means ± SEM of three to four independent experiments. *Significantly different from respective basal; §significantly different from respective insulin-stimulated control.

FIG. 7.

Chemerin signaling in skeletal muscle cells. A: Skeletal muscle cells from different donors were cultured with chemerin for 30 min and as a control with 2.5 nmol/l TNF-α for 10 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Representative blots are shown. B: Skeletal muscle cells from different donors were cultured with chemerin for 10–120 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Data are the means ± SEM of four to five independent experiments. *Significantly different from unstimulated control. C: Skeletal muscle cells from different donors were cultured with different concentrations of chemerin for 24 h. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Data are the means ± SEM of four to five independent experiments. *Significantly different from unstimulated control.

FIG. 8.

Prevention of chemerin-induced insulin resistance by ERK inhibition. A: Skeletal muscle cells from different donors were precultured with or without 50 μmol/l of the specific ERK inhibitor PD 98059 for 15 min before starting the administration with chemerin or TNF-α. The cells were then treated with chemerin for 30 min and as a control with 2.5 nmol/l TNF-α for 10 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with a phosphospecific antibody for ERK-1/2 and tubulin for loading control. Representative blots are shown. B and C: After pretreatment for 15 min with PD 98059 (50 μmol/l), skeletal muscle cells from different donors were treated with chemerin overnight. After insulin stimulation, total cell lysates were resolved by SDS-PAGE and immunoblotted with a phosphospecific antibody for Akt and tubulin for loading control. Representative blots are shown. Data are the means ± SEM of four independent experiments. Glucose uptake was measured as outlined in the research design and methods section. Data are the means ± SEM of three independent experiments. *Significantly different from respective insulin-stimulated control. ■, insulin; □, basal.