Clinically confirmed DEL-1 as a myokine attenuates lipid-induced inflammation and insulin resistance in 3T3-L1 adipocytes via AMPK/HO-1- pathway

Chang Hyuk Kwon, Jaw Long Sun, Myeong Jun Kim, A M Abd El-Aty, Ji Hoon Jeong, Tae Woo Jung, Chang Hyuk Kwon, Jaw Long Sun, Myeong Jun Kim, A M Abd El-Aty, Ji Hoon Jeong, Tae Woo Jung

Abstract

Regular exercise is the first line of therapy for treating obesity-mediated metabolic disorders, including insulin resistance. It has been reported that developmental endothelial locus-1 (DEL-1) enhances macrophage efferocytosis, resulting in inflammation clearance as well as improves insulin resistance in skeletal muscle. However, the relationship between exercise and DEL-1, and the effects of DEL-1 on insulin signalling in adipocytes have not been fully elucidated to date. Protein expression levels were determined by Western blot analysis. Cells were transfected with small interfering (si) RNA to suppress gene expression. Lipid accumulation levels were detected using Oil red-O staining. Proinflammatory cytokine secretion levels were measured using ELISA. DEL-1 expression levels were induced in the skeletal muscle of people who exercised using microarray analysis. Recombinant DEL-1 augmented AMP-activated protein kinase (AMPK) phosphorylation and haem oxygenase (HO)-1 expression to alleviating inflammation and impairment of insulin signalling in 3T3-L1 adipocytes treated with palmitate. siRNA of AMPK or HO-1 also mitigated the effects of DEL-1 on inflammation and insulin resistance. DEL-1 ameliorates inflammation and insulin resistance in differentiated 3T3-L1 cells via AMPK/HO-1 signalling, suggesting that DEL-1 may be the exercise-mediated therapeutic target for treating insulin resistance and type 2 diabetes.

Keywords: AMPK; DEL-1; HO-1; Myokine; inflammation; insulin resistance.

Conflict of interest statement

The authors declared no conflict of interest.

Figures

Graphical abstract
Graphical abstract
Graphical abstract
Graphical abstract
Figure 1.
Figure 1.
Exercise induces DEL-1 gene expression in human skeletal muscle. (a) Gene expression (TPM; transcripts per million) in resting and exercise for 15 min using RNAseq analysis. (b) gene expression of pre-exercise, 3 h post-exercise and 48 h post-exercise using microarray analysis. Gene expression of normal and obese (c)/diabetic (d) pre-exercise using microarray analysis. (e) gene expression of obese pre-exercise, 6 h post-exercise and 24 h post-exercise using microarray analysis. (f) gene expression of diabetic pre-exercise, 0 h post-exercise and 3 h post-exercise using microarray analysis. ***P < 0.001 and *P < 0.05, when compared to DEL-1 gene expression in control. !!!P < 0.001, when compared to 3 h post-exercise
Figure 1.
Figure 1.
Exercise induces DEL-1 gene expression in human skeletal muscle. (a) Gene expression (TPM; transcripts per million) in resting and exercise for 15 min using RNAseq analysis. (b) gene expression of pre-exercise, 3 h post-exercise and 48 h post-exercise using microarray analysis. Gene expression of normal and obese (c)/diabetic (d) pre-exercise using microarray analysis. (e) gene expression of obese pre-exercise, 6 h post-exercise and 24 h post-exercise using microarray analysis. (f) gene expression of diabetic pre-exercise, 0 h post-exercise and 3 h post-exercise using microarray analysis. ***P < 0.001 and *P < 0.05, when compared to DEL-1 gene expression in control. !!!P < 0.001, when compared to 3 h post-exercise
Figure 2.
Figure 2.
DEL-1 ameliorates palmitate-induced inflammation and insulin resistance in 3T3-L1 adipocytes. (a) Western blot analysis of NFκB and IκB phosphorylation in differentiated 3T3-L1 cells treated with palmitate (200 μM) and DEL-1 (0–1 μg/mL) for 24 h. (b) Western blot analysis of IRS-1 and Akt phosphorylation in differentiated 3T3-L1 cells treated with palmitate (200 μM) and DEL-1 (0–1 μg/mL) for 24 h. Human Insulin (10 nM) stimulates IRS-1 and Akt phosphorylation for 3 min. Means ± SEM were obtained from three independent experiments. ***P < 0.001 and **P < 0.01 when compared to control or insulin treatment. !!!P < 0.001 and !P < 0.05 when compared to palmitate or insulin plus palmitate treatment
Figure 2.
Figure 2.
DEL-1 ameliorates palmitate-induced inflammation and insulin resistance in 3T3-L1 adipocytes. (a) Western blot analysis of NFκB and IκB phosphorylation in differentiated 3T3-L1 cells treated with palmitate (200 μM) and DEL-1 (0–1 μg/mL) for 24 h. (b) Western blot analysis of IRS-1 and Akt phosphorylation in differentiated 3T3-L1 cells treated with palmitate (200 μM) and DEL-1 (0–1 μg/mL) for 24 h. Human Insulin (10 nM) stimulates IRS-1 and Akt phosphorylation for 3 min. Means ± SEM were obtained from three independent experiments. ***P < 0.001 and **P < 0.01 when compared to control or insulin treatment. !!!P < 0.001 and !P < 0.05 when compared to palmitate or insulin plus palmitate treatment
Figure 3.
Figure 3.
AMPK/HO-1 contributes to the attenuation of inflammation and insulin resistance in 3T3-L1 adipocytes. (a) Western blot analysis of AMPK phosphorylation and HO-1 expression in differentiated 3T3-L1 cells treated with DEL-1 (0–1 μg/mL) for 24 h. (b) Western blot analysis of AMPK phosphorylation and HO-1 expression in AMPK or HO-1 siRNA-transfected 3T3-L1 adipocytes treated with DEL-1 (1 μg/mL) for 24 h. Western blot analysis of NFκB and IκB phosphorylation in AMPK (c) or HO-1 (d) siRNA-transfected 3T3-L1 myocytes treated with palmitate (200 μM) and DEL-1 (1 μg/mL) for 24 h. (e) ELISA for TNFα and MCP-1 release by AMPK or HO-1 siRNA-transfected 3T3-L1 adipocytes treated with DEL-1 (1 μg/mL) for 24 h. Western blot analysis of phosphorylation of IRS-1 and Akt and glucose uptake measurement (f) in AMPK or HO-1 siRNA-transfected 3T3-L1 adipocytes treated with 200 μM palmitate and DEL-1 (1 μg/mL) for 24 h. Human insulin (10 nM) stimulates insulin signalling for 3 min. Means ± SEM were obtained from three independent experiments. ***P < 0.001 when compared to control or insulin treatment. !!!P < 0.001, !!P < 0.01 and !P < 0.05 when compared to palmitate or insulin plus palmitate treatment. ###P < 0.001, ##P < 0.01 and #P < 0.05 when compared to the insulin, palmitate plus DEL-1 or insulin, palmitate plus DEL-1 treatment
Figure 3.
Figure 3.
AMPK/HO-1 contributes to the attenuation of inflammation and insulin resistance in 3T3-L1 adipocytes. (a) Western blot analysis of AMPK phosphorylation and HO-1 expression in differentiated 3T3-L1 cells treated with DEL-1 (0–1 μg/mL) for 24 h. (b) Western blot analysis of AMPK phosphorylation and HO-1 expression in AMPK or HO-1 siRNA-transfected 3T3-L1 adipocytes treated with DEL-1 (1 μg/mL) for 24 h. Western blot analysis of NFκB and IκB phosphorylation in AMPK (c) or HO-1 (d) siRNA-transfected 3T3-L1 myocytes treated with palmitate (200 μM) and DEL-1 (1 μg/mL) for 24 h. (e) ELISA for TNFα and MCP-1 release by AMPK or HO-1 siRNA-transfected 3T3-L1 adipocytes treated with DEL-1 (1 μg/mL) for 24 h. Western blot analysis of phosphorylation of IRS-1 and Akt and glucose uptake measurement (f) in AMPK or HO-1 siRNA-transfected 3T3-L1 adipocytes treated with 200 μM palmitate and DEL-1 (1 μg/mL) for 24 h. Human insulin (10 nM) stimulates insulin signalling for 3 min. Means ± SEM were obtained from three independent experiments. ***P < 0.001 when compared to control or insulin treatment. !!!P < 0.001, !!P < 0.01 and !P < 0.05 when compared to palmitate or insulin plus palmitate treatment. ###P < 0.001, ##P < 0.01 and #P < 0.05 when compared to the insulin, palmitate plus DEL-1 or insulin, palmitate plus DEL-1 treatment
Figure 3.
Figure 3.
(Continued)
Figure 3.
Figure 3.
(Continued)
Figure 4.
Figure 4.
DEL-1 did not affect 3T3-L1 differentiation. (a) Oil-red O staining in differentiated 3T3-L1 cells in the presence of DEL-1 (0–1 μg/mL) for 6 d. TG accumulation was quantitated by modified TG assay kit. (b) Western blot analysis of processed SREBP1 and SCD1 expression in 3T3-L1 adipocytes treated with DEL-1 (0–1 μg/mL) for 6 d. (c) Western blot analysis of UCP-1 and PGC-1α expression in AMPK siRNA-transfected 3T3-L1 adipocytes treated with DEL-1 (0–1 μg/mL) for 24 h. Means ± SEM were obtained from three independent experiments. ***P < 0.001 and **P < 0.01 when compared to control. !P < 0.05 when compared to DEL-1 (1 μg/mL) treatment
Figure 4.
Figure 4.
DEL-1 did not affect 3T3-L1 differentiation. (a) Oil-red O staining in differentiated 3T3-L1 cells in the presence of DEL-1 (0–1 μg/mL) for 6 d. TG accumulation was quantitated by modified TG assay kit. (b) Western blot analysis of processed SREBP1 and SCD1 expression in 3T3-L1 adipocytes treated with DEL-1 (0–1 μg/mL) for 6 d. (c) Western blot analysis of UCP-1 and PGC-1α expression in AMPK siRNA-transfected 3T3-L1 adipocytes treated with DEL-1 (0–1 μg/mL) for 24 h. Means ± SEM were obtained from three independent experiments. ***P < 0.001 and **P < 0.01 when compared to control. !P < 0.05 when compared to DEL-1 (1 μg/mL) treatment
Figure 5.
Figure 5.
Schematic diagram for the effects of DEL-1 on inflammation and insulin resistance in adipocytes
Figure 5.
Figure 5.
Schematic diagram for the effects of DEL-1 on inflammation and insulin resistance in adipocytes

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