αMSH inhibits adipose inflammation via reducing FoxOs transcription and blocking Akt/JNK pathway in mice

Abstract

Alpha melanocyte stimulating hormone (αMSH) abates inflammation in multiple tissues, while Forkhead box proteins O (FoxOs) stimulate inflammatory cascade. However, the relationship between αMSH and FoxOs in adipose inflammation remains unclear. In this study, we used LPS-induced inflammation model, attempted to interpret the function of αMSH in inflammation and the interactions with FoxOs. Results indicated that upon inflammatory situation, the secretion of αMSH and the expression of its receptor MC5R were greatly decreased, but FoxOs expressions were elevated. After the treatment with αMSH, LPS-induced adipose inflammation together with FoxOs expressions was significantly reduced. Conversely, when Foxo1, Foxo3a or Foxo4 overexpressed in αMSH treated inflammatory mouse model, all the anti-inflammatory impacts of αMSH were found disappeared. We further studied the mechanisms by which αMSH exerts its anti-inflammatory impacts and how FoxOs reverse αMSH's function. Foxo4 was found as a negative regulator for MC5R transcription in αMSH inhibited inflammation. Moreover, a negative role was found of αMSH in regulating both Akt and JNK signal pathways by observing the enhanced the anti-inflammatory impacts of pathway-specific inhibitors with αMSH treatment. Our findings demonstrate αMSH plays a key role in the prevention of adipose inflammation and inflammatory diseases by down-regulating Akt/JNK signal pathway and negatively interacting with FoxOs, which brings up αMSH as a novel candidate factor in the adipose anti-inflammation process in obesity.

Keywords: Akt/JNK; FoxOs; adipose; inflammation; αMSH.

Figure 1. αMSH weakens LPS-induced adipose inflammation by inhibiting FoxOs expressions in mice

(A) Serum protein levels of IL-1β, IL-6, IL-4 and IL-10 after mice with LPS or saline intraperitoneal injection (n=6). (B) Measurement of serum αMSH level before and after LPS or saline intraperitoneal injection (n=6). Epididymis white adipose tissue was isolated after intraperitoneal injection with LPS or saline, then mRNA levels of MC5R (C), Foxo1, Foxo3a, Foxo4 and Foxo6 (D) were detected (n=6). Base on the LPS/saline injection, mice were injected with another 500 nM αMSH, then further detected for relative serum protein levels of IL-1β, IL-6, IL-4 (E) and mRNA levels of M1, M2 markers (F), MC5R (G), IL-6, MCP-1, Leptin (H), Foxo1, Foxo3a, Foxo4 and Foxo6 (I) in white adipose tissue (n=6). Values are means ± SD. vs. control group, * P < 0.05.

Figure 2. FoxOs reverse the inhibition of αMSH on adipose inflammation in mice

Mice white adipose tissue was isolated after intraperitoneal injection with pAd-Foxo1 and with another 500 nM αMSH, then mRNA levels of Foxo1, MC5R (A), M1, M2 markers (C), IL-6, MCP-1, Leptin (D) were measured (n=6). After intraperitoneal injection with pAd-Foxo1 and with another 500 nM αMSH, serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (B) were detected (n=6). After intraperitoneal injection with pAd-Foxo3a and with another 500 nM αMSH to mice, mRNA levels of Foxo3a, MC5R (E), M1, M2 markers (G), IL-6, MCP-1, Leptin (H) were measured in white adipose tissue (n=6). Serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (F) were also detected (n=6). Mice were intraperitoneal injected with pAd-Foxo4 and another 500 nM αMSH, serum protein levels of IL-1β, IL-6, IL-4 and IL-10 (J) and mRNA levels of Foxo4, MC5R (I), M1, M2 markers (K), IL-6, MCP-1, Leptin (L) were measured (n=6). Values are means ± SD. vs. control group, * P < 0.05.

Figure 3. FoxOs abolish the suppression of αMSH on inflammation in mice adipocytes

(A) Oil Red O staining for differentiated primary adipocytes isolated from epididymal white adipose tissue after administration of αMSH for 1 h (left). Relative concentration of TG in adipocytes (middle) and FFA in cell culture medium (right) were detected (n=3). With αMSH treatment, mRNA levels of MC5R (B), IL-6, TNFα, MCP-1 and Leptin (C), Foxo1, Foxo3a, Foxo4 and Foxo6 (D), protein levels of p-AKT and total Akt (E) were detected in adipocytes (n=3). (F) Adipocytes treated with pAd-Foxo1 and αMSH, protein levels of IL-6, TNFα, MCP-1 and Leptin were measured (n=3). (G) Expression levels for Foxo3a, p-Akt, Akt and IL-6 protein after cells treated with pAd-Foxo3a and αMSH (n=3). (H) Normalized mRNA levels of Foxo4, MC5R, Leptin and IL-6 with pAd-Foxo4 and αMSH treatments (n=3). Values are means ± SD. vs. control group, * P < 0.05.

Figure 4. Foxo1 and Foxo3a reverse the inhibition of αMSH on LPS-induced inflammation in mice adipocytes

(A) Primary adipocytes were cultured and incubated for 0 h, 12 h, 24 h and 48 h in the presence of 1 μg/mL LPS. Cell viability was detected by CCK-8 (n=3). (B) Relative mRNA expressions of IL-6, Leptin, MC5R, Foxo1, Foxo3a, Foxo4, Foxo6 and Caspase3 with αMSH treatment were analyzed (n=3). (C) After adipocytes treated with pAd-Foxo1 and incubated for LPS/saline and αMSH, mRNA levels of Foxo1, IL-6, MCP-1 and Leptin were detected (n=3). (D) Adipocytes were treated with pAd-Foxo3a and incubated for LPS/saline and αMSH, then measured mRNA levels of Foxo3a, IL-6, MCP-1 and Leptin (n=3). Values are means ± SD. vs. control group, * P < 0.05.

Figure 5. Foxo4 negatively regulate MC5R transcription in αMSH inhibited inflammation in mice adipocytes

(A) Fragments of MC5R promoter fused to a luciferase reporter plasmid or PGL3-basic (control) were co-transfected into cells together with Renlilla plasmid and pAd-Foxo4 (n=3). Luciferase activity was corrected for Renilla luciferase activity and normalized to control activity (n=3). (B) Chromatin immunoprecipitation (ChIP) analysis of Foxo4 and MC5R interaction. (C, D) After pAd-Foxo4 together with αMSH or pc-MC5R in LPS/saline treatment, MC5R mRNA level was determined in adipocytes (n=3). (E) When adipocytes were treated with pAd-Foxo4 and αMSH in LPS/saline treatment, mRNA levels of Foxo4, IL-6, TNFα, MCP-1 and Leptin were analyzed (n=3). Values are means ± SD. vs. control group, * P < 0.05.

Figure 6. Akt/JNK signal pathway is impaired in the inhibition of αMSH on adipocyte inflammation and FoxOs expressions

Mouse adipocytes were pretreated with αMSH and MK-2206 or SP600125, respectively. Relative protein levels of Akt, p-Akt^ser473, JNK, p-JNK^Thr183 (A), Foxo1, Foxo3a, Foxo4, IL-6 and Leptin (B) with or without MK-2206 (n=3). Representative immunoblots and densitometric quantification for Akt, p-Akt^ser473, JNK, p-JNK^Thr183 (C), Foxo1, Foxo3a, Foxo4, IL-6 and Leptin protein (D) with or without SP600125 (n=3). The level of total GAPDH was used as the loading control. Values are mean ± SD. *P < 0.05 compared with the control.

Figure 7. Proposed mechanism of the inhibition of αMSH on adipose inflammation

αMSH decreases adipose inflammation via blunting the phosphorylation of Akt/JNK. Akt phosphorylation activates Foxo1 and Foxo3a, while JNK phosphorylation activates Foxo4. What is more, Foxo4 acts via binding the promoter of MC5R, which is the receptor of αMSH.