Modulation of pro-inflammatory adipokines and myokines by capillarisin and alantolactone in the metabolic disorders

Abstract

Skeletal muscle and white adipose tissue are the largest organs in the human body and considered as endocrine organs. Skeletal muscle produces cytokines and chemokines that contribute to further immune system. Cytokines and chemokines that are produced and released by muscle fibers expressing paracine/endocrine effects are termed myokines. Adipocytes also release metabolites, lipids, and bioactive peptides

so-called adipokines. Released adipokines regulate biological process, such as inflammation, fat distribution, and insulin sensitivity. Both myokines and adipokines have profound effects on inflammation and metabolism, contributing to the pathogenesis of inflammation, obesity, insulin resistance, diabetes, and other diseases. In fact, many myokines are also produced by adipocytes and vice versa. Therefore, recent reports proposed the term adipo-myokines. IL-6, TNFα, and MCP-1 are the most studied pro-inflammatory adipo-myokines released by both skeletal muscle and adipose tissue. Therefore, it is likely that adipo-myokines may crosslink between the inflammation and metabolic homeostasis in the skeletal muscle and adipose tissues. Herein, this study investigated first, eccentric (downhill) exercise, second, cytokine (IL-6), and third, fatty acid (palmitate) induced metabolic disorders in the skeletal muscle and adipocytes in association with commonly expressed pro-inflammatory adipo-myokines IL-6, TNFα and MCP-1.

First, eccentric (downhill) exercise can lead to leucocyte infiltration, inflammation and reactive oxidative stress (ROS) in the skeletal muscle. Capillarisin isolated from Artemisia capillaris Thunberg is known to have antioxidant and anti-inflammatory effects. Eccentric exercise was conducted to induce muscle damage in mice skeletal muscle. Intense exercise increased the level of ROS production in the skeletal muscle, but capillarisin administration reduced these levels in a dose dependent manner (p<0.05). Muscle damage markers, CPK and LDH were also attenuated at plasma level from capillarisin treated groups. Exercise activated MAPK (ERK 1/2 and JNK but not p38) and NF-κB (nuclear p50 and p65, and cytosolic p-IκBα) subunits at protein level but capillarisin suppressed these increase. At the mRNA level, inflammation-associated chemokines CINC-1 and MCP-1, and cytokine IL-6 and TNFα in gastrocnemius muscle were increased by exercise, whereas capillarisin showed protective and inhibitive effects against these changes. Overall, our results indicate that capillarisin can attenuate muscle damage by exerting antioxidant and anti-inflammatory effects by regulating pro-inflammatory adipo-myokines.

Second, cytokines, such as IL-6 and TNFα are reported as a bridge between inflammation and insulin resistance. IL-6 has been proposed to be one of the mediators that link chronic inflammation to glucose intolerance and insulin resistance. In the present study, we observed protective effects of alantolactone, a sesquiterpene lactone isolated from Inula helenium against IL-6 induced inflammation and glucose intolerance in association with myokine expressions. Alantolactone has been reported to have anti-inflammatory and anti-cancer effects through IL-6-induced STAT3 signalling pathway. Prolonged IL-6 exposure also increased expression of TLR4, involved in inflammation in the skeletal muscle, thus the underlying mechanisms were investigated. We observed dysregulation of glucose uptake and suppression of AKT phosphorylation after prolonged IL-6 treatment

however, pretreatment with alantolactone activated AKT phosphorylation and improved glucose uptake. Alantolactone also attenuated IL-6-stimulated STAT3 phosphorylation, followed by an increase in expression of negative regulator SOCS3. Furthermore, IL-6-induced expression of pathogen recognition receptor, TLR4, was also suppressed by alantolactone pretreatment. Post-silencing of STAT3 using siRNA approach, IL-6-stimulated siRNA-STAT3 improved glucose uptake and suppressed TLR4 gene expression. Taken together, we propose that, as a STAT3-SOCS3 inhibitor, alantolactone, improves glucose uptake in the skeletal followed by inhibition of the TLR4 gene expression. Lastly, alantolactone suppressed IL-6 induced pro-inflammatory myokines, IL-6, TNFα and MCP-1. Therefore, alantolactone can be a promising candidate for the treatment of inflammation-associated glucose intolerance and insulin resistance.

Third, adipocytes, together with macrophages create a crosstalk between inflammation and insulin resistance. Resident macrophages are surrounded by adipocytes that constantly release free fatty acids (FFAs) via lipolysis. FFAs such as palmitate activate macrophages and pro-inflammatory adipokines such as IL-6, TNFα and MCP-1, consequently alter their function. This research aimed to evaluate the potency of alantolactone in reducing palmitate-induced glucose intolerance, fat accumulation, and inflammation in 3T3-L1 adipocytes. In vitro obese model was constructed by adipocyte-macrophage co-culture system (3T3-L1-RAW264.7). This study observed that palmitate and co-culture system reduced glucose uptake and increased fat accumulation, which indicated dysfunctional adipocytes. Alantolactone pretreatment reversed these changes by increasing glucose uptake and attenuating fat accumulation in a dose-dependent manner (P < 0.05). Palmitate and co-culture model activated JNK and IKKβ/α phosphorylation, and increased the levels of pro-inflammatory adipokines (TNFα, IL-6, and MCP-1). Alantolactone treatment selectively reduced JNK and TLR4 gene expression, suggesting inhibition of TLR4-JNK signalling. Alantolactone also reduced macrophage infiltration associated chemkines MCP-1 and cytokine IL-6 in both adipocyte and adipocyte-macrophage co-culture system. Our study showed that palmitate treatment led to adipocyte dysfunction and inflammation

however, alantolactone improved palmitate-induced glucose intolerance and inflammation. These findings suggest that alantolactone may inhibit obesity-induced insulin resistance and improve glucose homeostasis and inflammation in the adipose tissues.

Overall, this study suggests capillarisin and alantolactone, as pro-inflammatory adipokines and myokines regulators, are promising therapeutic agents for inflammation associated metabolic disorders in the skeletal muscle and adipose tissue.