Increasing incidence of metabolic syndrome-related diseases such as obesity, diabetes, and cardiovascular diseases has become a global health problem. Insulin resistance is the common pathophysiology basis of metabolic syndrome. All metabolic syndrome patients have insulin resistance in different degrees. Therefore, therapeutic strategies for metabolic syndrome need to ameliorate insulin resistance. In this study, we found that DHM activated the Ca2+-CaMKK-AMPK signal pathway by binding to the PLC. Activation of AMPK ameliorated adipocytes insulin resistance by blocking the inflammation factor-induced inflammatory response. Our study not only clarified the molecular mechanism of DHM in inflammation-induced insulin resistance but also discovered the target of DHM in this process. This study offers a new therapeutic strategy for patients with metabolic syndrome.
The increasing incidence of insulin resistance is inextricably related to the increase in the global obese population . A recent report showed that 2.1 billion people, about 30% of the world population were either overweight or obese. During the process of obesity, the proliferation and hypertrophy of adipocytes triggered hypoxic stress, causing the secretion of inflammatory cytokines . Our results showed that levels of serum and adipose tissue inflammatory cytokines were increased in HFD-induced obese mice.
The inflammatory cytokines secreted by adipose tissue activated NF-κB and JNK signal pathways in insulin-sensitive organs like adipose tissue, skeletal muscle, and liver [22–24]. The activation of NF-κB and JNK inhibited the phosphorylation of IRS-1, and, in turn, blocked the PI3K/AKT activation . Results from our in vivo experiments showed that the level of the inflammatory cytokines was increased in adipose tissue, the inflammatory cytokines induced the NF-κB and JNK-1 activation and insulin resistance in mice. Results from our in vitro experiments also showed that TNFα induced the NF-κB and JNK-1 activation and insulin resistance in 3T3-L1 cells.
AMPK is not only an important regulator in cellular energy metabolism but also plays an essential role in many physiological processes such as tumor growth, inflammation, and enhanced insulin sensitivity . The pharmacological function of DHM to activate AMPK has been demonstrated in various physiological processes. DHM improved skeletal muscle insulin sensitivity partially through inducing autophagy by activating the AMPK-PGC-1α-Sirt signaling pathway . DHM protected rats from developing Alzheimer's disease via the up-regulation of the AMPK-SIRT1 pathway to inhibit inflammation-induced apoptosis of hippocampal cells and ameliorate cognitive function . We demonstrated that DHM activated AMPK in vivo and in vitro, and we also demonstrated that the function of DHM in ameliorating insulin resistance depended on its activation effect on AMPK in vitro.
AMPK blocked the inflammatory response by inhibiting the NF-κB signaling pathway. AMPK activation blocked the phosphorylation of IKK and NF-κB directly . AMPK also activated SIRT1, then P65, the subunit of NF-κB, was deacetylated by SIRT1. Thus, the transcriptional activity of NF-κB was also inhibited . In this study, we found that DHM blocked NF-κB phosphorylation to ameliorate insulin resistance through AMPK in vivo and in vitro.
To our knowledge, most DHM-related research reported that DHM functions depend on its activation of AMPK ; but AMPK is not the direct target of DHM in cells, and the target of DHM in cells has not been identified. Two predominant upstream kinases are known to activate AMPK: LKB1 and CaMKK . Cynandione A, a Cynanchum wilfordii extract, inhibited lipogenesis by activating the LKB1-AMPK pathway in HepG2 cells . Apigenin, a natural flavonoid, induced autophagy in human keratinocytes via up-regulating CaMKK-AMPK . To investigate the pathway DHM used to activate AMPK, we blocked the AMPK upstream factors LKB1 and CaMKK. Results from our in vitro experiment showed that DHM ameliorated inflammation-induced insulin resistance through CaMKK-AMPK instead of the LKB1-AMPK pathway.
As far as we know, there is no published report on how DHM activates CaMKK. Triptolide, an extract from the Chinese herb thunder god vine, increased intracellular Ca2+ by stimulating the endoplasmic reticulum stress. The triptolide induced autophagy in human prostate cancer cells by activating the Ca2+-CaMKK-AMPK signaling pathway . Brosimone I, a flavonoid from Artocarpus heterophyllus, increased intracellular Ca2+ by stimulating the endoplasmic reticulum stress. Brosimone I induced cell cycle arrest and apoptosis via the activation of the Ca2+-CaMKK-AMPK signaling pathway . These reports showed that plant extracts could activate the CaMKK-AMPK signaling pathway through Ca2+. In this study, we found that the DHM activated CaMKK-AMPK signaling pathway is dependent on Ca2+. Dandelion root extract (10–400 µg/mL) dose-dependently increased intracellular Ca2+ level in the presence of external Ca2+. The effect of dandelion root extract-induced Ca2+ increase was inhibited in the absence of extracellular Ca2+ . However, in our study, DHM activated intracellular Ca2+ signals regardless of the presence or absence of extracellular Ca2+. The endoplasmic reticulum is the main storage organelle for Ca2+ in cells. The ryanodine receptor and IP3 receptor are the main channels for calcium release from the endoplasmic reticulum and play a central role in cellular Ca2+ signal transduction . In this study, we discovered that by blocking the PLC-IP3 receptor pathway, the DHM’s ability to activate Ca2+ and CaMKK disappeared, but blocking the ryanodine receptor had no effect on preventing the activation of Ca2+-CaMKK induced by DHM.
The identification of target protein for small molecules is critical for drug discovery and chemical metabolomics . DARTS is a quick and straightforward approach to identify potential target protein of small molecules. The mechanism of DARTS is that the interaction of small molecule and target protein resists proteolysis. The most significant advantage of this method is it allows the use of the small native molecule without any modification, such as the incorporation of biotin, photo-affinity labels, or radioisotope . In this study, we used DARTS to identify potential binding targets of DHM and used DARTS-Western blotting to test and validate the potential DHM target. Our results showed that DHM interacted with PLC to activate the Ca2+-CaMKK-AMPK signal pathway.