Cell lysis-free quantum dot multicolor cellular imaging-based mechanism study for TNF-α-induced insulin resistance

Background: TNF-alpha is an inflammatory cytokine that plays an important role in insulin resistance observed in obesity and chronic inflammation. Many cellular components involved in insulin signaling cascade are known to be inhibited by TNF-alpha. Insulin receptor substrate (IRS)-1 is one of the major targets in TNF-alpha-induced insulin resistance. The serine phosphorylation of IRS-1 enables the inhibition of insulin signaling. Until now, many studies have been conducted to investigate the mechanism of TNF-alpha-induced insulin resistance based on Western blot. Intracellular protein kinase crosstalk is commonly encountered in inflammation-associated insulin resistance. The crosstalk among the signaling molecules obscures the precise role of kinases in insulin resistance. We have developed a cell lysis-free quantum dots (QDots) multicolor cellular imaging to identify the biochemical role of multiple kinases (p38, JNK, IKK beta, IRS1(ser), IRS1(tyr), GSK3 beta, and FOXO1) in inflammation-associated insulin resistance pathway with a single assay in one run. QDot-antibody conjugates were used as nanoprobes to simultaneously monitor the activation/deactivation of the above seven intracellular kinases in HepG2 cells. The effect of the test compounds on the suppression of TNF-alpha-induced insulin resistance was validated through kinase monitoring. Aspirin, indomethacin, cinnamic acid, and amygdalin were tested. Results: Through the measurement of the glycogen level in HepG2 cell treated with TNF-alpha, it was found that aspirin and indomethacin increased glycogen levels by almost two-fold compared to amygdalin and cinnamic acid. The glucose production assay proved that cinnamic acid was much more efficient in suppressing glucose production, compared with MAP kinase inhibitors and non-steroidal anti-inflammatory drugs. QDot multicolor cellular imaging demonstrated that amygdalin and cinnamic acid selectively acted via the JNK1-dependent pathway to suppress the inflammation-induced insulin resistance and improve insulin sensitivity. Conclusion: The regulatory function of multiple kinases could be monitored concurrently at the cellular level. The developed cellular imaging assay provides a unique platform for the understanding of inflammation and insulin resistance signaling pathways in type II diabetes mellitus and how they regulate each other. The results showed that amygdalin and cinnamic acid inhibit serine phosphorylation of IRS-1 through targeting JNK serine kinase and enhance insulin sensitivity.