Previous posts have described some of the novel strategies and innovative technologies used to identify candidate biomarkers and therapeutic targets. But what comes next? How can researchers verify that putative biomarkers are biologically relevant and worthy of further research? Shannon Reilly et al.,1 deconstruct the signalling pathway that mediates obesity-induced inflammation, and elegantly demonstrate that kinases IKK-e and TBK1 are likely therapeutic targets in obesity.

Obesity leads to chronic, low-grade local inflammation in adipose tissue and the liver, possibly through the NF-kB signalling pathway. This inflammation can eventually result in insulin resistance and type 2 diabetes. In an insulin-resistant state, insulin no longer inhibits hepatic glucose production, among other things. Reilly and her team previously showed that IKK-e and TBK1 kinases, part of the NF-kB inflammatory signalling pathway, are inhibited by amlexanox, an anti-inflammatory compound. In this study, they explored how amlexanox-induced IKK-e and TBK1 inhibition suppresses liver gluconeogenesis, breaking down the pathway one step at a time. The following is a precis of their experiments and findings.

1 Amlexanox induces the liver Jak/Stat signalling pathways

Differential RNA expression assays demonstrated that 1700 RNA transcripts were upregulated in the liver in response to amlexanox, and on the basis of this information, pathway analysis was used to determine which signalling pathways were most likely to be involved.

2 Cytokine IL-6 likely stimulates the liver Jak/Stat pathway

After hypothesizing that serum cytokines activate the Jak/Stat pathway, researchers looked for upstream activators in the serum. They found that the levels of cytokine IL-6, a known upstream activator of the Jak/Stat pathway, peaked four hours after amlexanox stimulation.

3 Adipocytes produce IL-6 in response to amlexanox

Gene expression analysis revealed that only subcutaneous white adipose tissue showed a significant increase in IL-6 mRNA levels in response to amlexanox. The investigators sorted different subcutaneous tissue cells using fluorescence-activated cell sorting and cell surface markers. Of all the sorted cell types, only adipocytes and pre-adipocytes showed increased IL-6 mRNA expression. Increases in amlexanox-induced IL-6 mRNA expression in adipocytes correlated with the secretion of IL-6 protein.

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4 Adipocyte IL-6 mRNA expression is induced through the cAMP pathway and is cAMP dependent

In adipocytes, the upregulation of cAMP is thought to help mobilize glucose and fatty acid from reserves. Researchers expressed normal and mutated IKK-e proteins in adipocyte cell cultures to mimic the obese state, and then tested various inhibitors and activators to influence IL-6 mRNA expression. They found that amlexanox-induced activation of cAMP is dependent on p38.

5 Serum IL-6 leads to Stat3 phosphorylation at tyrosine 705

The team observed that increases in serum IL-6 levels led to increased phosphorylation and activation of Stat 3.  Using neutralizing antibodies, they confirmed that hepatic Stat3 activation is IL-6 dependent.

6 Activated Stat3 suppresses liver gluconeogenesis

Using promoter-binding assays, researchers determined that, in response to amlexanox, Stat3 binds to the G6pc promoter to suppress gluconeogenic gene expression.

This study demonstrates that there is a signalling pathway from adipose tissue to the liver. Amlexanox inhibits IKK-e and TKB1 in adipocytes leading to p38-dependent cAMP upregulation, which leads to IL-6 production and secretion into the serum. IL-6 stimulates phosphorylation and activation of hepatic Stat3, which suppresses liver glucose production.

While the researchers were unable to reproduce their experiments in humans, they did measure the levels of circulating IL-6 and insulin sensitivity in response to amlexanox in six obese, diabetic human subjects. While subjects exhibited varying levels of IL-6 in response to amlexanox, these IL-6 levels correlated with reductions in insulin resistance. Insulin resistance is a precursor for diabetes, so increasing insulin sensitivity, perhaps through IKK-e and TKB1 inhibitors such as amlexanox, may be a promising strategy for preventing this disease. This research team is currently exploring this approach in two ongoing clinical trials.2

  1. Reilly SM, Anhadian M, Zamarron BF et al. A subcutaneous adipose tissue-liver signalling axis controls hepatic gluconeogenesis. Nat Commun. 2015 12(6):6047. doi: 10.1038/ncomms7047.
  1. Trials NCT01975935 and NCT01842282 are listed on www.clinicaltrials.gov.