Abstract
An alarming trend of the 21st century is the increasing prevalence of insulin resistance, an underlying factor of type 2 diabetes, non-alcoholic fatty liver disease and cardiovascular disease. While the focus has been on treating these diseases, prevention would be a more effective strategy, and this requires a thorough understanding of how insulin resistance develops. A particular target of interest in this regard is the aryl hydrocarbon receptor (AHR), a transcription factor activated by a range of exogenous and endogenous ligands. The AHR has been implicated in regulating glucose and lipid metabolism as well as insulin sensitivity, but the findings have been conflicting as to whether ligand activation of the AHR has a beneficial or detrimental effect. Given the diversity of AHR ligands they are unlikely to all act in the same manner and a thorough comparison of how these ligands affect metabolic pathways is necessary. Therefore, this study compared the effects of 10 AHR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 6-formylindolo[3,2-b]carbazole (FICZ), β-naphthoflavone (BNF), GNF-351 (GNF), indole-3-carbinol (I3C), 4,7-dibromo-2,3-dichloroindole (4DBDCI), 7-bromo-2,3-dichloro-6-iodoindole (BDCII), 6,7-dibromo-2,3-dichloroindole (6DBDCI), 2,6,7-tribromo-3-chloroindole (TBCI) and 4-chloro-3-hydroxyl-3-(2-oxopropyl)-2-oxindole (CHOOI), representing a range of sources, in human liver HepG2 cells.
When tested for their ability to activate AHR signalling, determined by CYP1A1 activity, the 10 ligands displayed a range of efficacies, from GNF causing no change in CYP1A1 activity, to TCDD and FICZ inducing 15 – 20-fold increases in CYP1A1 activity. Similarly, the durations of AHR activation varied significantly, with CHOOI inducing maximal CYP1A1 activity at 6 hours, and TCDD and TBCI continuing to increase CYP1A1 activity over 72 hours. Furthermore, five new seaweed-derived indole compounds, 4DBDCI, BDCII, 6DBDCI, TBCI and CHOOI, were found to activate AHR signalling.
Examining how these differences in AHR activation translated into an effect on the expression of lipid (SREBF1, FASN and PPARA) and glucose (G6PC1) metabolism genes did not reveal any significant patterns. Furthermore, only two ligands, 4DBDCI and TBCI, were able to reduce lipid accumulation by 50% and 20%, respectively, determined by Oil Red O staining. However, in a model of hepatic insulin resistance, FICZ prevented the fructose-mediated reduction of insulin-stimulated pAKT/AKT, assessed via western blotting, while BNF, I3C and 4DBDCI worsened the fructose-induced insulin resistance a further 60%. The mechanism of FICZ’s protection was dependent on the AHR, as co-treatment with the AHR antagonist, GNF, completely abrogated the protective effect and further exploration of this mechanism determined it was not due to alterations in lipid accumulation, oxidative stress or AHR nuclear translocator protein expression. Additionally, the effect of AHR ligands on metabolic pathways were not associated with their efficacy or persistence of AHR stimulation, in contrast to previous reports.
Overall, this study found a protective effect of FICZ against insulin resistance in HepG2 cells, and also identified five new ligands that activate AHR signalling, and further investigation into the therapeutic potential of these ligands should be conducted. However, it is still unclear how these ligands produce such varying effects, and this is necessary to understand if targeting the AHR is to become a viable therapeutic strategy.