Down-Regulation of Erk and Sirt1 Signaling May Lead to Reduced Fgf-21 Sensitivity in a Mouse Model of Diabetes

Introduction Fibroblast Growth Factor 21 (FGF-21) belongs to the FGF family of growth factors. FGF-21 regulates various aspects of glucose and lipid metabolism and is anti-diabetic. For example, FGF-21 administration or transgenic overexpression alleviates hyperglycemia and hyperlipidemia in obese mice, reduces body weight, and protects against fatty liver disease. FGF-21 signals via the FGF receptors and signaling also requires an accessory protein called β-klotho. Downstream FGF-21 effectors include ERK and Sirt1. FGF-21 is primarily secreted by adipocytes and hepatocytes and its expression is increased by agonists of PPARα (e.g. fenofibrate) and AMPK (e.g. metformin). Here, we used a mouse model of diabetes and investigated changes in FGF-21 and related signaling proteins in liver and adipose tissue. The role of microRNAs as potential regulators of FGF-21 signaling was also assessed. Methods WT or leptin receptor mutant (db/db) mice were used in this study. Mice were identified by genotyping and blood glucose levels were monitored (to confirm hyperglycemia in db/db mice). All animal experiments were performed using an IACUC-approved protocol. Mice were euthanized and liver and perivascular adipose tissue were collected and snap-frozen for later analysis. Blood was collected from anesthetized mice. Western blotting and quantitative PCR were performed using standard protocols. Data were analyzed using GraphPad software and t-tests and ANOVA analysis were used for statistical comparisons. Results WT and leptin receptor mutant (db/db) mice were treated with metformin (100 mg/kg/day) for 6–8 weeks. Western blot experiments in liver lysates revealed that metformin treatment significantly increased FGF-21 protein expression in WT mice. However, metformin failed to elevate FGF-21 in db/db mice. Metformin also significantly increased the expression of β-klotho protein in the liver of WT mice, but not in db/db mice. There was also an increase in FGF-21 and β-klotho gene expression after metformin treatment in WT mice, but not in db/db mice. ERK and Sirt1 are downstream molecules required for FGF-21 signaling. Experiments revealed that there was a significant decrease in ERK phosphorylation in db/db liver. Metformin treatment of WT as well as db/db mice also further reduced ERK phosphorylation in liver. Expression of Sirt1 protein was also reduced in db/db liver and further decrease was observed after metformin treatment. To investigate mechanisms involved in down-regulation of ERK and Sirt1, experiments were focused on microRNA 34a (miR34a), which is known to regulate both ERK and Sirt1 expression. A significant increase in circulating plasma miR34a was observed in db/db mice. Short treatment with metformin (1–3 days) produced a further increase (>10-fold) in circulating plasma miR34a in db/db mice. To identify the tissue source for miR34a synthesis and release into circulation, experiments were performed in perivascular adipose tissue (PVAT). Experiments revealed a significant increase in miR34a, but no further increase after metformin treatment in PVAT from db/db mice, suggesting that PVAT was not a likely source for miR34a release. Conclusions The data suggest up-regulation of FGF-21 and β-klotho in livers of db/db mice but a concomitant reduction in its downstream effectors, ERK and Sirt1. This may lead to compromised FGF-21 sensitivity in diabetes. Down-regulation of ERK and Sirt1 may result from elevated circulating miR34a levels. Acknowledgements The project was funded by NPRP grants (6-428-3-113 and 5-149-3-040) from QNRF.