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CREB‐Regulated miR‐27b Is Linked to Hepatic Insulin Resistance by Targeting Insulin/Akt Signaling

Insulin resistance (IR) is a predisposed condition of type 2 diabetes and commonly leads to fatty liver, in which hepatocytes exhibit excessive lipogenesis and gluconeogenesis. The role of microRNAs (miR) in liver‐associated IR remains largely unknown. Among microRNAs that participate in metabolic signaling, miR‐27b is a critical regulator of lipogenesis in both liver and adipose tissues; however, it is not clear if dysregulation of miR‐27b contributes to IR in liver. To address this question, we established several IR experimental models, including palmitate‐treated mouse AML12 hepatocytes, and dietary‐induced mouse fatty liver; Interestingly, miR‐27b was upregulated in all of these IR models. Furthermore, bioinformatic analysis predicted that miR‐27b targets several genes involved in Akt phosphorylation, including Pdpk1, Pik3ca (P110), Pik3r1 (P85), and Pik3cd. Based on these data, we hypothesize that miR‐27b over‐expression results in defective Akt signaling upon insulin stimulation. Indeed, over‐expression of miR‐27 in human HepaRG liver cells and mouse primary hepatocytes significantly suppressed Akt phosphorylation at both T308 and S473 residues and down‐regulated Pdpk1, Pik3r1 (P85), and Pik3ca (P110) at protein level upon insulin stimulation. Our luciferase reporter assay using 3T3 cells further demonstrated that miR‐27 directly targeted Pdpk1 and Pik3r1 3′UTR. Next, we investigated the mechanism by which miR‐27 is regulated in insulin signaling. Interestingly, we found that insulin significantly reduced miR‐27 expression below 50% of control in both HepaRG and AML12 cells. Further study on the detail mechanisms revealed that miR‐27b expression is positively regulated by cAMP response element‐binding protein (CREB), which is an IR promoter and regulated by insulin. Using HepaRG and AML12 cells, we demonstrated that miR‐27 is significantly down‐regulated by siRNA‐ or metformin‐mediated knockdown of CREB, but up‐regulated by forksolin‐induced activation of CREB. Also, miR‐27b is significantly increased when CREB is up‐regulated in mouse livers by either overnight fasting or high‐fat‐diet feeding, whereas decreased when CREB is down‐regulated in livers of mice treated with metformin. Taken together, our in vitro and in vivo data demonstrated that insulin may negatively regulate miR‐27 by repressing CREB transcriptional activity and the abnormal activation of hepatic CREB in IR animals may cause the aberrant elevation of miR‐27b to further decrease insulin sensitivity.