miR-378b Regulates Insulin Sensitivity by Targeting Insulin Receptor and p110α in Alcohol-Induced Hepatic Steatosis

Abstract

Insulin resistance has been implicated in alcoholic liver disease. A previous study has shown that microRNAs (miRNAs) play a major role in the production, secretion, and function of insulin. MiRNAs are capable of repressing multiple target genes that in turn negatively regulate various physiological and pathological activities. However, current information on the biological function of miRNAs in insulin resistance is limited. The goal of the present study was to elucidate the role of miR-378b in alcohol-induced hepatic insulin resistance and its underlying mechanism. This study has observed that miR-378b is upregulated in National Institute on Alcohol Abuse and Alcoholism (NIAAA) alcoholic mouse models as well as in ethanol-induced L-02 cells in vitro. Furthermore, miR-378b overexpression impaired the insulin signaling pathway, and inhibition of miR-378b improved insulin sensitivity in vivo and in vitro. A mechanistic study revealed that IR and p110a are direct targets of miR-378b. Together, these results suggest that miR-378b controls insulin sensitivity by targeting the insulin receptor (IR) as well as p110a and possibly play an inhibitory role in the development of insulin resistance, thereby providing insights into the development of novel diagnostic and treatment methods.

microRNA-378b regulates ethanol-induced hepatic steatosis by targeting CaMKK2 to mediate lipid metabolism

“…The specific methods used for cell culture refer to previous protocols [ 23 ]. Moreover, preceding research showed that 200 mM EtOH successfully caused to happen ALD in L-02 cells [ 18 ], so our following experiments were performed with an EtOH concentration of 200 mM.…”

“…An miR-378b mimic and inhibitor (including their respective negative controls-NCs) were purchased from GeneChem (Shanghai, China). The plasmid was transferred into cells by electroporation (Bio-Rad Laboratories, Inc., Hercules, CA, USA) [ 18 ].…”

“…L-02 cells cultured in a 55 square centimeters petri dish under the nutrient conditions described above were divided into a control group, model group (200 mm EtOH treatment), miR-378b mimic, inhibitor and their corresponding NC group. Except for cells in the control group, others were cultured in a fresh medium containing 200 mM EtOH for 48 h [ 18 ]. After cells cultured in groups, intracellular lipids were extracted according to the previously described method [ 24 ].…”

“…The protein concentration was assessed using the Nanodrop 2000 spectrophotometer (Thermo Scientific, USA). According to the manufacturer’s instructions [ 18 ], the same amount of proteins (30 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then attached onto PVDF membranes (Millipore Corp,Billerica, MA, USA). In addition, the rotational conditions are slightly adjusted according to the molecular weight.…”

“…Increasing evidence shows that miR-378 plays an important role in the regulation of lipid metabolism and the occurrence and development of liver diseases [ 17 ]. Combined with high-throughput sequencing, we previously observed that miR-378b also modulates insulin signaling by specifically binding the insulin receptor and p110α in animal and cellular models of ALD [ 18 ]. Along with previous research, we predicted the target of miR-378b via starBase and found that CAMKK2 messenger RNA (mRNA) contains a miR-378b-binding site in its 3ʹ-untranslated region (UTR) which is the upstream of AMPK, suggesting that miR-378b may play an important role in regulating AMPK signal conduction.…”