The insulin receptor (INSR) has been shown to be hyperactive in hepatic stellate cells (HSCs) in humans and rodents with liver fibrosis. To explore HSC cellular mechanisms that INSR regulates during pro‐fibrotic stimulation, we used CRISPR‐Cas9 technology. We knocked out a portion of the INSR gene in human LX2 HSC cells ( INSR ^e5‐8 KO) that regulates insulin responsiveness but not the insulin‐like growth factor (IGF) or transforming growth factor‐β (TGFβ) signaling. The INSR ^e5‐8 KO HSCs had significantly higher cell growth, BrdU incorporation, and lower TP53 expression that suppresses growth, and they also exhibited increased migration compared to the Scramble control. We treated the scramble control and INSR ^e5‐8 KO HSCs with insulin or TGFβ and profiled hundreds of kinase activities using the PamGene PamStation kinome technology. Our analysis showed that serine/threonine kinase (STK) activities were reduced, and most of the protein‐tyrosine kinase (PTK) activities were increased in the INSR ^e5‐8 KO compared to the Scramble control HSCs. To study gene transcripts altered in activated Scramble control and INSR ^e5‐8 KO HSCs, we treated them with TGFβ for 24 h. We isolated RNA for sequencing and found that the INSR ^e5‐8 KO cells, compared to control HSCs, had altered transcriptional responsiveness to TGFβ stimulation, collagen‐activated signaling, smooth muscle cell differentiation pathways, SMAD protein signaling, collagen metabolic process, integrin‐mediated cell adhesion, and notch signaling. This study demonstrates that reduced INSR responsiveness enhances HSC growth and selectively mediates TGFβ‐induced HSC activation. These findings provide new insights into the development of more effective treatments for liver fibrosis.

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