Abstract
We have previously reported that Ildr2 knockdown via adenovirally-delivered shRNA causes hepatic steatosis in mice. In the present study we investigated hepatic biochemical and anatomic phenotypes of Cre-mediated Ildr2 knock-out mice. Liver-specific Ildr2 knock-out mice were generated in C57BL/6J mice segregating for a floxed (exon 1) allele of Ildr2, using congenital and acute (10-13-week-old male mice) Cre expression. In addition, Ildr2 shRNA was administered to Ildr2 knock-out mice to test the effects of Ildr2 shRNA, per se, in the absence of Ildr2 expression. RNA sequencing was performed on livers of these knockdown and knockout mice. Congenital and acute liver-specific and hepatocyte-specific knockout mice did not develop hepatic steatosis. However, administration of Ildr2 shRNA to Ildr2 knock-out mice did cause hepatic steatosis, indicating that the Ildr2 shRNA had apparent “off-target” effects on gene(s) other than Ildr2. RNA sequencing and BLAST sequence alignment revealed Dgka as a candidate gene mediating these “off-target” effects. Ildr2 shRNA is 63% homologous to the Dgka gene, and Dgka expression decreased only in mice displaying hepatic steatosis. Dgka encodes diacylglycerol kinase (DGK) alpha, one of a family of DGKs which convert diacylglycerides to phosphatidic acid for second messenger signaling. Dgka knockdown mice would be expected to accumulate diacylglyceride, contributing to the observed hepatic steatosis. We conclude that ILDR2 plays a negligible role in hepatic steatosis. Rather, hepatic steatosis observed previously in Ildr2 knockdown mice was likely due to shRNA targeting of Dgka and/or other “off-target” genes. We propose that the gene candidates identified in this follow-up study may lead to identification of novel regulators of hepatic lipid metabolism.
Highlights
Non-alcoholic fatty liver disease (NAFLD) is rapidly becoming the leading cause of liver failure and transplantation in the United States and is predicted to affect ~30% of adults in the US [1]
Since we showed that adenoviral treatment alone does not cause hepatic steatosis, so we turned our attention to the Ildr2 short hairpin RNA (shRNA)
These results suggest that the Ildr2 shRNA targets other gene(s) involved in hepatic lipid metabolism, and that KD of these gene(s) is primarily responsible for the gross steatosis in the original Ildr2 shRNA ADKD mice [4] as well as the less striking, but still significantly increased TG accumulation observed here
Summary
Non-alcoholic fatty liver disease (NAFLD) is rapidly becoming the leading cause of liver failure and transplantation in the United States and is predicted to affect ~30% of adults in the US [1]. While the simple steatosis that defines NAFLD is relatively benign, it can progress to non-alcoholic steatohepatitis (known as NASH) with inflammatory infiltration and fibrosis [3]. The physiologic and metabolic factors that cause NAFLD and trigger its progression to NASH remain poorly understood. Identified by positional genetics as a diabetes-susceptibility gene in mice [5], Ildr knockdown via adenovirally-delivered shRNA (ADKD) resulted in gross hepatic steatosis and inflammation within 10 days of infection [4]. Transcript analyses indicated initial increase in expression of genes mediating lipogenesis (3 days post-adenovirus infection), followed by decrease in expression of these transcripts after development of steatosis, and differential expression of genes involved in the unfolded protein response (ER stress) pathways [4]
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