ILDR2 has a negligible role in hepatic steatosis

<div><p>We have previously reported that <i>Ildr2</i> knockdown via adenovirally-delivered shRNA causes hepatic steatosis in mice. In the present study we investigated hepatic biochemical and anatomic phenotypes of Cre-mediated <i>Ildr2</i> knock-out mice. Liver-specific <i>Ildr2</i> knock-out mice were generated in C57BL/6J mice segregating for a floxed (exon 1) allele of <i>Ildr2</i>, using congenital and acute (10-13-week-old male mice) Cre expression. In addition, <i>Ildr2</i> shRNA was administered to <i>Ildr2</i> knock-out mice to test the effects of <i>Ildr2</i> shRNA, <i>per se</i>, in the absence of <i>Ildr2</i> 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 <i>Ildr2</i> shRNA to <i>Ildr2</i> knock-out mice did cause hepatic steatosis, indicating that the <i>Ildr2</i> shRNA had apparent “off-target” effects on gene(s) other than <i>Ildr2</i>. RNA sequencing and BLAST sequence alignment revealed <i>Dgka</i> as a candidate gene mediating these “off-target” effects. <i>Ildr2</i> shRNA is 63% homologous to the <i>Dgka</i> gene, and <i>Dgka</i> expression decreased only in mice displaying hepatic steatosis. <i>Dgka</i> encodes diacylglycerol kinase (DGK) alpha, one of a family of DGKs which convert diacylglycerides to phosphatidic acid for second messenger signaling. <i>Dgka</i> 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 <i>Ildr2</i> knockdown mice was likely due to shRNA targeting of <i>Dgka</i> 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.</p>