Targeting ACSS3 and IRF1 for the metabolic and inflammatory stages of liver disease
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Abstract
Metabolic diseases such as obesity, diabetes, and nonalcoholic fatty liver disease (NAFLD) are responsible for a staggering number of deaths within the United States and beyond. Given that the incidence of these conditions is only predicted to increase in the future, there is serious need for the improvement of current treatments. Although understated in the popular consciousness, the liver plays an enormous role in the development of these pathologies. This study constitutes a double-pronged investigation, pursuing two different players in pathological liver dysfunction. In hepatocytes, the parenchymal cells of the liver, acyl-CoA synthetase short chain family member 3 (ACSS3) has previously been suggested to promote potentially harmful metabolic outcomes like de novo lipogenesis, reactive oxygen species formation, hepatic glucose production, and hepatic insulin tolerance. It appears likely that silencing the ACSS3 gene could provide multiple metabolic benefits, including a reduction in deleterious lipotoxicity. Towards this end, this study evaluated multiple different short hairpin RNAs (shRNAs) to assess their effectiveness in knocking down the expression of the gene encoding for ACSS3 production. This process entailed cell culture, plasmid extraction and transfection, protein extraction, and Western blotting. Despite expectations, none of the tested shRNAs exhibited consistent and significant knockdown of the ACSS3 gene. Separately, interferon regulatory factor 1 (IRF1) exhibits increased expression in fibrotic livers, involved in conditions like NAFLD. A secondary investigation into IRF1 was also conducted, evaluating mouse models bred as homozygous (Irf1-/-) or heterozygous (Irf1+/-) knockouts for this gene alongside wild type (Irf1+/+) littermates, employing glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs). GTT results revealed that both male and female Irf1+/- mice of all different age ranges exhibited the greatest degree of glucose tolerance. On the other hand, homozygous deletion of IRF1 resulted in sex-dependent changes in glucose homeostasis: While male Irf1-/- mice displayed a tendency toward increased glucose tolerance, female Irf1-/- mice had impaired glucose clearance compared to their wild type counterparts for each gender. Additionally, when data from both sexes were merged, a significant difference still remained between the Het and WT genotypes. ITT findings across male and female mice lacked statistically significant differences within each sex, likely owing to their smaller sample size, but nonetheless present a similar trend of heterozygote IRF1 expression improving insulin tolerance over wild type, especially among female mice. Furthermore, when both sexes had their ITT data pooled together, point analysis did reveal a statistically significant difference between Het and WT at the end of the ITT. Although these results reveal a little sexual dimorphism worth further investigation, they suggest that partially suppressing the expression of IRF1 may hold potential in discouraging such conditions in human beings as well, perhaps through such means as siRNA or pharmacological inhibitor administration.
Description
2024