Schlafen 3 knockout mice display gender-specific differences in weight gain, food efficiency, and expression of markers of intestinal epithelial differentiation, metabolism, and immune cell function

Abstract

Self-renewal and differentiation are essential for intestinal epithelium absorptive functioning and adaptation to pathological states such as short gut syndrome, ulcers, and inflammatory bowel disease. The rodent Slfn3 and its human analog Slfn12 are critical in regulating intestinal epithelial differentiation. We sought to characterize intestinal function in Slfn3 knockout (KO) mice. Male and female pair-fed Slfn3KO mice gained less weight with decreased food efficiency than wild type (WT) mice, with more pronounced effects in females. RNA sequencing performed on intestinal mucosa of Slfn3KO and WT mice showed gene ontology decreases in cell adhesion molecule signaling, tumor necrosis factor receptor binding, and adaptive immune cell proliferation/functioning genes in Slfn3KO mice, with greater effects in females. qPCR analysis of fatty acid metabolism genes, Pla2g4c, Pla2g2f, and Cyp3c55 revealed an increase in Pla2g4c, and a decrease in Pla2g2f in Slfn3KO females. Additionally, adipogenesis genes, Fabp4 and Lpl were decreased and ketogenesis gene Hmgcs2 was increased in female Slfn3KO mice. Sequencing did not reveal significant changes in differentiation markers, so qPCR was utilized. Slfn3KO tended to have decreased expression of intestinal differentiation markers sucrase isomaltase, dipeptidyl peptidase 4, villin 1, and glucose transporter 1 (Glut1) vs. WT males, although these trends did not achieve statistical significance unless data from several markers was pooled. Differentiation markers, Glut2 and sodium-glucose transporter 1 (SGLT1), did show statistically significant sex-dependent differences. Glut2 mRNA was reduced in Slfn3KO females, while SGLT1 increased in Slfn3KO males. Notch2 and Cdx2 were only increased in female Slfn3KO mice. Although Slfn3KO mice gain less weight and decreased food efficiency, their biochemical phenotype is more subtle and suggests a complex interplay between gender effects, Slfn3, and another regulatory pathway yet to be identified that compensates for the chronic loss of Slfn3.

Fig 1. Decreased weight gain and food efficiency in Slfn3KO mice.

(A) Confirmed knockdown of Slfn3 in Slfn3KO mice. Total RNA was isolated from intestinal mucosa of WT and Slfn3KO mice. Slfn3 mRNA expression was analyzed by qPCR using RPLP0 as a reference control gene. (n = 37–47; *p<0.05 to respective WT; #p<0.05 to respective male genotype). Slfn3KO mice were pair-fed to WT mice over a 6 week period. (B) Weight was measured twice per week and (C) food intake was measured daily. (D) Food efficiency was calculated as weight gained divided by food intake. (Male n = 35–44, female n = 14–16; *p<0.05 to respective WT; #p<0.05 to respective male genotype).

Fig 2. Loss of Slfn3 changes the histology of the intestine.

Slfn3KO mice have differential (A) villus length, (B) crypt depth, (C) and muscularis externa thickness in comparison to WT mice. (n = 3 mice/group, n = 85–103 villi, n = 84–173 crypts, and n = 128–190 muscularis externa measurements taken; *p<0.05 to respective WT).

Fig 3. Meta-analysis heatmap of Slfn3KO males vs. WT males compared to Slfn3KO females vs. WT females.

Fig 4. Differentially expressed α-linoleic acid and arachidonic acid metabolism genes in Slfn3KO mice.

The mRNA expression of (A) Pla2g4c, Phospholipase A2, group IVC (cPLA2γ), (B) Pla2g2f, Phospholipase A2, group IIF (cPLA2α, IIF) and (C) Cyp2c55, Cytochrome P450, subfamily c, polypeptide 55 were analyzed by qPCR using RPLP0 as a reference control gene. (n = 33–56; *p<0.05 to respective WT).

Fig 5. Adipogenesis genes are decreased in female Slfn3KO mice.

The mRNA expression of (A) Fabp4, Fatty acid binding protein 4 and (B) Lpl, Lipoprotein lipase were analyzed by qPCR using RPLP0 as a reference control gene. (n = 33–56; *p<0.05 to respective WT).

Fig 6. Ketogenesis gene, Hmgc2 was increased in female Slfn3KO mice.

The mRNA expression of Hmgcs2, Hydroxy-methylglutaryl-CoA Synthase was analyzed by qPCR using RPLP0 as a reference control gene. (n = 33–56; *p<0.05 to respective WT).

Fig 7. Glut2 and SGLT1 mRNA expression levels in Slfn3KO mice is sex-dependent.

Total RNA was isolated from intestinal mucosa of WT and Slfn3KO mice and differentiation marker expression was analyzed by qPCR using RPLP0 as a reference control gene. mRNA expression of (A) SI, (B) Dpp4, (C) Vil1, (D) Glut1, (E) Glut2, and (F) SGLT1. (n = 37–47; *p<0.05 to respective WT; #p<0.05 to respective male genotype).

Fig 8. Increased mRNA expression of Notch2 and Cdx2 in Slfn3KO male mice.

Total RNA was isolated from intestinal mucosa of WT and Slfn3KO mice and mRNA expression of (A) Notch1, (B) Notch2 and (C) Cdx2 was analyzed by qPCR using HPRT as a reference control gene. (n = 28–41; *p<0.05 to respective WT).