Poster Topical Area: Nutrient-Gene Interactions
Location: Hall D
Poster Board Number: 436
Gene expression analyses require validated housekeeping genes (HKG) that maintain uniform expression under controlled and experimental conditions. Our objectives were to assess the effect of RS fermentation in rats on commonly used HKG and also gluconeogenesis regulatory enzymes essential for regulating glucose homeostasis in the cecum, specifically, phosphoenolpyruvate carboxykinase (PEPCK1). Currently, there is not universal consensus on the occurrence of intestinal gluconeogenesis (IGN).
In a 2x2x2 factorial arrangement, eight dietary treatments were fed to Sprague Dawley (SD) rats for six weeks. Treatment factors were RS (Present/Absent), Whole grain (Present/Absent), and Fat (High, Moderate). Using RT-qPCR, a commonly used housekeeping gene, cyclophilin F (PPIF), was tested to determine any statistically significant effect of dietary RS treatment. Gene expression (RT-qPCR) and protein produced (Western blot and ELISA) were determined for PEPCK1 in the cecum. In a second study (2x2 factorial), four dietary treatments were fed to SD rats for 12 weeks. Treatment factors were: Sodium Butyrate [SB] and RS (Present/Absent). To compare effects of possible dietary RS treatment effect on HKG expression to the first study, we then performed RT-qPCR and statistical analysis on 18S rRNA and PPIF genes.
In both studies, dietary RS fermentation had a significant effect to increase PPIF gene expression (p≤0.002) while all other dietary treatments were not significantly different. In study 2, 18S rRNA gene was not significantly different (p≥0.18) for dietary RS. Western blot produced no band for PEPCK1, while ELISA was effective for protein quantitation. A significant increase in PEPCK1 gene expression (p<0.0001) and protein (p≤0.001) produced was observed when dietary RS was present.
Increased PPIF gene expression, when used as a HKG, biases against a RS effect. However, despite PPIF bias, significance is still attained with dietary RS due to robust effects of fermentation, even in one rodent study with RS as low as 5% (w/w). We recommend using the 18S rRNA gene as the HKG to avoid overlooking subtle changes unlike robust RS fermentation. Dietary RS positively affects gene expression and protein production of PEPCK1, illustrating support for cecum as a site for IGN.
USDA–NIFA #2014–67017–21760, LSU AgCenter. Ingredion Incorporated gifted flour/starches.
Baton Rouge, Louisiana