Date of Completion
Karl L. Insogna; Yih-Woei C. Fridell
Field of Study
Master of Science
The effect of dietary protein on bone health remains controversial. We have shown that increasing dietary protein increases calcium absorption in both humans and rats with no change in bone resorption. We used a rat model in which dietary protein increases intestinal calcium absorption and whole body calcium retention, to explore the molecular basis for this effect. Female Sprague-Dawley rats were fed a low (5%) or high (40%) protein diet for 7 days. On day 7, duodenal mucosa was harvested and total RNA isolated. Microarray analyses using these RNA samples identified 208 genes whose expression was at least 1.5 fold different between groups. Of particular relevance, the calcium permissive tight junction protein, claudin-2 (Cldn-2), was found to be upregulated 2.9-fold by the high protein diet and a posited calcium intolerant tight junction protein, kallikrein-related peptidase-10 (KLK-10), was found to be downregulated 5.5-fold by the high protein diet. These results were confirmed at the mRNA level by qPCR and at the protein level by western blot analysis. To examine cellular expression of Cldn-2 and KLK-10, Caco-2 Bbe cells (14 days post-confluent) were incubated with control media (CM) or CM + 2X amino acids for 6 hrs, or 1,25(OH)2vitamin D for 24 hrs. Both Cldn-2 and KLK-10 were found to be expressed in the cytoplasm, as well as at the cell periphery, the latter consistent with tight junction expression. In at least some experiments amino acid and 1,25(OH)2vitamin D treatment increased Cldn-2 tight junction expression and its apparent colocalization with ZO-1, a known tight junction membrane protein. In preliminary experiments, amino acid treatment decreased KLK-10 tight junction expression and its apparent colocalization with ZO-1. These data suggest that amino acid-induced changes in the expression and/or subcellular distribution of Cldn-2 and KLK-10 may be part of the molecular mechanism by which dietary protein increases paracellular calcium flux.
Kotler, Belinda M., "Dietary Protein Influences Paracellular Calcium Transport: Two Molecular Targets" (2012). Master's Theses. 321.
Jane E. Kerstetter