Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies.

Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1-7) protective axis of renin-angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

Effects of long-term caloric restriction on glucose homeostasis and on the first steps of the insulin signaling system in skeletal muscle of normal and Ames dwarf (Prop1df/Prop1df) mice.

Ames dwarf mice are a model of retarded aging and extended longevity and display enhanced insulin sensitivity. Caloric restriction (CR) and the dwarf mutation have additive effects on lifespan. To begin to understand the mechanisms behind this effect, an analysis of the in vivo status of the insulin signaling system was performed in skeletal muscle from Ames dwarf (df/df) and normal mice fed ad libitum or subjected to long-term (over 1 year) CR. The response to CR was different in both groups of animals. In normal animals, CR induced a significant reduction in both circulating insulin and glucose levels, together with an increase in the in vivo insulin-stimulated phosphorylation of the IR, a trend towards an increase in the in vivo insulin-stimulated phosphorylation levels of IR substrate-1, and an increase in the abundance of GLUT4 in muscle. In contrast, CR did not modify none of these parameters in df/df mice. Interestingly, CR induced a reduction in the p85 subunit of phosphatidylinositol 3-kinase abundance in skeletal muscle in both groups of animals. These results suggest that in skeletal muscle, long-term CR induces different effects on the first steps of the insulin signaling system in normal mice than in df/df mice.