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Reproductive aging results in declines in female fertility and in slight declines in baseline serum inhibins in humans. The authors tested the hypothesis that exogenous ovarian stimulation as a noninvasive dynamic test, by amplifying inhibin levels, can define more accurately than baseline serum inhibin levels the ovarian age of female rats. Female rats--young (immature, 26 days old), adult (65-75 days old), and reproductively aged (8- to 9-month-old retired breeders)--were administered pregnant mare's serum gonadotropin to stimulate the ovaries and the serum inhibin A and inhibin B, and ovarian inhibins were measured. The young and adult females had at least a 4- to 10-fold increase in serum inhibin A and inhibin B after ovarian stimulation, but the reproductively aged females did not. Adult female serum inhibin measurements after stimulation at every stage of the estrous cycle revealed a consistent 4- to 5-fold increase in all stages, whereas the reproductively aged females did not. Western blot analyses of ovarian lysates were consistent with the serum inhibin level results and revealed that ovarian inhibin α, inhibin β A, and inhibin β B subunit levels in adult ovaries were elevated after ovarian stimulation but not in the reproductively aged ovaries. The data presented here demonstrate that young, adult, and reproductively aged female inhibin responses after exogenous ovarian stimulation were different. Inhibin measurement in serum after ovarian challenge in rats, therefore, is a noninvasive method that could be used to dissect ovarian function in aging in more detail. [ABSTRACT FROM AUTHOR]

Insulin-induced GLUT4 recruitment to the plasma membrane involves GLUT4 trafficking through multiple subcellular compartments regulated by multiple proteins, many of which are yet to be identified. Here we describe a 65 kDa protein found in purified GLUT4 vesicles of rat adipocytes as a potential GLUT4 traffic regulatory protein. On the basis of MALDI-TOF MS, RT-PCR, gene cloning, protein sequencing, and immunoreactivity data, we identified this protein as EHD2, a member of the EH domain-containing proteins that have been implicated in vesicle trafficking. EHD2 in rat adipocytes was 85% membrane-associated, including approximately 10% in immunopurified GLUT4 vesicles. This association of EHD2 with GLUT4 vesicles occurred in PM and three distinct endosomal fractions and was not significantly affected by cellular insulin treatment. In co-immunoprecipitation experiments, however, EHD2 physically interacted with GLUT4 in each of these fractions, and cellular insulin treatment selectively enhanced this interaction in an endosomal fraction thought to contain GLUT4 exocytic vesicles. EHD2 also interacted with the clathrin adaptor middle chain subunit μ1, μ2, and rCALM in GST pull-down experiments. Significantly, an affinity-purified EHD2 antibody and a peptide corresponding to the EHD2 sequence Glu428-Glu535 drastically (by 75% and 35%, respectively) suppressed the insulin-induced increase in the plasma membrane GLUT4 contents in SLO-permeabilized rat adipocytes without affecting the basal GLUT4 distribution. These findings strongly suggest that EHD2 interacts with GLUT4 in rat adipocytes and may play a key role in insulin-induced GLUT4 recruitment to the plasma membrane. [ABSTRACT FROM AUTHOR]