Ashwish, Nadya M., authorRoess, Deborah A., advisorBarisas, George, committee memberCrans, Debbie C., committee memberMiller, Charles W., committee member2019-06-142019-06-142019https://hdl.handle.net/10217/195241Changes in gene expression in granulosa cells from mares may result from aging or development of metabolic disease as well as from other causes. We used qRT-PCR to quantify expression of equine insulin receptor (IR), insulin-like growth factor receptor (IGF-1R), glucose transporter type 4 (GLUT4) and adenosine monophosphate-activated protein kinase (AMPK) subunit genes in granulosa cells from young (4-9 years), middle-aged (10-16 years) and older (>16 years) mares. Granulosa cells were isolated following follicular aspiration and incubated in the presence or absence of 10% fetal bovine serum (FBS) and 30mM glucose for 24 hrs. Cells were then treated for 1 hr with insulin (100nM), IGF-1 (10nM), epidermal growth factor (EGF; 1μM), progesterone (P4; 100 nM) or human chorionic gonadotropin (hCG; 100 nM). ∆Ct, where Δct is equal to the CT value for the gene of interest minus the CT value for a house keeping gene, values for IGF-1R or IR expression did not differ significantly for any age group. However, there were statistically significant differences in the ratio of ∆Ct for IR relative to ∆Ct for IGF-1R for individual young, middle-aged and old mares. Young animals expressed increased IGF-1R relative to IR after pre-incubation of cells in either – fetal bovine serum FBS/30mM glucose or + FBS/30mM glucose media followed by treatment with insulin, IGF-1, or EGF. Conversely, middle-aged animals expressed increased IR relative to IGF-1R following preincubation in both medium and following treatment with insulin or IGF-1. Finally, older animals expressed approximately equal amounts of IR relative to IGF-1R ( FBS/-30mM glucose) or increased IR relative to IGF-1R. When cells were pre-incubated in +FBS/+30mM glucose medium and treated with insulin or IGF-1, the ratio of IR expression relative to IGF1R expression changed and there was increased IGF-1R relative to IR. Changes in the relative numbers of IR and IGF-1R monomers expressed from IR and IGF-1R genes may affect the particular receptor dimers assembled from these monomers. In evaluating fold change 2-∆∆Ct in gene expression, we observed increases in IGF-1R expression (+FBS/+30mM glucose; p<0.03), GLUT4 expression (+FBS/+30mM glucose; p<0.05) and AMPKα2 expression (-FBS/-30mM glucose; p<0.03) in older mares relative to young animals. Together, these results indicate that young animals differ from older animals in several ways. Young animals maintain stable ratios of ∆CT for IGF-1R to ∆CT for IR and these ratios are not affected by treatment with insulin, IGF-1 or EGF. Thus, it is likely that young animals maintain higher levels of IGF-1R homodimers and hybrid receptors formed from IGF-1R and insulin receptor monomers and that receptor numbers, including insulin receptors, remain stable, particularly when circulating glucose and insulin levels are high. Older animals, on the other hand, are more labile with respect to ∆CT for IGF-1R relative to ∆CT for IR. Exposure of granulosa cells pre-incubated in +FBS/+30mM glucose medium to high levels of glucose, insulin, IGF-1 or EGF demonstrated increased IGF-1R expression. This may decrease numbers of insulin-responsive insulin homodimers, increasing hybrid receptors and IGF-1R homodimers and thus decreasing insulin responsiveness and insulin effects on metabolism. Moreover, when there are significant effects on expression of IR, IGF1R or GLUT4 in older animals, there are accompanying increases in expression of some AMPK subunit genes in older animals indicating additional effects of aging on overall metabolism.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.Text