title_logo menu_logo
twitter
menu_bg
DiaComp Funded Abstracts



Program Application Abstract
The nuclear and cytosolic role of androgen receptor in regulating insulin signaling and gluconeogenesis in hepatocyte cells of female mice.
Udochi, ChiChi   (Johns Hopkins University)
Hyperandrogenemia (HA) is a defining cause of PCOS, polycystic ovarian syndrome, and is associated with impaired metabolic functions. There have been studies of the effects of HA on insulin signaling in adipocytes and skeletal muscle tissue in rodents. However, there is a lack of study on liver tissue. In addition, the serum DHT levels in mice used in those studies were 6-­8 folds higher than controls whereas women with PCOS only display 2-­3 fold increase in serum DHT compared to non-­PCOS women. Our project uses DHT levels comparable to levels found in PCOS women. We seek to determine whether androgen receptors role in insulin resistance and gluconeogenesis is more prominent in the cytosol or in the nucleus. At low dose DHT levels, androgen receptor (AR) will decrease insulin signaling, upregulate gluconeogenesis and it performs these functions predominantly in a non-­genomic fashion. We started this project with three aims: Aim1 was to determine if DHT (at a concentration similar to that used in our mouse model) has similar effects on insulin signaling and gluconeogenic factors as in the low dose DHT mouse model. Aim2 was to determining the role of AR overexpression (OE) gene in basal state or DHT-­induced effects on insulin signaling and gluconeogenic factors. The idea being to mimic what was seen in the low dose DHT mouse model. Aim3 was to determine if AR’s DNA binding function plays a role in basal state or DHT-­induced effects on insulin signaling and gluconeogenic factors. H2.35, a hepatocyte cell line derived from female mice, were cultured and set to the conditions as basal state, DHT, Insulin and DHT+ Insulin. In addition, we transfected hepatocytes with plasmids that contain an AR overexpression (OE) gene and another set of hepatocytes with mutant AR that is unable to bind to DNA (ARmut). The transfected cells were also prepared under the same conditions as above. QPCR data showed that AR-­OE increased the mRNA expression for AR (400-­fold) and the gluconeogenic rate-­ limiting enzyme, glucose 6 phosphatase, G6P. Cells treated with insulin displayed increased IRS2, PI3K, GLUT1 and GLUT2 mRNA. Low dose DHT decreased PI3K and GLUT1 and increased GLUT2 and G6P in the basal state. Interestingly, DHT blunted the insulin effect on GLUT1 but potentiated the effect on GLUT2 mRNA expression. Western blotting results showed that AR-­OE and ARmut increased AR protein levels. As expected, insulin increased p-­Foxo1;; however, DHT decreased insulin stimulated p-­Foxo1 indicating insulin resistance. Insulin resistance was not displayed in the presence of the ARmut. Due to a lack in change in insulin action in the AR binding mutants, it can deduced that DHT induced insulin resistance is primarily due to cytosolic AR functions.