Date of Award
Open Access Thesis
Medical Doctor (MD)
Insulin resistance and obesity are two conditions associated with hypertension. Although the causes of hypertension are likely to be multifactorial, dysregulation of renal distal tubule sodium reabsorption may play a role. It has been shown that insulin increases sodium reabsorption in the distal tubule through the increased expression of epithelial sodium channels (ENaC); and obesity has been shown to cause insulin resistance in various tissues. Thiazolidinediones (TZDs) are insulin-sensitizing drugs whose use can be limited by a side effect of fluid-retention leading to edema and congestive heart failure. We set out to determine if the expression of ENaC in distal tubule cells, as modeled by the A6 (frog nephron) cells, became resistant to insulin following exposure to various concentrations of free-fatty acids (FFAs). We then set out to determine if TZDs had an insulin-sensitizing effect on distal tubule cells in both a FFA naive environment and after being exposed to FFAs. We exposed monolayers of A6 nephron cells to palmitic acid (PA) in concentrations of 0, 0.25, 0.5, 1, and 1.5 mM. After an incubation time of 20 hours we stimulated the cells with insulin. Measuring the cells with equivalent short-circuit current we found that as the PA concentration increased from 0 to 0.5 mM there was an increase in current (Isc), implying an increase in ENaC activity, followed by a marked decrease in Isc at PA concentrations of 1 mM or greater. Consistent with an increase in Isc, the calculated transepithelial resistance decreased with 0.25 and 0.5 mM PA, however, at higher concentrations of PA we observed a marked drop in resistance, implying probable apoptosis. After insulin treatment we found that in control cells (0 mM PA) the Isc increased 100% whereas with increasing concentrations of PA, there was a smaller fractional increase of 46%, 12% and 0% at 0.25, 0.5 and 1.0 mM PA, respectively. After verifying that PPARα, β, and γ were expressed in these cells we treated A6 cells with 10 μM of Rosiglitazone (PPARg agonist) or 3 mM of Clofibrate (PPARa agonist). We found there was no change in Isc following exposure to these drugs. We next pretreated the cells with both 0.5 mM PA with Clofibrate or Rosiglitazone. Again, there was no statistically-significant difference between these conditions. Overall we found that exposure to PA caused resistance to the insulin induced ENaC expression in A6 cells; at small concentrations of PA there was increased base-line ENaC expression but higher concentrations were toxic to the cells. We also found that TZDs did not have any effect on ENaC expression at base-line, with insulin, or with exposure to PA.
Sullivan, Timmy, "Palmitic Acid Increases Enac Expression and Contributes to Insulin Resistance in Renal Distal Tubule" (2009). Yale Medicine Thesis Digital Library. 463.