Figure 7

Transient overexpression of Ddit4 is sufficient to induce lipolysis in cultured adipocytes. (A)-(D) C3H10T1/2 cells were differentiated to adipocytes for 7 days. Cells were detached and electroporated in the presence of either empty vector (pMSCV) or Ddit4 overexpression vector (pDdit4). (A) 48 hours later cells were harvested to measure mRNA (left qPCR) and protein (right western blots), and supernatants were collected for glycerol and FFA assay. qPCR measurements (n = 3) were normalized to 36b4 expression and related to control cells and a two-tailed, unpaired student’s t-test was used to determine statistical significance (***p < 0.001). Western blot shows 2 biological replicates and a control treated with 100 nM rapamycin (Rapa). (B) and (C) Glycerol and FFA in cell supernatants were determined, normalized to protein content and related to measurements from control cells to yield the relative release caused by Ddit4 overexpression in unstimulated (B) and isoproterenol-stimulated (1 μM, 1 hour) (C) conditions. Significance was determined by a one-sample t-test (* p < 0.05). (D) Electroporated C3H10T1/2 adipocytes were incubated overnight with medium containing ¹⁴C-labeled deoxy-glucose. Incorporated radioactivity was counted in the lipid extract and related to controls (n = 3). A statistically significant difference was not detected by a one-sample t-test.