Figure 3. GLT25D1 knockdown promotes lipid accumulation.

(A) GLT25D1/D2 and LH3 mRNA expression were examined throughout differentiation in SGBS adipocytes. There were no significant differences between the time points of GLT25D1 mRNA expression throughout differentiation (two-way ANOVA, n=5, P>0.05). However, GLT25D1 mRNA expression was significantly greater than GLT25D2, being expressed at levels approximately 10-fold greater on the days indicated (t-test, n=5, *P<0.05). LH3 was expressed at levels similar to GLT25D2. siRNA was used to knockdown GLT25D1 in SGBS adipocytes, prior to differentiation. After 72 h knockdown, cells were given 2 days of rest, then differentiation was induced using standard protocols. For RT-qPCR, the statistics were determined using a t-test relative to the scrambled control (n=4, *P<0.05). (B) GLT25D1 mRNA expression was significantly decreased during the 4–6 day period. (C) On day 4 and 6, cells were fixed and stained with Oil Red O. GLT25D1 knockdown showed greater Oil Red O staining than scrambled. Scale bar reflects 20 μm. (D) Oil Red O was extracted and quantitated by reading the absorbance at 490 nm. At day 6, there was a significant increase in the amount of Oil Red O present when GLT25D1 was knocked down. Statistics were determined using a t-test relative to the scrambled control (n=4, *P<0.05). (E) Peroxisome proliferator-activated receptor-γ (PPARγ) was significantly decreased from day 5–6. (F) Adiponectin was significantly increased at day 4, which then reversed at day 5–6. (G) Adipose triglyceride lipase (ATGL) was significantly decreased from day 4 onwards. Therefore, changes in adiponectin and ATGL were independent of PPARγ mRNA expression.