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1.
Figure 7

Figure 7. NAC abrogated forskolin-induced HSL translocation from the cytosol to the lipid droplet.. From: Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes.

Human differentiated adipocytes were treated as described in figure 6 except with or without 5 mM NAC for 15 minutes. Cell were then fixed with paraformaldehyde and subsequently incubated with anti-total HSL antibody (green); cells were also labeled with Lipotox red for lipid droplet detection (red). Confocal pictures were taken (A) and analyzed using image J software. (B) The panel shows normalized mean values ± S.E.M. for three independent experiments with at least 50 cells per condition per experiment; * p <0.05 vs. basal, # p<0.05 vs. forskolin.

Sarah A. Krawczyk, et al. PLoS One. 2012;7(4):e34904.
2.
Figure 8

Figure 8. Resveratrol abrogated forskolin-induced HSL translocation from the cytosol to the lipid droplet.. From: Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes.

Human differentiated adipocytes were treated as described in figure 6 except with or without 100 µM resveratrol for 15 minutes. Cell were then fixed with paraformaldehyde and subsequently incubated with anti-total HSL antibody (green), cells were also labeled with Lipotox red for lipid droplet detection (red). Confocal pictures were taken (A) and analyzed using image J software. (B) The panel shows normalized mean values ± S.E.M. for three independent experiments with at least 50 cells per condition per experiment; * p <0.05 vs. basal, # p<0.05 vs. forskolin.

Sarah A. Krawczyk, et al. PLoS One. 2012;7(4):e34904.
3.
Figure 2

Figure 2. DPI, NAC and resveratrol decrease lipolysis.. From: Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes.

Human differentiated adipocytes were treated with or without (A) 10 µM DPI, (B) 5 mM NAC, or 100 µM resveratrol in the absence or presence of 5 µM forskolin for 4 hours in KRB containing 1% BSA and 0.5 mM oleate. Media were collected and assayed for glycerol using an enzyme-linked luciferase assay. Results for glycerol are normalized to the basal condition and represented as fold increase. Average basal value was 16.7 ± 1.7 nmols per million cells per hour, average forskolin-stimulated value was 85.8 ± 9.1 nmols per million cells per hour. Values ± S.E.M. for three independent experiments; * p <0.05 vs. basal control, # p<0.05 vs. forskolin control.

Sarah A. Krawczyk, et al. PLoS One. 2012;7(4):e34904.
4.
Figure 4

Figure 4. HSL phosphorylation was decreased by DPI treatment under lipolytic stimulation after 15 min, 1 hour and 4 hours.. From: Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes.

Human differentiated adipocytes were treated with or without 10 µM DPI in the absence or presence of 5 µM forskolin for (A) 4 hours (B) 1 hour or (C) 15 min in KRB with 1% BSA and 0.5 mM oleate. Individual proteins were analyzed by western blotting with specific antibodies. Signals were visualized with an enhanced chemiluminescence substrate kit (Thermo Scientific, Rockford IL) as described in materials and methods. Immunoblots shown are representative of those obtained from three separate experiments.

Sarah A. Krawczyk, et al. PLoS One. 2012;7(4):e34904.
5.
Figure 5

Figure 5. Regulation of HSL by phosphorylation.. From: Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes.

As reviewed by Lampidonis et al [46]. (A) HSL can be phosphorylated by a number of kinases. PKA is able to phosphorylate human HSL at Ser563, Ser649 and Ser650. ERK can phosphorylate HSL at Ser589. AMPK can phosphorylate HSL at Ser554, which in some tissues reduces HSL phosphorylation at Ser552 by PKA and inhibits HSL activity. Phosphorylation at HSL Ser650 and Ser554 is unaltered by DPI treatment. (B) Human differentiated adipocytes were treated with or without 10 µM DPI in the absence or presence of 5 µM forskolin for 15 minutes in KRB with 1% BSA and 0.5 mM oleate. Individual proteins were analyzed by western blotting with specific antibodies. Signals were visualized with an enhanced chemiluminescence substrate kit (Thermo Scientific, Rockford IL) as described in materials and methods. Immunoblots shown are representative of those obtained from three separate experiments.

Sarah A. Krawczyk, et al. PLoS One. 2012;7(4):e34904.
6.
Figure 6

Figure 6. DPI abrogated forskolin-induced HSL translocation from the cytosol to the lipid droplet.. From: Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes.

Human differentiated adipocytes were grown and differentiated in 35 mm coated glass bottom dishes and then treated with or without 10 µM DPI in the absence or presence of 5 µM forskolin for 15 min in KRB containing 1% BSA and 0.5 mM oleate. Cells were then fixed with paraformaldehyde and subsequently incubated with anti-total HSL antibody (green), cells were also labeled with Lipotox red for lipid droplet detection (red), as described in the methods section. Cells were imaged in these 35 mm dishes in an inverted microscope. Confocal pictures were taken (A) Images were analyzed using ImageJ software. (B) The panel shows normalized mean values ± S.E.M. for three independent experiments with at least 50 cells per condition per experiment; * p <0.05 vs. basal, # p<0.05 vs. forskolin.

Sarah A. Krawczyk, et al. PLoS One. 2012;7(4):e34904.
7.
Figure 3

Figure 3. The lipolytic pathway is unaltered by DPI treatment.. From: Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes.

Human differentiated adipocytes were treated with or without 10 µM DPI in the absence or presence of 5 µM forskolin. (A) 15 minutes in KRB with 1% BSA and 0.5 mM oleate. Cell lysates were collected and assayed for cAMP using an ELISA-based assay. (B, C) Cells were treated under the same conditions for 4 hours. Individual proteins were analyzed by western blotting with specific antibodies. Signals were visualized with an enhanced chemiluminescence substrate kit (Thermo Scientific, Rockford IL) as described in materials and methods. Results for cAMP are normalized to the basal condition and represented as fold increase. Average basal value was 3.9 ± 0.5 pmols per million cells, average forskolin-stimulated value was 68.9 ± 13.9 pmols per million cells. Values ± S.E.M. for three independent experiments. Immunoblots shown are representative of those obtained in three separate experiments.

Sarah A. Krawczyk, et al. PLoS One. 2012;7(4):e34904.
8.
Figure 1

Figure 1. Antioxidant Structures and effect on ROS generation.. From: Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes.

(A) Each antioxidant scavenges ROS as a consequence of their chemical structures. Each also has specific additional mechanisms: Diphenyliodonium (DP) inhibits NADPH oxidase, NAC increases cellular glutathione levels and resveratrol is a sirtuin activator. (B) Human differentiated adipocytes were preincubated for half an hour in DMEM with 1% BSA and 5 µM of the dye, CM-H2DCFDA. Cells were then washed and media were changed to HBSS. Compounds were added (10 µM DPI, 5 mM NAC or100 µM resveratrol) in the absence or presence of 5 µM forskolin and fluorescent readings were taken for 1.5 hours at 37°C at wavelengths 485 nm excitation, 538 nm emission. Data are expressed as the relative change of the final value minus the initial baseline reading. The panel shows normalized mean values ± S.E.M. for three independent experiments; *p <0.05 vs. basal control, # p<0.05 vs. forskolin control.

Sarah A. Krawczyk, et al. PLoS One. 2012;7(4):e34904.

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