Tamoxifen reduces fat mass by boosting reactive oxygen species

Tam promoted apoptosis and autophagy in adipose tissue

The regulators of apoptosis and autophagy were implicated in the regulation of fat mass.^{18, 19, 20, 21, 22, 23} To examine whether Tam had effects on apoptosis and autophagy, we used eWAT at weeks 2 and 6 after Tam administration and analyzed the mediators of apoptosis and autophagy—the activated or cleaved caspase 3 (Cas3(c)) and microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate (LC3), respectively.^{24, 25} As shown in Figures 2a and b, Tam treatment increased the level of Cas3(c) by 6.8-fold (P<0.0001) and the autophagosomal marker LC3-II by 1.9-fold (P<0.05) at week 2. However, these changes were largely reversed at week 6 and showed no statistical significance (Figures 2c and d).

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Figure 2

Tam increased apoptotic and autophagic regulators in adipose tissue. (a and b) At week 2 after Tam administration, western blotting (a) was performed to analyze Cas3(c) and LC3 with densitometric analysis (b) of western blotting images using the NIH ImageJ software; n=5–7. (c and d) At week 6 after Tam administration, western blotting (c) was performed to analyze Cas3 (c) and LC3, and densitometric analysis (d) of western blotting images with the NIH ImageJ software; n=5–7. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was probed as a loading control. *P<0.05; ***P<0.0001; NS, not significant

Tam promoted the production of ROS

ROS and the resultant oxidative stress have an important role in apoptosis and autophagy.^{26, 27, 28} To examine whether ROS and oxidative stress was involved in Tam-induced effects, we analyzed heme oxygenase 1 (HO1), the sensitive indicator of cellular oxidative stress.²⁹ Tam treatment upregulated HO1 protein levels in adipose tissue by over 7-fold (P<0.0001) in mice at week 2; however, at week 6, the HO1 abundance in the treated mice was comparable to that in untreated mice, showing no statistical significance (Figures 3a and b). Interestingly, the HO1 levels in untreated mice were higher at week 6 than at week 2, supporting the notion that HO1 expression increases with age.^{30, 31} Measurement of ROS levels in adipose tissue indicated a 2.3-fold elevation in Tam-treated mice than in vehicle-treated mice at week 2 (Figure 3c), but they became statistically insignificant at week 6 (Figure 3d). Of note, the upregulation and downregulation of ROS and HO1 levels seems to coincide well with the changes in Cas3(c), LC3-II and fat mass (Figures 1, ​,2,2, ​,33).

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Figure 3

Tam promoted ROS and oxidative stress. (a) Western blotting analysis of HO1 in mouse adipose tissues at weeks 2 and 6, respectively, after Tam administration, with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) probed as a loading control. (b) Densitometric analysis of western blotting images using the NIH ImageJ software; n=6–8. (c) Measurement of ROS in adipose tissue at week 2 after Tam administration (n=3–4). (d) Measurement of ROS in adipose tissue at week 6 after Tam administration (n=3–4). **P<0.01; ***P<0.0001; NS, not significant

Antioxidant abolished Tam-induced ROS, apoptosis and autophagy

To map the interaction between ROS and other Tam-induced cellular events, we treated 3T3L1 adipocytes with Tam combined with a potent ROS-scavenger NAC.^{32, 33} As observed in adipose tissue, Tam induced significant upregulation of HO1 in 3T3L1 adipocytes, and the effect was dose dependent in the tested range of 0–128 μM (Supplementary Figure S2). Tam also promoted ROS production by 2.5-fold (P<0.01) in 3T3L1 adipocytes (Figure 4a) and significantly upregulated the apoptosis regulators Cas3(c) and autophagosomal marker LC3-II (Figures 4b and c). However, inclusion of NAC in the treatments suppressed Tan-induced elevation of ROS and HO1 and normalized the protein levels of Cas3(c) and LC3-II (Figures 4a–c).

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Figure 4

Antioxidant NAC attenuated ROS level and reversed Tam effects. (a) Measurement of ROS in 3T3L1 adipocytes. (b and c) Western blotting analysis (b) of HO, Cas3 (c) and LC3 in 3T3L1 adipocytes after 48-h treatment with Tam (128 μM) or Tam (128 μM) plus NAC (1 mM), with densitometric analysis (c) of western blotting images using the NIH ImageJ software; GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was probed as a loading control. n=3–5; *P<0.05; **P<0.01

Antioxidant reversed Tam-induced reduction in cell density and adipocyte population

Because alteration in adipocyte number affects adiposity,² we asked whether Tam treatment influenced cell density. Compared with the vehicle-treated adipocytes, Tam-treated cells showed a significant decrease in cell density (24%, P<0.05), consistent with the upregulation of apoptotic marker (Figures 5a, b and d, Figures 4b and c). Interestingly, the population of lipid-droplet-containing cells (i.e., mature adipocytes) also declined (36%, P<0.05), implying a process of ‘dedifferentiation' might be induced by Tam (Figures 5a, b and e).^{34, 35, 36} Regardless, addition of the ROS-scavenger NAC largely restored the cell density and population of mature adipocytes (Figures 5c and e).

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Figure 5

Antioxidant NAC mitigates Tam effect on cell density and mature adipocyte population. (a–c) Microscopy imaging of 3T3L1 adipocytes treated with vehicle (a), 128 μM Tam (b) and Tam (128 μM) plus NAC (1 mM) (c). The microscope was set at × 100. (d and e) Measurement of cell density and population of mature adipocytes using the NIH ImageJ software; n=6–8. *P<0.05

Mice

The FoxO1 floxed mice (f-FoxO1) and Irs1/Irs2 double floxed mice (df-Irs) were bred and housed as previously described.^{15, 16, 52} Briefly, the mice were housed in plastic cages on a 12-h light–dark photocycle, with free access to water and regular chow diet. Before Tam treatment experiments, male mice (14–16-week old) were weighed, and fat mass was measured with a Bruker Minispec LF90 NMR Analyzer (Bruker Optics, Billerica, MA, USA). Then the mice were transferred to a biosafety level 2 (BSL2) room and administered with Tam (1 mg/20 g body weight) or the vehicle (sunflower oil) by I.P. injection (once a day for 5 consecutive days). After Tam administration, the cages were changed every 2 days until week 2, when the mice were transferred into BSL1 room, and the measurement of body fat mass was resumed. Depending on the experimental design, the mice were weighed and killed to harvest tissue for snap freezing in liquid nitrogen, at week 2 or week 6 after Tam treatment. All the procedures followed the NIH guideline and were approved by the Virginia Tech Institutional Animal Care and Use Committee.

Cell culture and treatment

3T3L1 preadipocytes (ATCC CL-173, Manassas, VA, USA) were cultured in basal media (i.e., DMEM media supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin (1 × P/S)), at 37 °C in a humidified atmosphere of 5% CO₂. The media were replaced every 2 days. Differentiation of 3T3L1 cells was induced as described previously with minor modifications.⁵³ Briefly, 3T3L1 cells were grown to confluence (day 0) and maintained in fresh basal media (BMI) for 2 days (days 1–2). At the end of day 2, BMI medium was changed to differentiation medium I (DMI): DMEM supplemented with 10% FBS, P/S (1 × ), IBMX (0.5 mM), dexamethasone (1 μM), insulin (1 μg/ml), and rosiglitazone (2 μM). At the end of day 4, DMI medium was changed to differentiation medium II (DMII): DMEM supplemented with 10% FBS, P/S (1 × ), and insulin (1 μg/ml). At the end of day 6, DMII medium was changed to basal media (BMII), and the cells were maintained in BMII (replaced with fresh basal medium every 2 days) until fully differentiated (day 12). As a control, preadipocytes were maintained in BMI till day 12 and supplied with fresh medium every other day. Upon full differentiation, 3T3L1 adipocytes were treated with Tam for 48 h at the concentrations of 0, 8, 16, 32, 64 and 128 μM and the vehicle 0.1% sunflower as a treatment control.¹² When applicable, NAC was added at a concentration of 1 mM with Tam for a 48-h treatment to study the role of ROS.³² Images of the cells were captured on day 12 with a Nikon ECLIPSE TS100 microscope (Melville, NY, USA), and the cell counting and population analysis was conducted with the NIH ImageJ software (Bethesda, MD, USA).

ROS measurement

ROS in adipocytes and adipose tissue was measured as previously described,^{54, 55} with a cell-permeable dye 5,6-carboxy-2′,7′-dichlorofluorescein diacetate (Carboxy-DCFDA, Molecular Probes, Grand Island, NY, USA). Snap-frozen adipose tissues were weighed and transferred into buffered medium (5 mmol/l HEPES in PBS) for quick thawing to improve the probe diffusion. After rapid thawing, the medium was discarded. Samples were exposed to 8 μM Carboxy-DCFDA in fresh medium and were incubated at 37 °C for 45 min under agitation. Medium was then removed, and samples were further incubated in a lysis buffer (0.1% SDS, Tris-HCl, pH 7.4) for 15 min at 4 °C. After homogenization, samples were centrifuged at 16 000 × g for 20 min at 4 °C. Supernatants were collected and subjected to fluorescence analysis at 530 nm under excitation at 485 nm using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA).

To measure ROS in 3T3L1 adipocytes, 1–5 × 10⁶ cells were harvested with typsin and washed three times with cold PBS, followed by incubation with 8 μM Carboxy-DCFDA in fresh medium (5 mmol/l HEPES in PBS) and were incubated at 37 °C for 45 min under agitation. Medium was then removed, and samples were further incubated in PLC lysis buffer:^{15, 52} (30 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na₃VO₄) supplemented with protease inhibitor cocktail (Roche, Branchburg, NJ, USA) and 1 mM PMSF for 15 min at 4 °C. After homogenization, samples were centrifuged at 16 000 × g for 20 min at 4 °C. Supernatants were collected and subjected to fluorescence analysis at 530 nm under excitation at 485 nm, and the total protein was determined with DC protein assay (Bio-Rad, Hercules, CA, USA) on a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc.). The ROS levels were normalized to the total protein for each cell dish.

Western blotting

To prepare tissue lysates, snap-frozen adipose tissues were weighed and homogenized with a Bullet Blender (Next Advance, Averill Park, NY, USA) in PLC lysis buffer supplemented with protease inhibitor cocktail (Roche), 1 mM PMSF, 10 μM TSA (Trichostatin A, Selleckchem, Houston, TX, USA) and 5 mM Nicotinamide (Alfa Aesar, Ward Hill, MA, USA).^{15, 52} For cell lysates, the 3T3L1 adipocytes were washed with ice-cold PBS and homogenized with a Bullet Blender. Total protein concentrations of the lysates were determined using the DC protein assay (Bio-Rad). Western blotting and image analysis were conducted as described previously.¹⁵ Antibody catalog numbers and vendors are as follows: cleaved caspase-3 Rabbit mAb (9664) and LC3B antibody (no. 2775) from Cell Signaling Technology (Beverly, MA, USA); PPAR-gamma antibody (MA5-14889) and GAPDH antibody (MA5-15738) from Pierce (Rockford, IL, USA) or Thermo Fisher Scientific (Waltham, MA, USA); and HO1 antibody (3391-100) from Biovision (Milpitas, CA, USA).

We thank Dr. Ronald Depinho (University of Texas MD Anderson Cancer Center) and Dr. Morris F White (Children's Hospital Boston, Harvard Medical School) for providing the f-FoxO1 and df-Irs mice, respectively. Funding for this work was provided, in part, by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, US Department of Agriculture (to ZC), and by grant 1R01AT007077 from the National Center for Complementary and Alternative Medicine in the National Institutes of Health (to DL). Publication of this article was supported by Virginia Tech's Open Access Subvention Fund.