Mol Cell Biochem DOI 10.1007/s11010-014-2047-x
Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis Sabrina Krautbauer • Kristina Eisinger Yvonne Hader • Markus Neumeier • Christa Buechler
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Received: 7 November 2013 / Accepted: 2 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis.
S. Krautbauer K. Eisinger Y. Hader M. Neumeier C. Buechler (&) Department of Internal Medicine I, University Hospital of Regensburg, 93042 Regensburg, Germany e-mail: [email protected]
Keywords Adipocyte Glutathione Antioxidants Triglycerides
Introduction Adipose tissue has a central role in whole body energy homeostasis, and lipodystrophy as well as obesity are associated with impaired glucose and lipid metabolism [1, 2]. Adipocytes originate from pluripotent mesenchymal stem cells committed to the adipocyte lineage, which become preadipocytes and subsequently differentiate into adipocytes when appropriately stimulated [3, 4]. Adipogenesis happens throughout the life to replace dying adipocytes with a half-life of approximately 8 years and is enhanced in obesity where adipocyte hypertrophy as well as hyperplasia contributes to adipose tissue growth [5–7]. Differentiation of preadipocytes to adipocytes is a complex and tightly regulated process and much of the current knowledge has been obtained using the cell lines 3T3-L1 and 3T3-F442A [4]. During adipogenesis, these cells acquire the ability to store enormous amounts of triglycerides in lipid droplets [4]. Differentiation of adipocytes depends on metabolites synthesized in mitochondria, and biogenesis of these organelles and expression of mitochondrial proteins are increased [8]. Mitochondria metabolize oxygen, and production of reactive oxygen species (ROS) is enhanced [9]. Induction of the antioxidative enzymes catalase, manganese superoxide dismutase (MnSOD), and Cu/Zn SOD [8, 10] counterbalances cellular ROS levels in maturating adipocytes [8, 11]. The increased generation of superoxide in adipocytes is a characteristic trait of insulin resistance which is reversed upon overexpression of MnSOD and MnSOD mimetics [12]. MnSOD deficiency in mice is lethal [13], and heterozygous mice are
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glucose intolerant, even when kept on a standard chow [12]. MnSOD-deficient bone marrow stromal cells show enhanced lipid storage when kept in basal, non-adipogenic cell culture media. Lipid accumulation in adipogenic differentiation medium is, however, similar to wild-type cells [14]. Reduced adipose tissue mass has nevertheless been described in a second line of MnSOD-deficient mice which survive for about 3 weeks [15]. L-Buthionine-(S,R)-sulfoximine (BSO) which depletes cellular glutathione enhances mitotic clonal expansion of 3T3-L1 preadipocytes and subsequently adipogenesis [16]. However, when BSO is added to confluent 3T3-L1 cells, adipogenesis is impaired [17]. Similarly, supplementation of medium with antimycin, a pharmacological inhibitor of complex III which increases ROS, reduces the adipocyte maturation of confluent 3T3-F442A cells. This effect is blocked by the MnSOD mimetic MnTBAP [18]. These findings suggest that ROS have a dual function in adipogenesis. They promote the clonal expansion of preadipocytes, while exposure of maturating adipocytes to the increased ROS seems to impair adipogenesis. The aim of the current work was to study the effect of MnSOD knockdown using small-interfering RNA (siRNA) and BSO on adipogenesis of pre-differentiated 3T3-L1 cells.
Materials and methods Culture media and reagents MnSOD antibody was from Thermo Fisher Scientific (Schwerte, Germany). Antibodies to the phosphorylated form of Akt (Ser473) and the corresponding antibody to the total form, antibodies to cytochrome C oxidase-4 (Cox-4), fatty acid synthase (FAS), fatty acid-binding protein 4 (FABP4), GAPDH, hormone-sensitive lipase (HSL), peroxisome proliferator-activated receptor c (PPARc), and poly ADP ribose polymerase (PARP) were from New England Biolabs GmbH (Frankfurt, Germany). Heme oxygenase 1 antibody was from Novus Biologicals (Cambridge, UK). Antibody to detect murine chemerin by immunoblot was from R&D Systems (Wiesbaden-Nordenstadt, Germany). Kit to measure lactate dehydrogenase (LDH) was purchased from Roche (Mannheim, Germany). OxiSelectTM Oxygen Radical Antioxidant Capacity (ORAC) Activity Assay was from Cell Biolabs Inc. (San Diego, CA, USA). Triglyceride concentrations were measured using GPO-PAP micro-test (purchased from Roche, Mannheim, Germany). L-Buthionine-(S,R)-sulfoximine (BSO) was from ENZO Life Sciences (Lo¨rrach, Germany). Staurosporine and hydrogen peroxide (H2O2) were purchased from Merck (Darmstadt, Germany), and Oil Red O
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was from Sigma Bioscience (Deisenhofen, Germany). CellTiter-BlueÒ Cell Viability Assay was from Promega (Mannheim, Germany), and reagent was diluted to 10-fold in cell culture medium. Nitro Blue Tetrazolium Chloride was from Life Technologies (Darmstadt, Germany). Cells were incubated for 120 min in PBS containing 0.2 % NBT. Formazan was dissolved in 50 % acetic acid, and the absorbance was determined at 570 nm. Knock-down of MnSOD MnSOD siRNAs (s74129: 5´ GCU CUA AUC AGG ACC CAU Utt 3´ and s74130 5´ AGG GAG AUG UUA CAA CUC Att 3´) and Silencer Negative Control siRNA were from Applied Biosystems (Darmstadt, Germany). Preadipocytes were transfected using the siRNA Xtreme transfection reagent from Roche (Mannheim, Germany) or using Endo-Porter (Gene Toola LLC, Philomath, Oregon, USA) and subsequently differentiated to adipocytes. Adipocyte cell culture 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured at 37 °C and 5 % CO2 in DMEM (Biochrom, Berlin, Germany) supplemented with 10 % newborn calf serum (Sigma Bioscience, Deisenhofen, Germany) and 1 % penicillin/streptomycin (PAN, Aidenbach, Germany). For adipogenesis, 3T3-L1 preadipocytes were grown to 80 % confluence and differentiated into adipocytes in DMEM/ F12/glutamate-medium (Lonza, Basel, Switzerland) supplemented with 20 lM 3-isobutyl-methyl-xanthine (Serva, Heidelberg, Germany), 10-6 M corticosterone, 10-7 M insulin, 200 lM ascorbate, 2 lg/ml transferrin, 1 lM biotin, 17 lM pantothenate (all procured from Sigma Bioscience, Deisenhofen, Germany), 5 % fetal bovine serum (Biochrom, Berlin, Germany), 1 % penicillin/streptomycin (PAN, Aidenbach, Germany), and 300 lg/l Pedersen-fetuin (MP Biomedicals, Illkirch, France) for 7 days. Differentiation medium was replaced at days 3 and 6 of adipogenesis. Thereafter, the cells were exposed to DMEM/F12/glutamate-medium with 10-6 M insulin for 24 h. This was followed by incubation with DMEM/F12/glutamate-medium for 24 h until the cells reached the fully differentiated phenotype which was controlled by light microscopy for the existence of a more rounded cell shape and the typical appearance of extensive lipid droplet accumulation. SDS-PAGE and immunoblotting Adipocytes were solubilized in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl (both from Merck, Darmstadt, Germany), 1 % v/v
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Statistical analysis Data are represented as mean ± standard deviation (SPSS 19.0). Statistical differences were analyzed by Student´s t test (MS Excel), and a value of p \ 0.05 was regarded as significant.
Results Fig. 1 MnSOD knock-down. a Knock-down of MnSOD using two different siRNAs was performed in preadipocytes. After siRNA transfection, cells were immediately differentiated, and MnSOD was analyzed by immunoblot in the 9-d maturated cells. b Quantification of MnSOD in the cells described in a, data of 5 different experiments are shown. * Indicates a p value \0.05
Nonidet P-40, 0.5 % v/v sodium desoxycholate (both from Sigma Bioscience, Deisenhofen, Germany), and 0.1 % v/v SDS (Serva, Heidelberg, Germany)). 10–20 lg protein was separated by SDS–polyacrylamide gel electrophoresis and was transferred to PVDF membranes (Bio-Rad, Germany). Incubations with antibodies were performed in 1.5 % BSA (Roche, Mannheim, Germany) in TBS or PBS, 0.1 % Tween (all purchased from Sigma Bioscience, Deisenhofen, Germany) overnight. Detection of the immune complexes was carried out with the ECL Western blot detection system (Amersham Pharmacia, Deisenhofen, Germany).
MnSOD in adipocytes differentiated from preadipocytes with MnSOD knock-down Preadipocytes were transfected with two different MnSOD siRNAs or non-silencing control siRNA in medium with the appropriate adipogenic cocktail (day 0 of differentiation), and 9 days later, MnSOD was analyzed by immunoblot. Cells transfected with both MnSOD siRNAs showed significantly and similarly reduced MnSOD protein (Fig. 1a, b). This showed that both siRNAs efficiently knocked-down MnSOD and demonstrated that MnSOD was still reduced after 9 days of differentiation. Knock-down of MnSOD does not reduce cell viability
Adiponectin ELISA was performed as recommended by the distributor (R&D Systems, Wiesbaden-Nordenstadt, Germany). Supernatant was diluted 1 to 250-fold for determination of adiponectin.
Light microscopy of adipocytes differentiated from preadipocytes transfected with MnSOD siRNA revealed that the number of differentiated adipocytes was reduced (Fig. 2a). To find out whether apoptosis is increased, poly ADP ribose polymerase (PARP) was analyzed by immunoblot. Cleavage of PARP is not enhanced in cells with low MnSOD (Fig. 2b). Heme oxygenase 1 (HO-1) which is upregulated upon oxidative stress [19] is not induced. LDH measured in the supernatants is even significantly reduced in MnSOD siRNA-treated cells (Fig. 2c).
Fig. 2 Effect of MnSOD knock-down on cell viability of 3T3-L1 cells. a Light microscopy of mature 3T3-L1 cells differentiated from preadipocytes treated with control siRNA and MnSOD siRNA. b MnSOD, PARP 116 (full-length) and PARP 89 (cleaved form), HO1 and GAPDH in cells differentiated from preadipocytes treated with
control siRNA and MnSOD siRNA. ST and H2O2 indicate lysates of staurosporine or H2O2-treated 3T3-L1 cells as positive control analyzed on the identical gel in a nonadjacent lane. c LDH in the supernatants of these cells; data of 4 experiments are shown. * Indicates a p value \0.05
Elisa
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Knock-down of MnSOD impairs differentiation of preadipocytes MnSOD was knocked-down by siRNA in 3T3-L1 preadipocytes which were subsequently differentiated to mature adipocytes. Oil Red O staining revealed reduced accumulation of lipids in these cells (Fig. 3a). In accordance with these findings, levels of triglycerides in the cell lysates and the supernatants were significantly lower (Fig. 3b, c). FAS and Cox-4 were expressed in preadipocytes and were modestly induced at days 2 and 3, respectively, after hormonal induction of differentiation (Fig. 3d), and were similarly expressed in MnSOD and control siRNA-treated cells (Fig. 3e). PPARc was induced in 2-d maturated cells and was further upregulated in 7-d maturated cells [20] (Fig. 3d), and was also not affected (Fig. 3e, f). Chemerin [21], fatty acid binding protein 4 (FABP4), and hormone sensitive lipase (HSL) were upregulated between day 3 and day 7 of differentiation (Fig. 3g and data not shown) in accordance with the published data [21] and were strongly reduced in adipocytes with low MnSOD (Fig. 3h, i, j). Adiponectin was markedly elevated between day 6 and day 9 of differentiation [20] and was also diminished in the supernatants of cells with MnSOD knock-down (Fig. 3k). Of note, antioxidative capacity was similar in control and MnSOD siRNA-treated cells (Fig. 3l). Superoxide was measured at days 2, 3, 7, and 9 after hormonal induction of adipogenesis. Levels were not modified during adipogenesis of preadipocytes transfected with control or MnSOD siRNA. Superoxides were not induced in MnSOD siRNAtransfected cells (Fig. 3m). Knock-down of MnSOD does not affect insulin signaling Insulin strongly increased phosphorylation of Akt in adipocytes differentiated from preadipocytes treated with MnSOD and control siRNA (Fig. 4a, b). BSO causes adipocyte de-differentiation BSO reduces cellular glutathione and thereby increases ROS [17]. To find out which concentrations of BSO significantly increase cellular ROS, mature 3T3-L1 adipocytes were incubated with 5, 10, and 20 lM BSO for 24 h, and antioxidative capacity was significantly reduced by 10 and 20 lM BSO (Fig. 5a). Confluent 3T3-L1 cells were induced to differentiate, and 3 days later, 20 and 40 lM BSO were added, and the treatment was continued until day 9 of differentiation. BSO at both concentrations impaired triglyceride accumulation (Fig. 5b) and chemerin, FABP4, HSL, and adiponectin were lower in cells exposed to BSO (40 lM) (Fig. 5 c, f
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and data not shown). FAS, PPARc, and Cox-4 were also markedly reduced (Fig. 5d, g). Of note, MnSOD was not affected by BSO treatment (Fig. 5d, h). BSO and MnSOD siRNA modestly reduce viability of preadipocytes Preadipocytes were transfected with MnSOD siRNA or control siRNA and 24 h later CellTiter-BlueÒ Cell Viability reagent was added for 24 h. Percentage of viable cells was modestly but significantly reduced in MnSOD siRNA -treated preadipocytes while LDH was increased (Fig. 6a, b). BSO (10 and 20 lM) did not affect number of viable cells, and LDH was only very modestly increased (Fig. 6c, d).
Discussion Current work shows that knock-down of MnSOD impairs adipogenesis of 3T3-L1 cells. Adipocytes differentiated from preadipocytes, where MnSOD has been knockeddown, have impaired triglyceride storage and low expression of proteins which are induced in late adipogenesis [20, 21]. Proteins which are already expressed in preadipocytes and only modestly induced in mature cells [20] are similarly abundant compared with cells transfected with control siRNA. Therefore, MnSOD which is upregulated during adipogenesis [8] is essential for lipid storage and expression of proteins and adipokines characteristic for mature adipocytes. PPARc regulates the abundance of adipocyte specific proteins [22] but is similarly expressed in control and MnSOD siRNA-transfected cells. This suggests that the levels of endogenous PPARc agonists [22] may be reduced in adipocytes with low MnSOD. MnSOD is modestly increased during adipogenesis [8, 23] and is markedly reduced in MnSOD siRNA transfected cells demonstrating that MnSOD siRNA is still functional 9 days after transfection of the preadipocytes. Cell viability and insulin-mediated phosphorylation of Akt were not impaired in adipocytes with low MnSOD excluding that increased cell death or insulin resistance contribute to the phenotype of mature adipocytes with reduced MnSOD. In preadipocytes, suppression of MnSOD reduces number of viable cells by about 5 %, and LDH is modestly increased. While this effect on viability may contribute to the reduced number of mature adipocytes, it is for sure not the principle explanation for the strongly impaired differentiation observed. Surprisingly, antioxidative capacity of adipocytes differentiated from preadipocytes treated with MnSOD siRNA is similar to control-transfected adipocytes, and superoxide measured during adipogenesis is not elevated in
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Fig. 3 Effect of MnSOD knock-down on adipogenesis of 3T3-L1 cells. a Light microscopy of mature 3T3-L1 cells differentiated from preadipocytes treated with control siRNA and MnSOD siRNA. Lipid accumulation was determined by Oil Red O staining. b Triglycerides (ng/lg cellular protein) in the cell lysates of these cells: data of 6 experiments have been calculated. c Triglycerides (ng/lg cellular protein) in the supernatants (SN) of these cells: data of 6 experiments have been calculated. d FAS, PPARc, and Cox-4 in preadipocytes and 2, 3, 7, and 9 d after hormonal induction of adipogenesis. e Knockdown of MnSOD was performed in preadipocytes and MnSOD, FAS, PPARc, Cox-4, and GAPDH were analyzed in 9-d-differentiated cells. f Quantification of PPARc partly shown in e. Data of 5 experiments are shown. g HSL and FABP4 in preadipocytes and 2, 3, 7, and 9 d after hormonal induction of adipogenesis. h Knock-down of
MnSOD was performed in preadipocytes, and MnSOD, chemerin, HSL, FABP4, and GAPDH were analyzed in 9-d-differentiated cells. i Quantification of HSL partly shown in h. Data of 5 experiments have been calculated. j Quantification of FABP4 partly shown in h. Data of 4 experiments are shown. k Adiponectin in the supernatants of these cells: data of 5 experiments have been calculated. l Antioxidative capacity of mature 3T3-L1 cells differentiated from preadipocytes treated with control siRNA and MnSOD siRNA. Data of 12 experiments are shown. m Superoxides were measured by NBT during differentiation of 3T3-L1 cells from preadipocytes treated with control siRNA and MnSOD siRNA. Data of one out of three experiments with comparable results are shown. * Indicates a p value \0.05
MnSOD siRNA-treated cells. This is in agreement with several studies which have shown that ROS are not altered in cells where MnSOD is overexpressed or blocked until these cells are exposed to agents increasing oxidative stress [24–28]. It is therefore possible that other ROS-detoxifying
enzymes are induced to compensate for low MnSOD during differentiation. Importantly, knock-down of MnSOD in mature adipocytes is associated with modestly reduced antioxidative capacity [23]. MnSOD is upregulated during differentiation
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[8, 23] surmising that this enzyme may be more important for ROS homeostasis in mature cells. The issues concerning whether related superoxide detoxifying enzymes are
Fig. 4 Insulin-mediated phosphorylation of Akt. a Akt, pAkt, and MnSOD in control siRNA- and MnSOD siRNA-treated preadipocytes which were differentiated to mature cells and subsequently stimulated with 50 and 200 nM insulin for 20 min. b Quantification of the results partly shown in a; data of 3 experiments have been calculated. * Indicates a p value \0.05 for comparison with cells not treated with insulin
Fig. 5 Effect of L-Buthionine-(S,R)-sulfoximine (BSO) in 3T3-L1 cells. a Antioxidative capacity of mature 3T3-L1 cells which were incubated with increasing concentrations of BSO for 24 h. b Light microscopy of mature 3T3-L1 cells differentiated from preadipocytes treated with BSO (20, 40 lM) since day 3 of differentiation. c Chemerin, HSL, FABP4, PARP, and GAPDH in the cells described in b. d MnSOD, FAS, PPARc, Cox-4, and GAPDH in the cells
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not at all or insufficiently induced in the mature cells need further studies. Anyhow, the findings described above exclude that the elevated ROS impair adipogenesis and triglyceride storage in cells with low MnSOD. Therefore, the mechanisms linking adipogenesis and MnSOD have to be identified in future studies. Previous data show that superoxides are transiently induced during differentiation or are higher in mature adipocytes compared to preadipocytes [11, 29, 30]. In the cell model used herein superoxides are not upregulated during adipogenesis. Therefore, either production of superoxides is not induced during adipogenesis or respective antioxidant enzymes are increased early enough. Discrepant findings may be at least in part explained by the many different protocols used for adipogenic differentiation of 3T3-L1 cells. Current data show that adipogenesis is not in general associated with higher superoxide levels. Pharmaceutical depletion of glutathione by BSO lowers antioxidative capacity of mature cells indicating the increased formation of ROS in accordance with the
described in b. e Quantification of HSL partly shown in c. Data of 3 experiments are shown. f Adiponectin in the supernatants of these cells: data of 4 experiments have been calculated. g Quantification of FABP4 partly shown in c. Data of 3 experiments are shown. h Quantification of MnSOD partly shown in d. Data of 3 experiments are shown. * Indicates a p value \0.05
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Fig. 6 Effect of MnSOD knock-down and L-Buthionine-(S,R)sulfoximine (BSO) in 3T3-L1 fibroblasts. a Viable cells in preadipocytes treated with control and MnSOD siRNA: data of 3 experiments have been calculated. b LDH in the supernatants of
these cells. c Viable cells in preadipocytes treated with BSO: data of one out of three experiments are shown. d LDH in the supernatants of these cells. * Indicates a p value \0.05
literature [17]. During differentiation, BSO not only blocks the upregulation of late adipogenesis markers but even lowers expression of proteins which are induced early during differentiation. Therefore, in contrast to cells with low MnSOD, BSO downregulates FAS, Cox-4, and PPARc, causing a de-differentiation of the cells. BSO at a concentration of 20 lM lowers AOC of mature adipocytes by about 4 % and nearly completely blocks adipogenesis and triglyceride storage when added during differentiation, surmising that adipogenesis is highly sensitive to oxidative stress. MnSOD is about twofold upregulated during adipogenesis [8] and subsequently should be reduced in BSO-treated cells. Nevertheless, MnSOD is similarly abundant in control- and BSO-exposed 3T3-L1 cells. MnSOD is induced by ROS [31, 32], and this mechanism may also account for relatively high abundance of this enzyme. Antiadipogenic effects of the increased ROS on preadipocytes have already been shown by other groups [17, 18] in accordance with current findings. In contrast, Nacetyl-L-cysteine (NAC) and EUK-8, a catalytic mimetic of superoxide dismutase (SOD) and catalase even inhibit adipogenesis of mesenchymal stem cells [33, 34]. ROS are essential for mitotic clonal expansion of preadipocytes and induction of C/EBPb which is expressed early in differentiation [35, 36]. BSO, however, does not promote mitosis of preadipocytes in the cell culture model used herein and in a recent study [37]. Those authors show that BSO lowers total but not nuclear glutathione and only the latter has a role in the cell–cycle regulation. In summary, current work shows that glutathione depletion and low MnSOD block adipogenesis of 3T3-L1 fibroblasts. The essential function of MnSOD for adipocyte triglyceride accumulation is, however, independent of its effect on cellular ROS homeostasis.
Conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis Sabrina Krautbauer • Kristina Eisinger Yvonne Hader • Markus Neumeier • Christa Buechler
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Received: 7 November 2013 / Accepted: 2 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis.
S. Krautbauer K. Eisinger Y. Hader M. Neumeier C. Buechler (&) Department of Internal Medicine I, University Hospital of Regensburg, 93042 Regensburg, Germany e-mail: [email protected]
Keywords Adipocyte Glutathione Antioxidants Triglycerides
Introduction Adipose tissue has a central role in whole body energy homeostasis, and lipodystrophy as well as obesity are associated with impaired glucose and lipid metabolism [1, 2]. Adipocytes originate from pluripotent mesenchymal stem cells committed to the adipocyte lineage, which become preadipocytes and subsequently differentiate into adipocytes when appropriately stimulated [3, 4]. Adipogenesis happens throughout the life to replace dying adipocytes with a half-life of approximately 8 years and is enhanced in obesity where adipocyte hypertrophy as well as hyperplasia contributes to adipose tissue growth [5–7]. Differentiation of preadipocytes to adipocytes is a complex and tightly regulated process and much of the current knowledge has been obtained using the cell lines 3T3-L1 and 3T3-F442A [4]. During adipogenesis, these cells acquire the ability to store enormous amounts of triglycerides in lipid droplets [4]. Differentiation of adipocytes depends on metabolites synthesized in mitochondria, and biogenesis of these organelles and expression of mitochondrial proteins are increased [8]. Mitochondria metabolize oxygen, and production of reactive oxygen species (ROS) is enhanced [9]. Induction of the antioxidative enzymes catalase, manganese superoxide dismutase (MnSOD), and Cu/Zn SOD [8, 10] counterbalances cellular ROS levels in maturating adipocytes [8, 11]. The increased generation of superoxide in adipocytes is a characteristic trait of insulin resistance which is reversed upon overexpression of MnSOD and MnSOD mimetics [12]. MnSOD deficiency in mice is lethal [13], and heterozygous mice are
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glucose intolerant, even when kept on a standard chow [12]. MnSOD-deficient bone marrow stromal cells show enhanced lipid storage when kept in basal, non-adipogenic cell culture media. Lipid accumulation in adipogenic differentiation medium is, however, similar to wild-type cells [14]. Reduced adipose tissue mass has nevertheless been described in a second line of MnSOD-deficient mice which survive for about 3 weeks [15]. L-Buthionine-(S,R)-sulfoximine (BSO) which depletes cellular glutathione enhances mitotic clonal expansion of 3T3-L1 preadipocytes and subsequently adipogenesis [16]. However, when BSO is added to confluent 3T3-L1 cells, adipogenesis is impaired [17]. Similarly, supplementation of medium with antimycin, a pharmacological inhibitor of complex III which increases ROS, reduces the adipocyte maturation of confluent 3T3-F442A cells. This effect is blocked by the MnSOD mimetic MnTBAP [18]. These findings suggest that ROS have a dual function in adipogenesis. They promote the clonal expansion of preadipocytes, while exposure of maturating adipocytes to the increased ROS seems to impair adipogenesis. The aim of the current work was to study the effect of MnSOD knockdown using small-interfering RNA (siRNA) and BSO on adipogenesis of pre-differentiated 3T3-L1 cells.
Materials and methods Culture media and reagents MnSOD antibody was from Thermo Fisher Scientific (Schwerte, Germany). Antibodies to the phosphorylated form of Akt (Ser473) and the corresponding antibody to the total form, antibodies to cytochrome C oxidase-4 (Cox-4), fatty acid synthase (FAS), fatty acid-binding protein 4 (FABP4), GAPDH, hormone-sensitive lipase (HSL), peroxisome proliferator-activated receptor c (PPARc), and poly ADP ribose polymerase (PARP) were from New England Biolabs GmbH (Frankfurt, Germany). Heme oxygenase 1 antibody was from Novus Biologicals (Cambridge, UK). Antibody to detect murine chemerin by immunoblot was from R&D Systems (Wiesbaden-Nordenstadt, Germany). Kit to measure lactate dehydrogenase (LDH) was purchased from Roche (Mannheim, Germany). OxiSelectTM Oxygen Radical Antioxidant Capacity (ORAC) Activity Assay was from Cell Biolabs Inc. (San Diego, CA, USA). Triglyceride concentrations were measured using GPO-PAP micro-test (purchased from Roche, Mannheim, Germany). L-Buthionine-(S,R)-sulfoximine (BSO) was from ENZO Life Sciences (Lo¨rrach, Germany). Staurosporine and hydrogen peroxide (H2O2) were purchased from Merck (Darmstadt, Germany), and Oil Red O
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was from Sigma Bioscience (Deisenhofen, Germany). CellTiter-BlueÒ Cell Viability Assay was from Promega (Mannheim, Germany), and reagent was diluted to 10-fold in cell culture medium. Nitro Blue Tetrazolium Chloride was from Life Technologies (Darmstadt, Germany). Cells were incubated for 120 min in PBS containing 0.2 % NBT. Formazan was dissolved in 50 % acetic acid, and the absorbance was determined at 570 nm. Knock-down of MnSOD MnSOD siRNAs (s74129: 5´ GCU CUA AUC AGG ACC CAU Utt 3´ and s74130 5´ AGG GAG AUG UUA CAA CUC Att 3´) and Silencer Negative Control siRNA were from Applied Biosystems (Darmstadt, Germany). Preadipocytes were transfected using the siRNA Xtreme transfection reagent from Roche (Mannheim, Germany) or using Endo-Porter (Gene Toola LLC, Philomath, Oregon, USA) and subsequently differentiated to adipocytes. Adipocyte cell culture 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured at 37 °C and 5 % CO2 in DMEM (Biochrom, Berlin, Germany) supplemented with 10 % newborn calf serum (Sigma Bioscience, Deisenhofen, Germany) and 1 % penicillin/streptomycin (PAN, Aidenbach, Germany). For adipogenesis, 3T3-L1 preadipocytes were grown to 80 % confluence and differentiated into adipocytes in DMEM/ F12/glutamate-medium (Lonza, Basel, Switzerland) supplemented with 20 lM 3-isobutyl-methyl-xanthine (Serva, Heidelberg, Germany), 10-6 M corticosterone, 10-7 M insulin, 200 lM ascorbate, 2 lg/ml transferrin, 1 lM biotin, 17 lM pantothenate (all procured from Sigma Bioscience, Deisenhofen, Germany), 5 % fetal bovine serum (Biochrom, Berlin, Germany), 1 % penicillin/streptomycin (PAN, Aidenbach, Germany), and 300 lg/l Pedersen-fetuin (MP Biomedicals, Illkirch, France) for 7 days. Differentiation medium was replaced at days 3 and 6 of adipogenesis. Thereafter, the cells were exposed to DMEM/F12/glutamate-medium with 10-6 M insulin for 24 h. This was followed by incubation with DMEM/F12/glutamate-medium for 24 h until the cells reached the fully differentiated phenotype which was controlled by light microscopy for the existence of a more rounded cell shape and the typical appearance of extensive lipid droplet accumulation. SDS-PAGE and immunoblotting Adipocytes were solubilized in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris HCl, pH 7.5, 150 mM NaCl (both from Merck, Darmstadt, Germany), 1 % v/v
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Statistical analysis Data are represented as mean ± standard deviation (SPSS 19.0). Statistical differences were analyzed by Student´s t test (MS Excel), and a value of p \ 0.05 was regarded as significant.
Results Fig. 1 MnSOD knock-down. a Knock-down of MnSOD using two different siRNAs was performed in preadipocytes. After siRNA transfection, cells were immediately differentiated, and MnSOD was analyzed by immunoblot in the 9-d maturated cells. b Quantification of MnSOD in the cells described in a, data of 5 different experiments are shown. * Indicates a p value \0.05
Nonidet P-40, 0.5 % v/v sodium desoxycholate (both from Sigma Bioscience, Deisenhofen, Germany), and 0.1 % v/v SDS (Serva, Heidelberg, Germany)). 10–20 lg protein was separated by SDS–polyacrylamide gel electrophoresis and was transferred to PVDF membranes (Bio-Rad, Germany). Incubations with antibodies were performed in 1.5 % BSA (Roche, Mannheim, Germany) in TBS or PBS, 0.1 % Tween (all purchased from Sigma Bioscience, Deisenhofen, Germany) overnight. Detection of the immune complexes was carried out with the ECL Western blot detection system (Amersham Pharmacia, Deisenhofen, Germany).
MnSOD in adipocytes differentiated from preadipocytes with MnSOD knock-down Preadipocytes were transfected with two different MnSOD siRNAs or non-silencing control siRNA in medium with the appropriate adipogenic cocktail (day 0 of differentiation), and 9 days later, MnSOD was analyzed by immunoblot. Cells transfected with both MnSOD siRNAs showed significantly and similarly reduced MnSOD protein (Fig. 1a, b). This showed that both siRNAs efficiently knocked-down MnSOD and demonstrated that MnSOD was still reduced after 9 days of differentiation. Knock-down of MnSOD does not reduce cell viability
Adiponectin ELISA was performed as recommended by the distributor (R&D Systems, Wiesbaden-Nordenstadt, Germany). Supernatant was diluted 1 to 250-fold for determination of adiponectin.
Light microscopy of adipocytes differentiated from preadipocytes transfected with MnSOD siRNA revealed that the number of differentiated adipocytes was reduced (Fig. 2a). To find out whether apoptosis is increased, poly ADP ribose polymerase (PARP) was analyzed by immunoblot. Cleavage of PARP is not enhanced in cells with low MnSOD (Fig. 2b). Heme oxygenase 1 (HO-1) which is upregulated upon oxidative stress [19] is not induced. LDH measured in the supernatants is even significantly reduced in MnSOD siRNA-treated cells (Fig. 2c).
Fig. 2 Effect of MnSOD knock-down on cell viability of 3T3-L1 cells. a Light microscopy of mature 3T3-L1 cells differentiated from preadipocytes treated with control siRNA and MnSOD siRNA. b MnSOD, PARP 116 (full-length) and PARP 89 (cleaved form), HO1 and GAPDH in cells differentiated from preadipocytes treated with
control siRNA and MnSOD siRNA. ST and H2O2 indicate lysates of staurosporine or H2O2-treated 3T3-L1 cells as positive control analyzed on the identical gel in a nonadjacent lane. c LDH in the supernatants of these cells; data of 4 experiments are shown. * Indicates a p value \0.05
Elisa
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Knock-down of MnSOD impairs differentiation of preadipocytes MnSOD was knocked-down by siRNA in 3T3-L1 preadipocytes which were subsequently differentiated to mature adipocytes. Oil Red O staining revealed reduced accumulation of lipids in these cells (Fig. 3a). In accordance with these findings, levels of triglycerides in the cell lysates and the supernatants were significantly lower (Fig. 3b, c). FAS and Cox-4 were expressed in preadipocytes and were modestly induced at days 2 and 3, respectively, after hormonal induction of differentiation (Fig. 3d), and were similarly expressed in MnSOD and control siRNA-treated cells (Fig. 3e). PPARc was induced in 2-d maturated cells and was further upregulated in 7-d maturated cells [20] (Fig. 3d), and was also not affected (Fig. 3e, f). Chemerin [21], fatty acid binding protein 4 (FABP4), and hormone sensitive lipase (HSL) were upregulated between day 3 and day 7 of differentiation (Fig. 3g and data not shown) in accordance with the published data [21] and were strongly reduced in adipocytes with low MnSOD (Fig. 3h, i, j). Adiponectin was markedly elevated between day 6 and day 9 of differentiation [20] and was also diminished in the supernatants of cells with MnSOD knock-down (Fig. 3k). Of note, antioxidative capacity was similar in control and MnSOD siRNA-treated cells (Fig. 3l). Superoxide was measured at days 2, 3, 7, and 9 after hormonal induction of adipogenesis. Levels were not modified during adipogenesis of preadipocytes transfected with control or MnSOD siRNA. Superoxides were not induced in MnSOD siRNAtransfected cells (Fig. 3m). Knock-down of MnSOD does not affect insulin signaling Insulin strongly increased phosphorylation of Akt in adipocytes differentiated from preadipocytes treated with MnSOD and control siRNA (Fig. 4a, b). BSO causes adipocyte de-differentiation BSO reduces cellular glutathione and thereby increases ROS [17]. To find out which concentrations of BSO significantly increase cellular ROS, mature 3T3-L1 adipocytes were incubated with 5, 10, and 20 lM BSO for 24 h, and antioxidative capacity was significantly reduced by 10 and 20 lM BSO (Fig. 5a). Confluent 3T3-L1 cells were induced to differentiate, and 3 days later, 20 and 40 lM BSO were added, and the treatment was continued until day 9 of differentiation. BSO at both concentrations impaired triglyceride accumulation (Fig. 5b) and chemerin, FABP4, HSL, and adiponectin were lower in cells exposed to BSO (40 lM) (Fig. 5 c, f
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and data not shown). FAS, PPARc, and Cox-4 were also markedly reduced (Fig. 5d, g). Of note, MnSOD was not affected by BSO treatment (Fig. 5d, h). BSO and MnSOD siRNA modestly reduce viability of preadipocytes Preadipocytes were transfected with MnSOD siRNA or control siRNA and 24 h later CellTiter-BlueÒ Cell Viability reagent was added for 24 h. Percentage of viable cells was modestly but significantly reduced in MnSOD siRNA -treated preadipocytes while LDH was increased (Fig. 6a, b). BSO (10 and 20 lM) did not affect number of viable cells, and LDH was only very modestly increased (Fig. 6c, d).
Discussion Current work shows that knock-down of MnSOD impairs adipogenesis of 3T3-L1 cells. Adipocytes differentiated from preadipocytes, where MnSOD has been knockeddown, have impaired triglyceride storage and low expression of proteins which are induced in late adipogenesis [20, 21]. Proteins which are already expressed in preadipocytes and only modestly induced in mature cells [20] are similarly abundant compared with cells transfected with control siRNA. Therefore, MnSOD which is upregulated during adipogenesis [8] is essential for lipid storage and expression of proteins and adipokines characteristic for mature adipocytes. PPARc regulates the abundance of adipocyte specific proteins [22] but is similarly expressed in control and MnSOD siRNA-transfected cells. This suggests that the levels of endogenous PPARc agonists [22] may be reduced in adipocytes with low MnSOD. MnSOD is modestly increased during adipogenesis [8, 23] and is markedly reduced in MnSOD siRNA transfected cells demonstrating that MnSOD siRNA is still functional 9 days after transfection of the preadipocytes. Cell viability and insulin-mediated phosphorylation of Akt were not impaired in adipocytes with low MnSOD excluding that increased cell death or insulin resistance contribute to the phenotype of mature adipocytes with reduced MnSOD. In preadipocytes, suppression of MnSOD reduces number of viable cells by about 5 %, and LDH is modestly increased. While this effect on viability may contribute to the reduced number of mature adipocytes, it is for sure not the principle explanation for the strongly impaired differentiation observed. Surprisingly, antioxidative capacity of adipocytes differentiated from preadipocytes treated with MnSOD siRNA is similar to control-transfected adipocytes, and superoxide measured during adipogenesis is not elevated in
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Fig. 3 Effect of MnSOD knock-down on adipogenesis of 3T3-L1 cells. a Light microscopy of mature 3T3-L1 cells differentiated from preadipocytes treated with control siRNA and MnSOD siRNA. Lipid accumulation was determined by Oil Red O staining. b Triglycerides (ng/lg cellular protein) in the cell lysates of these cells: data of 6 experiments have been calculated. c Triglycerides (ng/lg cellular protein) in the supernatants (SN) of these cells: data of 6 experiments have been calculated. d FAS, PPARc, and Cox-4 in preadipocytes and 2, 3, 7, and 9 d after hormonal induction of adipogenesis. e Knockdown of MnSOD was performed in preadipocytes and MnSOD, FAS, PPARc, Cox-4, and GAPDH were analyzed in 9-d-differentiated cells. f Quantification of PPARc partly shown in e. Data of 5 experiments are shown. g HSL and FABP4 in preadipocytes and 2, 3, 7, and 9 d after hormonal induction of adipogenesis. h Knock-down of
MnSOD was performed in preadipocytes, and MnSOD, chemerin, HSL, FABP4, and GAPDH were analyzed in 9-d-differentiated cells. i Quantification of HSL partly shown in h. Data of 5 experiments have been calculated. j Quantification of FABP4 partly shown in h. Data of 4 experiments are shown. k Adiponectin in the supernatants of these cells: data of 5 experiments have been calculated. l Antioxidative capacity of mature 3T3-L1 cells differentiated from preadipocytes treated with control siRNA and MnSOD siRNA. Data of 12 experiments are shown. m Superoxides were measured by NBT during differentiation of 3T3-L1 cells from preadipocytes treated with control siRNA and MnSOD siRNA. Data of one out of three experiments with comparable results are shown. * Indicates a p value \0.05
MnSOD siRNA-treated cells. This is in agreement with several studies which have shown that ROS are not altered in cells where MnSOD is overexpressed or blocked until these cells are exposed to agents increasing oxidative stress [24–28]. It is therefore possible that other ROS-detoxifying
enzymes are induced to compensate for low MnSOD during differentiation. Importantly, knock-down of MnSOD in mature adipocytes is associated with modestly reduced antioxidative capacity [23]. MnSOD is upregulated during differentiation
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[8, 23] surmising that this enzyme may be more important for ROS homeostasis in mature cells. The issues concerning whether related superoxide detoxifying enzymes are
Fig. 4 Insulin-mediated phosphorylation of Akt. a Akt, pAkt, and MnSOD in control siRNA- and MnSOD siRNA-treated preadipocytes which were differentiated to mature cells and subsequently stimulated with 50 and 200 nM insulin for 20 min. b Quantification of the results partly shown in a; data of 3 experiments have been calculated. * Indicates a p value \0.05 for comparison with cells not treated with insulin
Fig. 5 Effect of L-Buthionine-(S,R)-sulfoximine (BSO) in 3T3-L1 cells. a Antioxidative capacity of mature 3T3-L1 cells which were incubated with increasing concentrations of BSO for 24 h. b Light microscopy of mature 3T3-L1 cells differentiated from preadipocytes treated with BSO (20, 40 lM) since day 3 of differentiation. c Chemerin, HSL, FABP4, PARP, and GAPDH in the cells described in b. d MnSOD, FAS, PPARc, Cox-4, and GAPDH in the cells
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not at all or insufficiently induced in the mature cells need further studies. Anyhow, the findings described above exclude that the elevated ROS impair adipogenesis and triglyceride storage in cells with low MnSOD. Therefore, the mechanisms linking adipogenesis and MnSOD have to be identified in future studies. Previous data show that superoxides are transiently induced during differentiation or are higher in mature adipocytes compared to preadipocytes [11, 29, 30]. In the cell model used herein superoxides are not upregulated during adipogenesis. Therefore, either production of superoxides is not induced during adipogenesis or respective antioxidant enzymes are increased early enough. Discrepant findings may be at least in part explained by the many different protocols used for adipogenic differentiation of 3T3-L1 cells. Current data show that adipogenesis is not in general associated with higher superoxide levels. Pharmaceutical depletion of glutathione by BSO lowers antioxidative capacity of mature cells indicating the increased formation of ROS in accordance with the
described in b. e Quantification of HSL partly shown in c. Data of 3 experiments are shown. f Adiponectin in the supernatants of these cells: data of 4 experiments have been calculated. g Quantification of FABP4 partly shown in c. Data of 3 experiments are shown. h Quantification of MnSOD partly shown in d. Data of 3 experiments are shown. * Indicates a p value \0.05
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Fig. 6 Effect of MnSOD knock-down and L-Buthionine-(S,R)sulfoximine (BSO) in 3T3-L1 fibroblasts. a Viable cells in preadipocytes treated with control and MnSOD siRNA: data of 3 experiments have been calculated. b LDH in the supernatants of
these cells. c Viable cells in preadipocytes treated with BSO: data of one out of three experiments are shown. d LDH in the supernatants of these cells. * Indicates a p value \0.05
literature [17]. During differentiation, BSO not only blocks the upregulation of late adipogenesis markers but even lowers expression of proteins which are induced early during differentiation. Therefore, in contrast to cells with low MnSOD, BSO downregulates FAS, Cox-4, and PPARc, causing a de-differentiation of the cells. BSO at a concentration of 20 lM lowers AOC of mature adipocytes by about 4 % and nearly completely blocks adipogenesis and triglyceride storage when added during differentiation, surmising that adipogenesis is highly sensitive to oxidative stress. MnSOD is about twofold upregulated during adipogenesis [8] and subsequently should be reduced in BSO-treated cells. Nevertheless, MnSOD is similarly abundant in control- and BSO-exposed 3T3-L1 cells. MnSOD is induced by ROS [31, 32], and this mechanism may also account for relatively high abundance of this enzyme. Antiadipogenic effects of the increased ROS on preadipocytes have already been shown by other groups [17, 18] in accordance with current findings. In contrast, Nacetyl-L-cysteine (NAC) and EUK-8, a catalytic mimetic of superoxide dismutase (SOD) and catalase even inhibit adipogenesis of mesenchymal stem cells [33, 34]. ROS are essential for mitotic clonal expansion of preadipocytes and induction of C/EBPb which is expressed early in differentiation [35, 36]. BSO, however, does not promote mitosis of preadipocytes in the cell culture model used herein and in a recent study [37]. Those authors show that BSO lowers total but not nuclear glutathione and only the latter has a role in the cell–cycle regulation. In summary, current work shows that glutathione depletion and low MnSOD block adipogenesis of 3T3-L1 fibroblasts. The essential function of MnSOD for adipocyte triglyceride accumulation is, however, independent of its effect on cellular ROS homeostasis.
Conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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