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The adaptor protein alpha-syntrophin regulates adipocyte lipid droplet growth
Experimental Cell Research 345 (2016) 100–107
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Research article
The adaptor protein alpha-syntrophin regulates adipocyte lipid droplet growth Kristina Eisinger, Lisa Rein-Fischboeck, Rebekka Pohl, Elisabeth M. Meier, Sabrina Krautbauer, Christa Buechler n Department of Internal Medicine I, University Hospital of Regensburg, Regensburg, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 8 January 2016 Received in revised form 4 May 2016 Accepted 25 May 2016 Available online 27 May 2016
The scaffold protein alpha-syntrophin (SNTA) regulates lipolysis indicating a role in lipid homeostasis. Adipocytes are the main lipid storage cells in the body, and here, the function of SNTA has been analyzed in 3T3-L1 cells. SNTA is expressed in preadipocytes and is induced early during adipogenesis. Knockdown of SNTA in preadipocytes increases their proliferation. Proteins which are induced during adipogenesis like adiponectin and caveolin-1, and the inflammatory cytokine IL-6 are at normal levels in the mature cells differentiated from preadipocytes with low SNTA. This suggests that SNTA does neither affect differentiation nor inflammation. Expression of proteins with a role in cholesterol and triglyceride homeostasis is unchanged. Consequently, basal and epinephrine induced lipolysis as well as insulin stimulated phosphorylation of Akt and ERK1/2 are normal. Importantly, adipocytes with low SNTA form smaller lipid droplets and store less triglycerides. Stearoyl-CoA reductase and MnSOD are reduced upon SNTA knock-down but do not contribute to lower lipid levels. Oleate uptake is even increased in cells with SNTA knock-down. In summary, current data show that SNTA is involved in the expansion of lipid droplets independent of adipogenesis. Enhanced preadipocyte proliferation and capacity to store surplus fatty acids may protect adipocytes with low SNTA from lipotoxicity in obesity. & 2016 Elsevier Inc. All rights reserved.
Keywords: Adipogenesis Triglycerides Lipolysis Insulin
1. Introduction Obesity presents a growing health problem and a major risk factor for metabolic diseases. Inappropriate fat storage in adipose tissues causes triglyceride deposition in the liver and muscle which contributes to insulin resistance [1–4]. A feature of insulin resistant patients is prolonged postprandial hyperglycemia due to a decreased glucose uptake by muscle and fat tissues [5]. Muscle mass and strength is reduced in diabetic patients further contributing to obesity and metabolic inflexibility [6]. Consequently, most skeletal muscle pathologies including muscular dystrophies are associated with metabolic abnormalities [7]. The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle integrity. The DAPC contributes to the structural stability of the plasma membrane and further is a platform for the formation of signaling complexes [8,9]. DAPC components are dystrophin, the dystroglycans, sarcoglycans, sarcospan, dystrobrevin and the syntrophins [9]. The domain organization of the five syntrophin family members is the same: a pleckstrin homology (PH) domain at the N-terminus with an embedded PDZ n
Corresponding author. E-mail address: [email protected] (C. Buechler).
http://dx.doi.org/10.1016/j.yexcr.2016.05.020 0014-4827/& 2016 Elsevier Inc. All rights reserved.
domain, a second PH domain and a C-terminal syntrophin unique domain [8]. The split PH domain of alpha-syntrophin (SNTA) binds inositol phospholipids while the second PH domain interacts with dystrophin. The PDZ domain is involved in lipid binding and further interacts with proteins like neuronal nitric oxide synthase [8,10,11]. SNTA is diminished in skeletal muscle of diabetic rats and patients with gestational diabetes and it is suggested that this protein is involved in the insulin response of skeletal muscle [12,13]. Members of the DAPC are all expressed in adipocytes [14] indicating a role in fat cell biology. Beta-sarcoglycan deficient mice do not form a sarcoglycan complex in adipocytes and skeletal muscle cells. These mice are insulin resistant and glucose intolerant. Interestingly, weight of intraabdominal adipose tissues is reduced in beta-sarcoglycan null mice [15]. Catecholaminergic stimulation, subsequent raise of cAMP levels and protein kinase A mediated phosphorylation of hormone sensitive lipase (HSL) and perilipin stimulates triglyceride breakdown in adipocytes in the fasting state [16]. This is a crucial pathway regulating the energy homeostasis of the whole organism [16]. SNTA binds guanine nucleotide-binding protein alpha-subunits which are downstream of various G-protein coupled receptors including β-adrenergic receptors [17]. Knock-down of SNTA in COS-7 cells enhances beta-adrenergic agonist induced cAMP
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production showing that SNTA inhibits lipolysis in these cells [17]. SNTA also binds to the C-terminal amino acids of the ATP-binding cassette transporter A1 (ABCA1) to stabilize the protein [18]. Adipocyte specific deletion of ABCA1 is linked to increased lipid storage and enlarged fat pad weights in rodents [19]. ABCA1 is, however, normally expressed in the liver of mice deficient in SNTA and beta 2 syntrophin (SNTB2) arguing against a role of SNTA in stabilizing hepatic ABCA1 [20]. Further, ABCA1 mediated cholesterol and phosphatidylcholine efflux is unchanged in macrophages with deleted SNTA and SNTB2 [21]. The cellular function of SNTA depends on its association with different binding partners [8] suggesting tissue specific roles of this adaptor protein. In the current study the function of SNTA in adipocytes was analyzed to evaluate whether this adaptor protein affects adipogenesis and lipid homeostasis.
2. Materials and methods 2.1. ELISA Adiponectin ELISA and IL-6 ELISA were from R&D Systems (Wiesbaden, Germany). 2.2. Immunoblot Immunoblot was performed as recently published [22]. The annexin A6 antibody has been described [23]. SNTA antibody was kindly provided by Prof. Adams and was described recently. The antibody recognizes a peptide sequence in the PH1b domain of SNTA and was raised in rabbits [24,25]. Antibodies for detection of ACC, pACC, Akt, pAkt, AMPK, pAMPK, ATGL, β-actin, caveolin-1, Cox IV, ERK1/2, pERK1/2, FABP4, FAS, GAPDH, HSL, pHSL, PARP1, perilipin, PPARγ, Rab5 and SCD1 were from New England Biolabs GmbH (Frankfurt am Main, Germany). Heme oxygenase 1 antibody was from Novus Biologicals (Cambridge, UK). SREBP2 antibody was from Cayman Chemicals (IBL International GmbH, Hamburg, Germany). ABCA1, adipophilin, alpha 1 adrenergic receptor and PGC1α antibodies were from Abcam (Cambridge, UK). Antibodies to eNOS and phosphorylated eNOS were from Merck Millipore (Schwalbach, Germany). Chemerin antibody was from R&D Systems (Wiesbaden, Germany). MnSOD antibody was from Thermo Fisher Scientific (Schwerte, Germany). 2.3. Transfection with siRNAs 3T3-L1 preadipocytes were from the ATCC (Manassas, VA, USA) and differentiated as described [22]. Transfection of preadipocytes with siRNAs was performed with XtremeGene transfection reagent (Roche, Mannheim, Germany) or Endoporter (Gene Tools LLC, Philomath, Oregon, USA). Silencers Select Pre-Designed siRNAs and Negative Control siRNA were from Applied Biosystems (Darmstadt, Germany). The siRNA s74114 (CGAUGGUCUUUAUCAUCCAtt) was used to knock down SNTA. SCD1 was knocked down with s73339 (GGGAUUUUCUACUACAUGAtt) and s73340 (CCGCGCAUCUCUAUGGAUAtt).
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2.5. Immunofluorescence 3T3-L1 adipocytes cultivated on cover slips (VWR, Darmstadt, Germany) were fixed with methanol at 20 °C for 15 min. After incubation with blocking solution (3% BSA, 0.05% Tween 20), and washing with PBS and PBS Tween (0.1%), cells were incubated with SNTA antibody (1:100-fold diluted) and adipophilin antibody (1:10-fold diluted) at 4 °C overnight. After washing, cells were incubated with secondary antibodies and DAPI (Roche) for 1 h at 37 °C. Slides were mounted with fluorescent mounting medium (DAKO, Glostrup, Denmark). 2.6. Cell viability CellTiter-Blue Cell Viability Assay was from Promega (Mannheim, Germany). LDH assay was from Roche (Mannheim, Germany). 2.7. Quantification of lipids Triglycerides were measured using GPO-PAP micro-test (Roche, Mannheim, Germany). Glycerol and free fatty acids were determined by an assay from BioCat (Berlin, Germany) and cholesterol by an assay from Diaglobal (Berlin, Germany). 2.8. Statistical analysis Data are given as box plots showing the median, lower and upper quartiles and range of the values (SPSS Statistics 21.0 program). Statistical differences were analyzed by Student's t-test (Ms Excel). A value of p o0.05 was regarded as significant.
3. Results 3.1. SNTA increases during adipogenesis SNTA was expressed in 3T3-L1 preadipocytes and increased two days after initiation of adipogenesis. Levels were not further changed during adipocyte maturation (Fig. 1A and B). Immunofluorescence revealed cytoplasmic localization of SNTA (Fig. 1C). SNTA did not colocalize with adipophilin in mature adipocytes excluding a close association of the adaptor protein with this lipid droplet associated protein. Differentiation of the cells in medium supplemented with oleate increased lipid storage (data not shown) and fatty acid binding protein 4 (FABP4) but did not affect SNTA levels (Fig. 1D). Similarly, palmitate and linoleate added during differentiation had no effect on SNTA protein (Fig. 1E). Chemerin is shown as positive control and is induced in adipocytes differentiated in the presence of the fatty acids as described [22] (Fig. 1E). The peroxisome proliferator activated receptor (PPAR) γ agonist pioglitazone did not upregulate SNTA in adipocytes (data not shown). This suggests that induction of SNTA early during adipogenesis is not regulated by the main adipogenic transcription factor PPARγ. 3.2. SNTA knock-down increases proliferation of preadipocytes and hinders formation of large lipid droplets in mature cells
2.4. Fatty acid treatment Oleate, linoleate and palmitate were ordered from Sigma (Deisenhofen, Germany). Fatty acids were complexed to fatty acidfree bovine serum albumin (Roche, Mannheim, Germany) with a molar ratio of 1:1. Equal amounts of bovine serum albumin were added to control cells. Fatty acids were added to the culture medium during differentiation or for 24 h.
Knock-down of SNTA in preadipocytes increased the number of viable cells suggesting enhanced proliferation (Fig. 2A). When the 3T3-L1 siRNA transfected preadipocytes were differentiated to adipocytes the cells with low SNTA formed only small lipid droplets (Fig. 2B). The number of lipid droplets with a diameter below 6 mM tended to be increased while the number of very large lipid droplets was significantly reduced (Fig. 2C). Adipocytes with low
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Fig. 1. Expression and regulation of SNTA in 3T3-L1 cells. A. SNTA, FABP4 and β-actin in preadipocytes (pre) and during adipogenesis. B. Protein densitometry of SNTA during adipogenesis (4 values per time point were calculated). C. SNTA and adipophilin staining in mature 3T3-L1 cells. DNA was visualized with DAPI. D. SNTA and FABP4 protein in preadipocytes (Pre) and cells differentiated for 1 d, 2 d, 3 d, 6 d and 9 d in the presence ( þ) or absence ( ) of 400 mM oleate (OA). E. SNTA, chemerin and GAPDH in 3T3-L1 cells differentiated in the presence of 200 or 400 mM palmitate (PA), oleate (OA) or linoleate (LA). * p o 0.05,*** p o 0.001, (*) indicates a trend.
Fig. 2. Effect of SNTA on lipid droplet size in 3T3-L1 cells. A. Proliferation of fibroblasts treated with scrambled (Scr) and SNTA siRNA (arbitrary units, au; n ¼ 32). B. 3T3-L1 cells differentiated from preadipocytes treated with scrambled and SNTA siRNA. Oil Red O stained cells are shown in the right pictures. C. Distribution of lipid droplets stratified for diameter in 3T3-L1 cells differentiated from preadipocytes treated with scrambled and SNTA siRNA (n¼ 4–5, at least 250 cells per experiment were measured). D. Cellular triglycerides. E. Cellular cholesterol. F. Soluble LDH. G. Adiponectin in the supernatants. H. IL-6 in the supernatants of cells differentiated from preadipocytes treated with scrambled and SNTA siRNA. Data of at least four experiments have been used for calculations. * p o 0.05, ** po 0.01, (*) indicates a trend.
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Fig. 3. Effect of SNTA on the levels of different proteins in 3T3-L1 cells. A. SNTA, PARP1, Rab5, alpha 1 adrenergic receptor (AR). B. SNTA, ABCA1, caveolin-1, annexin A6 and active SREBP2. C. AMPK, ACC and their phosphorylated forms. D. Perilipin, eNOS and its phosphorylated form, ATGL and HSL. E. MnSOD, HO-1, Cox IV and PGC1α. F. SNTA, FAS, SCD1 and FABP4 in cells differentiated from preadipocytes treated with scrambled and SNTA siRNA. G. Protein densitometry of SNTA, SCD1 and MnSOD (%). The dotted line represents 100%. Data of at least four experiments have been used for calculations. * p o 0.05.
SNTA stored less triglycerides while cholesterol concentrations were normal (Fig. 2D and E). Released lactate dehydrogenase, a marker of cell death, was even reduced showing that low SNTA protects cells from cytotoxicity (Fig. 2F). 3.3. SNTA knock-down does not affect adipogenesis Adiponectin is induced late in adipogenesis [26] and was similar in scrambled and SNTA siRNA transfected cells (Fig. 2G). Level of the inflammatory cytokine IL-6 measured in the supernatants was comparable in both cell types (Fig. 2H). Immunoblot analysis confirmed reduced SNTA protein in the mature adipocytes differentiated from preadipocytes which were treated with SNTA siRNA (Fig. 3A, B, F, G) demonstrating that SNTA is still knockeddown. The adipogenic transcription factor PPARγ was similarly expressed in scrambled and SNTA siRNA transfected cells (Fig. 5B). PARP1 is induced during adipogenesis and regulates PPARγ activity [27] and was comparable in both cell types (Fig. 3A). Rab5 is a marker of early endosomes which are involved in lipid transport [28] and was also unchanged (Fig. 3A). SNTA affects the stability of alpha 1-adrenergic receptors [29] but analysis of alpha 1 receptors in control and SNTA siRNA treated cells revealed similar levels in both cell types (Fig. 3A). Proteins with a role in cholesterol homeostasis (ABCA1, caveolin-1, annexin A6 and sterol regulatory element binding protein 2 (SREBP2)) were normally abundant in the SNTA siRNA transfected cells (Fig. 3B). Phosphorylated AMP activated protein kinase (AMPK) and its downstream substrate acetyl-CoA carboxylase (ACC) were not changed (Fig. 3C). Proteins which regulate lipolysis namely perilipin, endothelial nitric oxid synthase (eNOS), hormone sensitive lipase (HSL), and adipose triglyceride lipase (ATGL) were normally expressed in both cell types (Fig. 3D). Manganese superoxide dismutase (MnSOD) was significantly lower in the cells
with SNTA knock-down (Fig. 3E and G). Heme oxygenase-1 (HO-1) which is induced upon oxidative stress [30] was not regulated (Fig. 3E). Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and cytochrome oxidase IV (Cox IV) were not changed (Fig. 3E). Fatty acid binding protein 4 (FABP4) and fatty acid synthase (FAS) levels were comparable between control and SNTA siRNA treated cells (Fig. 3F). Stearoyl CoA Desaturase 1 (SCD1) was markedly reduced in adipocytes with low SNTA (Fig. 3F and G).
3.4. SCD1 knock-down does not affect triglyceride storage MnSOD is induced by fatty acids in adipocytes [31] suggesting that lower expression in cells with SNTA knock-down was related to reduced triglyceride storage. MnSOD knock-down in differentiated and pre-differentiated cells did not affect cellular triglycerides [31] excluding that this mitochondrial enzyme regulates cellular triglycerides independent of adipogenesis. SCD1 was reduced in cells with low SNTA (Fig. 3F and G) and may contribute to diminished triglyceride levels. To clarify a potential function of SCD1 in lipid droplet formation during adipogenesis SCD1 was knocked down in preadipocytes and cells were subsequently differentiated to mature adipocytes. SCD1 was markedly reduced as expected (Fig. 4A and B). HSL and PPARγ which are upregulated during adipogenesis were normally expressed. Further, low SCD1 did not affect SNTA levels (Fig. 4A and B). Lipid droplet size, cellular triglycerides and cholesterol were not changed upon SCD1 knock-down (Fig. 4C–E). This indicates that reduced SCD1 did not contribute to lower triglycerides and smaller lipid droplets in cells treated with SNTA siRNA.
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Fig. 4. Role of SCD1 in lipid droplet formation of 3T3-L1 cells. A. FAS, HSL, PPARγ, SNTA and SCD1 in cells differentiated from preadipocytes treated with scrambled or SCD1 siRNA. B. Protein densitometry of SNTA and SCD1 in these cells. Values are given as % of control and the dotted line represents 100%. C. Adipocytes differentiated from preadipocytes treated with scrambled or SCD1 siRNA. D. Cellular triglycerides. E. Cholesterol levels of these cells. * p o0.05.
3.5. SNTA knock-down does not affect basal adipocyte functions Adipocytes forming smaller lipid droplets may have a differential response to metabolic stimuli. Insulin mediated phosphorylation of Akt and ERK1/2 and expression of these proteins was nevertheless comparable in both cell types (Fig. 5A). SNTA has been described to regulate lipolysis in COS-7 cells [17]. Epinephrine induced phosphorylation of HSL was normal in the SNTA siRNA treated adipocytes (Fig. 5B). Basal and epinephrine stimulated
fatty acid and glycerol release was comparable in both cell types (Fig. 5C and D). 3.6. SNTA knock-down cells have improved lipid storage Cells with smaller lipid droplets may have an improved capacity to store excess fatty acids. Cellular lipids increased in scrambled and SNTA siRNA treated cells incubated with 200 mM oleate for 24 h (Fig. 5E). Triglycerides tended to be higher (Fig. 5E) and Oil
Fig. 5. Role of SNTA in 3T3-L1 cell function. A. Insulin (20 min incubation time) induced phosphorylation of Akt and ERK1/2 in cells differentiated from preadipocytes treated with scrambled and SNTA siRNA. B. HSL and its phosphorylated form and PPARγ in cells differentiated from preadipocytes transfected with scrambled and SNTA siRNA. Cells were treated with 0, 100 and 500 nM epinephrine for two hours. C. Effect of epinephrine on fatty acids in the supernatants of cells differentiated from preadipocytes treated with scrambled and SNTA siRNA (n¼ 4). D. Effect of epinephrine on glycerol in the supernatants of cells differentiated from preadipocytes treated with scrambled and SNTA siRNA (n ¼ 6). E. Triglycerides in adipocytes differentiated from preadipocytes treated with scrambled and SNTA siRNA and cultivated in the presence of 200 mM oleate for 24 h (n ¼3). F. Oil Red O Staining of the cells described in E (n ¼ 11). G. LDH in the supernatants of cells described in E (n ¼6). * p o 0.05, (*) trend.
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Red O staining (Fig. 5F) was significantly increased in cells with low SNTA. Improved lipid storage was not linked to increased cytotoxicity in cells with low SNTA (Fig. 5G).
4. Discussion The current study demonstrates a role of the adaptor protein SNTA in lipid droplet growth. Differentiation of 3T3-L1 cells with low SNTA hinders the formation of large lipid droplets. This is not a secondary effect of impaired adipogenesis. Proteins like adiponectin, FABP4 and HSL which are induced during adipocyte maturation [22,26] are normally expressed in the adipocytes differentiated from preadipocytes with SNTA knock-down. Small lipid droplets in cells with SNTA knock-down are accompanied by reduced triglyceride storage. SNTA does not regulate proteins involved in fatty acid synthesis, uptake and release. Further, these cells form multiple small lipid droplets excluding that formation of these structures is generally impaired. SNTA reduces β-adrenergic stimulated cAMP production in COS-7 cells indicating that increased lipolysis may contribute to reduced triglyceride storage in 3T3-L1 adipocytes with low SNTA [17]. Basal and epinephrine induced fatty acid and glycerol release is nevertheless normal in adipocytes with low SNTA. This argues against a function of this scaffold protein in adipocyte lipolysis. Further, insulin mediated phosphorylation of Akt and ERK1/2 are unchanged in these cells suggesting normal insulin-induced lipid storage. Lower triglyceride levels may be related to reduced lipid uptake during adipogenesis which has not been analyzed herein. In mature cells cellular pathways involved in lipid homeostasis are nevertheless normal. White adipocytes are mostly completely filled with a single lipid droplet which thereby determines the cell size [32]. Small and large adipocytes within the same fat depot have been shown to exert comparable insulin response but are also found to differ in insulin sensitivity with smaller cells being more sensitive [33,34]. Smaller adipocytes may have a higher capacity to store surplus lipids but may also represent inappropriately differentiated adipocytes [33,35]. Adipocytes with low SNTA display normal insulin sensitivity and have increased fatty acid storage when cells are challenged with oleate. This indicates that improved deposition of fat in these cells may protect from the harmful effects of lipid surplus in obesity. SNTA is an adaptor protein [8] thus indicating that the inability of cells with low SNTA to form large lipid droplets is mediated by SNTA binding proteins. With the exception of MnSOD and SCD1 all of the proteins analyzed are normally expressed in the adipocytes with low SNTA. Reduced MnSOD in 3T3-L1 cells treated with SNTA siRNA is most likely related to lower triglyceride levels [31]. Fatty acids induce MnSOD in adipocytes while knock-down of this mitochondrial enzyme in mature cells has no effect on triglyceride storage [31]. Expression of further mitochondrial proteins and heme oxygenase are normal when SNTA is reduced arguing against impaired mitochondrial biogenesis and increased oxidative stress. SCD1 converts palmitate and stearate into palmitoleate and oleate [36]. SCD1 is reduced in adipocytes with insufficient SNTA. The transcription factor SREBP1c regulates SCD1, FAS and ACC [37]. Expression of the latter two proteins is, however, not changed in SNTA depleted 3T3-L1 cells. This excludes that SREBP1c is involved herein. Further studies are needed to clarify the mechanisms contributing to reduced SCD1 levels in adipocytes with low SNTA. 3T3-L1 preadipocytes treated with an SCD1 inhibitor throughout differentiation have decreased triglycerides [38]. SCD1 downregulation by siRNA has no effect on lipid droplet size and triglyceride levels in our experiments. Current data are in agreement with findings published previously by Jennifer L. Christianson et al.
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[39]. In their work fibroblasts were transfected with SCD1 or SCD2 siRNA and subsequently differentiated to adipocytes. They found no effect of SCD1 siRNA on adipogenesis or lipid loading. SCD2 knock-down markedly reduced adipogenesis and lipid storage. This shows that SNTA seems not to regulate SCD2 levels because adipogenic markers are normal. Adipocyte specific deletion of SCD1 in mice does not protect from obesity [40] further arguing against a role of SCD1 in fat depot growth. Therefore, SCD1 does not regulate adipocyte triglyceride levels and consequently does not contribute to small lipid droplets in 3T3-L1 cells with low SNTA. Current experiments show that SNTA deficiency during adipogenesis results in reduced triglycerides and small lipid droplets in the mature cells. Expression of several proteins regulating lipid droplet size is not changed. SNTA is a scaffold for signaling complexes suggesting that cellular localization and not absolute levels of proteins involved in lipid droplet formation and/or triglyceride synthesis may be changed. We do, however, not have additional evidence to support this conclusion. The SNTA regulated proteins in adipocytes have still to be identified. The so far described SNTA interacting proteins aquaporin 4 and neuronal nitirc oxid synthase are not expressed in adipocytes [41,42], ABCA1 is a further SNTA interacting protein but its levels are normal in adipocytes with SNTA knock-down. This is in agreement with recent findings in the liver and macrophages [20,21]. TAPP1 interacts with SNTA and this complex has a role in the remodeling of the cytoskeleton [43] and insulin response [44]. Normal insulin sensitivity of 3T3-L1 cells with low SNTA argues against a role of TAPP1 herein. Alpha 1 adrenergic receptor levels are regulated by SNTA in HEK293 cells [29] but protein is not changed in 3T3-L1 cells with low SNTA. Recently our group excluded that adiponectin receptor 1 signaling is affected in SNTA deficient mice [45]. SNTA is an actin-binding protein [46]. The actin cytoskeleton is involved in the expansion of lipid droplets and enhanced actin cytoskeletal remodeling promotes lipid accumulation in adipocytes [47]. Whether cytoskeletal remodeling is altered in cells with low SNTA has not been investigated yet. Rac1 regulates cytoskeletal organization and cell proliferation [48]. SNTA activates Rac1 and thereby increases cellular reactive oxygen species and proliferation of breast cancer cell lines [49]. In preadipocytes proliferation is nevertheless induced upon SNTA knock-down and HO-1 which is a sensitive marker of cellular ROS levels [30] is normally expressed in adipocytes with low SNTA. A role for SNTA in adipocyte Rac1 activation is, therefore, unlikely indicating cell-type specific functions of SNTA. Phosphatidylinositol 4,5-bisphosphate and cholesterol can bind to SNTA [50,51], and it may be assumed that association of SNTA with lipids contributes to lipid droplet growth.
5. Conclusion The current investigations demonstrate a function of the adaptor protein SNTA in lipid droplet growth. SNTA does neither affect adipogenesis nor adipocyte function. Importantly, adipocytes with low SNTA are able to more effectively store surplus triglycerides suggesting a protective role under conditions of fatty acid excess.
Acknowledgments The study was supported by a Grant from the German Research Foundation (BU 1141/8-1). We thank Prof. Dr. Charalampos Aslanidis for helpful suggestions.
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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
Research article
The adaptor protein alpha-syntrophin regulates adipocyte lipid droplet growth Kristina Eisinger, Lisa Rein-Fischboeck, Rebekka Pohl, Elisabeth M. Meier, Sabrina Krautbauer, Christa Buechler n Department of Internal Medicine I, University Hospital of Regensburg, Regensburg, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 8 January 2016 Received in revised form 4 May 2016 Accepted 25 May 2016 Available online 27 May 2016
The scaffold protein alpha-syntrophin (SNTA) regulates lipolysis indicating a role in lipid homeostasis. Adipocytes are the main lipid storage cells in the body, and here, the function of SNTA has been analyzed in 3T3-L1 cells. SNTA is expressed in preadipocytes and is induced early during adipogenesis. Knockdown of SNTA in preadipocytes increases their proliferation. Proteins which are induced during adipogenesis like adiponectin and caveolin-1, and the inflammatory cytokine IL-6 are at normal levels in the mature cells differentiated from preadipocytes with low SNTA. This suggests that SNTA does neither affect differentiation nor inflammation. Expression of proteins with a role in cholesterol and triglyceride homeostasis is unchanged. Consequently, basal and epinephrine induced lipolysis as well as insulin stimulated phosphorylation of Akt and ERK1/2 are normal. Importantly, adipocytes with low SNTA form smaller lipid droplets and store less triglycerides. Stearoyl-CoA reductase and MnSOD are reduced upon SNTA knock-down but do not contribute to lower lipid levels. Oleate uptake is even increased in cells with SNTA knock-down. In summary, current data show that SNTA is involved in the expansion of lipid droplets independent of adipogenesis. Enhanced preadipocyte proliferation and capacity to store surplus fatty acids may protect adipocytes with low SNTA from lipotoxicity in obesity. & 2016 Elsevier Inc. All rights reserved.
Keywords: Adipogenesis Triglycerides Lipolysis Insulin
1. Introduction Obesity presents a growing health problem and a major risk factor for metabolic diseases. Inappropriate fat storage in adipose tissues causes triglyceride deposition in the liver and muscle which contributes to insulin resistance [1–4]. A feature of insulin resistant patients is prolonged postprandial hyperglycemia due to a decreased glucose uptake by muscle and fat tissues [5]. Muscle mass and strength is reduced in diabetic patients further contributing to obesity and metabolic inflexibility [6]. Consequently, most skeletal muscle pathologies including muscular dystrophies are associated with metabolic abnormalities [7]. The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle integrity. The DAPC contributes to the structural stability of the plasma membrane and further is a platform for the formation of signaling complexes [8,9]. DAPC components are dystrophin, the dystroglycans, sarcoglycans, sarcospan, dystrobrevin and the syntrophins [9]. The domain organization of the five syntrophin family members is the same: a pleckstrin homology (PH) domain at the N-terminus with an embedded PDZ n
Corresponding author. E-mail address: [email protected] (C. Buechler).
http://dx.doi.org/10.1016/j.yexcr.2016.05.020 0014-4827/& 2016 Elsevier Inc. All rights reserved.
domain, a second PH domain and a C-terminal syntrophin unique domain [8]. The split PH domain of alpha-syntrophin (SNTA) binds inositol phospholipids while the second PH domain interacts with dystrophin. The PDZ domain is involved in lipid binding and further interacts with proteins like neuronal nitric oxide synthase [8,10,11]. SNTA is diminished in skeletal muscle of diabetic rats and patients with gestational diabetes and it is suggested that this protein is involved in the insulin response of skeletal muscle [12,13]. Members of the DAPC are all expressed in adipocytes [14] indicating a role in fat cell biology. Beta-sarcoglycan deficient mice do not form a sarcoglycan complex in adipocytes and skeletal muscle cells. These mice are insulin resistant and glucose intolerant. Interestingly, weight of intraabdominal adipose tissues is reduced in beta-sarcoglycan null mice [15]. Catecholaminergic stimulation, subsequent raise of cAMP levels and protein kinase A mediated phosphorylation of hormone sensitive lipase (HSL) and perilipin stimulates triglyceride breakdown in adipocytes in the fasting state [16]. This is a crucial pathway regulating the energy homeostasis of the whole organism [16]. SNTA binds guanine nucleotide-binding protein alpha-subunits which are downstream of various G-protein coupled receptors including β-adrenergic receptors [17]. Knock-down of SNTA in COS-7 cells enhances beta-adrenergic agonist induced cAMP
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production showing that SNTA inhibits lipolysis in these cells [17]. SNTA also binds to the C-terminal amino acids of the ATP-binding cassette transporter A1 (ABCA1) to stabilize the protein [18]. Adipocyte specific deletion of ABCA1 is linked to increased lipid storage and enlarged fat pad weights in rodents [19]. ABCA1 is, however, normally expressed in the liver of mice deficient in SNTA and beta 2 syntrophin (SNTB2) arguing against a role of SNTA in stabilizing hepatic ABCA1 [20]. Further, ABCA1 mediated cholesterol and phosphatidylcholine efflux is unchanged in macrophages with deleted SNTA and SNTB2 [21]. The cellular function of SNTA depends on its association with different binding partners [8] suggesting tissue specific roles of this adaptor protein. In the current study the function of SNTA in adipocytes was analyzed to evaluate whether this adaptor protein affects adipogenesis and lipid homeostasis.
2. Materials and methods 2.1. ELISA Adiponectin ELISA and IL-6 ELISA were from R&D Systems (Wiesbaden, Germany). 2.2. Immunoblot Immunoblot was performed as recently published [22]. The annexin A6 antibody has been described [23]. SNTA antibody was kindly provided by Prof. Adams and was described recently. The antibody recognizes a peptide sequence in the PH1b domain of SNTA and was raised in rabbits [24,25]. Antibodies for detection of ACC, pACC, Akt, pAkt, AMPK, pAMPK, ATGL, β-actin, caveolin-1, Cox IV, ERK1/2, pERK1/2, FABP4, FAS, GAPDH, HSL, pHSL, PARP1, perilipin, PPARγ, Rab5 and SCD1 were from New England Biolabs GmbH (Frankfurt am Main, Germany). Heme oxygenase 1 antibody was from Novus Biologicals (Cambridge, UK). SREBP2 antibody was from Cayman Chemicals (IBL International GmbH, Hamburg, Germany). ABCA1, adipophilin, alpha 1 adrenergic receptor and PGC1α antibodies were from Abcam (Cambridge, UK). Antibodies to eNOS and phosphorylated eNOS were from Merck Millipore (Schwalbach, Germany). Chemerin antibody was from R&D Systems (Wiesbaden, Germany). MnSOD antibody was from Thermo Fisher Scientific (Schwerte, Germany). 2.3. Transfection with siRNAs 3T3-L1 preadipocytes were from the ATCC (Manassas, VA, USA) and differentiated as described [22]. Transfection of preadipocytes with siRNAs was performed with XtremeGene transfection reagent (Roche, Mannheim, Germany) or Endoporter (Gene Tools LLC, Philomath, Oregon, USA). Silencers Select Pre-Designed siRNAs and Negative Control siRNA were from Applied Biosystems (Darmstadt, Germany). The siRNA s74114 (CGAUGGUCUUUAUCAUCCAtt) was used to knock down SNTA. SCD1 was knocked down with s73339 (GGGAUUUUCUACUACAUGAtt) and s73340 (CCGCGCAUCUCUAUGGAUAtt).
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2.5. Immunofluorescence 3T3-L1 adipocytes cultivated on cover slips (VWR, Darmstadt, Germany) were fixed with methanol at 20 °C for 15 min. After incubation with blocking solution (3% BSA, 0.05% Tween 20), and washing with PBS and PBS Tween (0.1%), cells were incubated with SNTA antibody (1:100-fold diluted) and adipophilin antibody (1:10-fold diluted) at 4 °C overnight. After washing, cells were incubated with secondary antibodies and DAPI (Roche) for 1 h at 37 °C. Slides were mounted with fluorescent mounting medium (DAKO, Glostrup, Denmark). 2.6. Cell viability CellTiter-Blue Cell Viability Assay was from Promega (Mannheim, Germany). LDH assay was from Roche (Mannheim, Germany). 2.7. Quantification of lipids Triglycerides were measured using GPO-PAP micro-test (Roche, Mannheim, Germany). Glycerol and free fatty acids were determined by an assay from BioCat (Berlin, Germany) and cholesterol by an assay from Diaglobal (Berlin, Germany). 2.8. Statistical analysis Data are given as box plots showing the median, lower and upper quartiles and range of the values (SPSS Statistics 21.0 program). Statistical differences were analyzed by Student's t-test (Ms Excel). A value of p o0.05 was regarded as significant.
3. Results 3.1. SNTA increases during adipogenesis SNTA was expressed in 3T3-L1 preadipocytes and increased two days after initiation of adipogenesis. Levels were not further changed during adipocyte maturation (Fig. 1A and B). Immunofluorescence revealed cytoplasmic localization of SNTA (Fig. 1C). SNTA did not colocalize with adipophilin in mature adipocytes excluding a close association of the adaptor protein with this lipid droplet associated protein. Differentiation of the cells in medium supplemented with oleate increased lipid storage (data not shown) and fatty acid binding protein 4 (FABP4) but did not affect SNTA levels (Fig. 1D). Similarly, palmitate and linoleate added during differentiation had no effect on SNTA protein (Fig. 1E). Chemerin is shown as positive control and is induced in adipocytes differentiated in the presence of the fatty acids as described [22] (Fig. 1E). The peroxisome proliferator activated receptor (PPAR) γ agonist pioglitazone did not upregulate SNTA in adipocytes (data not shown). This suggests that induction of SNTA early during adipogenesis is not regulated by the main adipogenic transcription factor PPARγ. 3.2. SNTA knock-down increases proliferation of preadipocytes and hinders formation of large lipid droplets in mature cells
2.4. Fatty acid treatment Oleate, linoleate and palmitate were ordered from Sigma (Deisenhofen, Germany). Fatty acids were complexed to fatty acidfree bovine serum albumin (Roche, Mannheim, Germany) with a molar ratio of 1:1. Equal amounts of bovine serum albumin were added to control cells. Fatty acids were added to the culture medium during differentiation or for 24 h.
Knock-down of SNTA in preadipocytes increased the number of viable cells suggesting enhanced proliferation (Fig. 2A). When the 3T3-L1 siRNA transfected preadipocytes were differentiated to adipocytes the cells with low SNTA formed only small lipid droplets (Fig. 2B). The number of lipid droplets with a diameter below 6 mM tended to be increased while the number of very large lipid droplets was significantly reduced (Fig. 2C). Adipocytes with low
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Fig. 1. Expression and regulation of SNTA in 3T3-L1 cells. A. SNTA, FABP4 and β-actin in preadipocytes (pre) and during adipogenesis. B. Protein densitometry of SNTA during adipogenesis (4 values per time point were calculated). C. SNTA and adipophilin staining in mature 3T3-L1 cells. DNA was visualized with DAPI. D. SNTA and FABP4 protein in preadipocytes (Pre) and cells differentiated for 1 d, 2 d, 3 d, 6 d and 9 d in the presence ( þ) or absence ( ) of 400 mM oleate (OA). E. SNTA, chemerin and GAPDH in 3T3-L1 cells differentiated in the presence of 200 or 400 mM palmitate (PA), oleate (OA) or linoleate (LA). * p o 0.05,*** p o 0.001, (*) indicates a trend.
Fig. 2. Effect of SNTA on lipid droplet size in 3T3-L1 cells. A. Proliferation of fibroblasts treated with scrambled (Scr) and SNTA siRNA (arbitrary units, au; n ¼ 32). B. 3T3-L1 cells differentiated from preadipocytes treated with scrambled and SNTA siRNA. Oil Red O stained cells are shown in the right pictures. C. Distribution of lipid droplets stratified for diameter in 3T3-L1 cells differentiated from preadipocytes treated with scrambled and SNTA siRNA (n¼ 4–5, at least 250 cells per experiment were measured). D. Cellular triglycerides. E. Cellular cholesterol. F. Soluble LDH. G. Adiponectin in the supernatants. H. IL-6 in the supernatants of cells differentiated from preadipocytes treated with scrambled and SNTA siRNA. Data of at least four experiments have been used for calculations. * p o 0.05, ** po 0.01, (*) indicates a trend.
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Fig. 3. Effect of SNTA on the levels of different proteins in 3T3-L1 cells. A. SNTA, PARP1, Rab5, alpha 1 adrenergic receptor (AR). B. SNTA, ABCA1, caveolin-1, annexin A6 and active SREBP2. C. AMPK, ACC and their phosphorylated forms. D. Perilipin, eNOS and its phosphorylated form, ATGL and HSL. E. MnSOD, HO-1, Cox IV and PGC1α. F. SNTA, FAS, SCD1 and FABP4 in cells differentiated from preadipocytes treated with scrambled and SNTA siRNA. G. Protein densitometry of SNTA, SCD1 and MnSOD (%). The dotted line represents 100%. Data of at least four experiments have been used for calculations. * p o 0.05.
SNTA stored less triglycerides while cholesterol concentrations were normal (Fig. 2D and E). Released lactate dehydrogenase, a marker of cell death, was even reduced showing that low SNTA protects cells from cytotoxicity (Fig. 2F). 3.3. SNTA knock-down does not affect adipogenesis Adiponectin is induced late in adipogenesis [26] and was similar in scrambled and SNTA siRNA transfected cells (Fig. 2G). Level of the inflammatory cytokine IL-6 measured in the supernatants was comparable in both cell types (Fig. 2H). Immunoblot analysis confirmed reduced SNTA protein in the mature adipocytes differentiated from preadipocytes which were treated with SNTA siRNA (Fig. 3A, B, F, G) demonstrating that SNTA is still knockeddown. The adipogenic transcription factor PPARγ was similarly expressed in scrambled and SNTA siRNA transfected cells (Fig. 5B). PARP1 is induced during adipogenesis and regulates PPARγ activity [27] and was comparable in both cell types (Fig. 3A). Rab5 is a marker of early endosomes which are involved in lipid transport [28] and was also unchanged (Fig. 3A). SNTA affects the stability of alpha 1-adrenergic receptors [29] but analysis of alpha 1 receptors in control and SNTA siRNA treated cells revealed similar levels in both cell types (Fig. 3A). Proteins with a role in cholesterol homeostasis (ABCA1, caveolin-1, annexin A6 and sterol regulatory element binding protein 2 (SREBP2)) were normally abundant in the SNTA siRNA transfected cells (Fig. 3B). Phosphorylated AMP activated protein kinase (AMPK) and its downstream substrate acetyl-CoA carboxylase (ACC) were not changed (Fig. 3C). Proteins which regulate lipolysis namely perilipin, endothelial nitric oxid synthase (eNOS), hormone sensitive lipase (HSL), and adipose triglyceride lipase (ATGL) were normally expressed in both cell types (Fig. 3D). Manganese superoxide dismutase (MnSOD) was significantly lower in the cells
with SNTA knock-down (Fig. 3E and G). Heme oxygenase-1 (HO-1) which is induced upon oxidative stress [30] was not regulated (Fig. 3E). Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and cytochrome oxidase IV (Cox IV) were not changed (Fig. 3E). Fatty acid binding protein 4 (FABP4) and fatty acid synthase (FAS) levels were comparable between control and SNTA siRNA treated cells (Fig. 3F). Stearoyl CoA Desaturase 1 (SCD1) was markedly reduced in adipocytes with low SNTA (Fig. 3F and G).
3.4. SCD1 knock-down does not affect triglyceride storage MnSOD is induced by fatty acids in adipocytes [31] suggesting that lower expression in cells with SNTA knock-down was related to reduced triglyceride storage. MnSOD knock-down in differentiated and pre-differentiated cells did not affect cellular triglycerides [31] excluding that this mitochondrial enzyme regulates cellular triglycerides independent of adipogenesis. SCD1 was reduced in cells with low SNTA (Fig. 3F and G) and may contribute to diminished triglyceride levels. To clarify a potential function of SCD1 in lipid droplet formation during adipogenesis SCD1 was knocked down in preadipocytes and cells were subsequently differentiated to mature adipocytes. SCD1 was markedly reduced as expected (Fig. 4A and B). HSL and PPARγ which are upregulated during adipogenesis were normally expressed. Further, low SCD1 did not affect SNTA levels (Fig. 4A and B). Lipid droplet size, cellular triglycerides and cholesterol were not changed upon SCD1 knock-down (Fig. 4C–E). This indicates that reduced SCD1 did not contribute to lower triglycerides and smaller lipid droplets in cells treated with SNTA siRNA.
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Fig. 4. Role of SCD1 in lipid droplet formation of 3T3-L1 cells. A. FAS, HSL, PPARγ, SNTA and SCD1 in cells differentiated from preadipocytes treated with scrambled or SCD1 siRNA. B. Protein densitometry of SNTA and SCD1 in these cells. Values are given as % of control and the dotted line represents 100%. C. Adipocytes differentiated from preadipocytes treated with scrambled or SCD1 siRNA. D. Cellular triglycerides. E. Cholesterol levels of these cells. * p o0.05.
3.5. SNTA knock-down does not affect basal adipocyte functions Adipocytes forming smaller lipid droplets may have a differential response to metabolic stimuli. Insulin mediated phosphorylation of Akt and ERK1/2 and expression of these proteins was nevertheless comparable in both cell types (Fig. 5A). SNTA has been described to regulate lipolysis in COS-7 cells [17]. Epinephrine induced phosphorylation of HSL was normal in the SNTA siRNA treated adipocytes (Fig. 5B). Basal and epinephrine stimulated
fatty acid and glycerol release was comparable in both cell types (Fig. 5C and D). 3.6. SNTA knock-down cells have improved lipid storage Cells with smaller lipid droplets may have an improved capacity to store excess fatty acids. Cellular lipids increased in scrambled and SNTA siRNA treated cells incubated with 200 mM oleate for 24 h (Fig. 5E). Triglycerides tended to be higher (Fig. 5E) and Oil
Fig. 5. Role of SNTA in 3T3-L1 cell function. A. Insulin (20 min incubation time) induced phosphorylation of Akt and ERK1/2 in cells differentiated from preadipocytes treated with scrambled and SNTA siRNA. B. HSL and its phosphorylated form and PPARγ in cells differentiated from preadipocytes transfected with scrambled and SNTA siRNA. Cells were treated with 0, 100 and 500 nM epinephrine for two hours. C. Effect of epinephrine on fatty acids in the supernatants of cells differentiated from preadipocytes treated with scrambled and SNTA siRNA (n¼ 4). D. Effect of epinephrine on glycerol in the supernatants of cells differentiated from preadipocytes treated with scrambled and SNTA siRNA (n ¼ 6). E. Triglycerides in adipocytes differentiated from preadipocytes treated with scrambled and SNTA siRNA and cultivated in the presence of 200 mM oleate for 24 h (n ¼3). F. Oil Red O Staining of the cells described in E (n ¼ 11). G. LDH in the supernatants of cells described in E (n ¼6). * p o 0.05, (*) trend.
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Red O staining (Fig. 5F) was significantly increased in cells with low SNTA. Improved lipid storage was not linked to increased cytotoxicity in cells with low SNTA (Fig. 5G).
4. Discussion The current study demonstrates a role of the adaptor protein SNTA in lipid droplet growth. Differentiation of 3T3-L1 cells with low SNTA hinders the formation of large lipid droplets. This is not a secondary effect of impaired adipogenesis. Proteins like adiponectin, FABP4 and HSL which are induced during adipocyte maturation [22,26] are normally expressed in the adipocytes differentiated from preadipocytes with SNTA knock-down. Small lipid droplets in cells with SNTA knock-down are accompanied by reduced triglyceride storage. SNTA does not regulate proteins involved in fatty acid synthesis, uptake and release. Further, these cells form multiple small lipid droplets excluding that formation of these structures is generally impaired. SNTA reduces β-adrenergic stimulated cAMP production in COS-7 cells indicating that increased lipolysis may contribute to reduced triglyceride storage in 3T3-L1 adipocytes with low SNTA [17]. Basal and epinephrine induced fatty acid and glycerol release is nevertheless normal in adipocytes with low SNTA. This argues against a function of this scaffold protein in adipocyte lipolysis. Further, insulin mediated phosphorylation of Akt and ERK1/2 are unchanged in these cells suggesting normal insulin-induced lipid storage. Lower triglyceride levels may be related to reduced lipid uptake during adipogenesis which has not been analyzed herein. In mature cells cellular pathways involved in lipid homeostasis are nevertheless normal. White adipocytes are mostly completely filled with a single lipid droplet which thereby determines the cell size [32]. Small and large adipocytes within the same fat depot have been shown to exert comparable insulin response but are also found to differ in insulin sensitivity with smaller cells being more sensitive [33,34]. Smaller adipocytes may have a higher capacity to store surplus lipids but may also represent inappropriately differentiated adipocytes [33,35]. Adipocytes with low SNTA display normal insulin sensitivity and have increased fatty acid storage when cells are challenged with oleate. This indicates that improved deposition of fat in these cells may protect from the harmful effects of lipid surplus in obesity. SNTA is an adaptor protein [8] thus indicating that the inability of cells with low SNTA to form large lipid droplets is mediated by SNTA binding proteins. With the exception of MnSOD and SCD1 all of the proteins analyzed are normally expressed in the adipocytes with low SNTA. Reduced MnSOD in 3T3-L1 cells treated with SNTA siRNA is most likely related to lower triglyceride levels [31]. Fatty acids induce MnSOD in adipocytes while knock-down of this mitochondrial enzyme in mature cells has no effect on triglyceride storage [31]. Expression of further mitochondrial proteins and heme oxygenase are normal when SNTA is reduced arguing against impaired mitochondrial biogenesis and increased oxidative stress. SCD1 converts palmitate and stearate into palmitoleate and oleate [36]. SCD1 is reduced in adipocytes with insufficient SNTA. The transcription factor SREBP1c regulates SCD1, FAS and ACC [37]. Expression of the latter two proteins is, however, not changed in SNTA depleted 3T3-L1 cells. This excludes that SREBP1c is involved herein. Further studies are needed to clarify the mechanisms contributing to reduced SCD1 levels in adipocytes with low SNTA. 3T3-L1 preadipocytes treated with an SCD1 inhibitor throughout differentiation have decreased triglycerides [38]. SCD1 downregulation by siRNA has no effect on lipid droplet size and triglyceride levels in our experiments. Current data are in agreement with findings published previously by Jennifer L. Christianson et al.
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[39]. In their work fibroblasts were transfected with SCD1 or SCD2 siRNA and subsequently differentiated to adipocytes. They found no effect of SCD1 siRNA on adipogenesis or lipid loading. SCD2 knock-down markedly reduced adipogenesis and lipid storage. This shows that SNTA seems not to regulate SCD2 levels because adipogenic markers are normal. Adipocyte specific deletion of SCD1 in mice does not protect from obesity [40] further arguing against a role of SCD1 in fat depot growth. Therefore, SCD1 does not regulate adipocyte triglyceride levels and consequently does not contribute to small lipid droplets in 3T3-L1 cells with low SNTA. Current experiments show that SNTA deficiency during adipogenesis results in reduced triglycerides and small lipid droplets in the mature cells. Expression of several proteins regulating lipid droplet size is not changed. SNTA is a scaffold for signaling complexes suggesting that cellular localization and not absolute levels of proteins involved in lipid droplet formation and/or triglyceride synthesis may be changed. We do, however, not have additional evidence to support this conclusion. The SNTA regulated proteins in adipocytes have still to be identified. The so far described SNTA interacting proteins aquaporin 4 and neuronal nitirc oxid synthase are not expressed in adipocytes [41,42], ABCA1 is a further SNTA interacting protein but its levels are normal in adipocytes with SNTA knock-down. This is in agreement with recent findings in the liver and macrophages [20,21]. TAPP1 interacts with SNTA and this complex has a role in the remodeling of the cytoskeleton [43] and insulin response [44]. Normal insulin sensitivity of 3T3-L1 cells with low SNTA argues against a role of TAPP1 herein. Alpha 1 adrenergic receptor levels are regulated by SNTA in HEK293 cells [29] but protein is not changed in 3T3-L1 cells with low SNTA. Recently our group excluded that adiponectin receptor 1 signaling is affected in SNTA deficient mice [45]. SNTA is an actin-binding protein [46]. The actin cytoskeleton is involved in the expansion of lipid droplets and enhanced actin cytoskeletal remodeling promotes lipid accumulation in adipocytes [47]. Whether cytoskeletal remodeling is altered in cells with low SNTA has not been investigated yet. Rac1 regulates cytoskeletal organization and cell proliferation [48]. SNTA activates Rac1 and thereby increases cellular reactive oxygen species and proliferation of breast cancer cell lines [49]. In preadipocytes proliferation is nevertheless induced upon SNTA knock-down and HO-1 which is a sensitive marker of cellular ROS levels [30] is normally expressed in adipocytes with low SNTA. A role for SNTA in adipocyte Rac1 activation is, therefore, unlikely indicating cell-type specific functions of SNTA. Phosphatidylinositol 4,5-bisphosphate and cholesterol can bind to SNTA [50,51], and it may be assumed that association of SNTA with lipids contributes to lipid droplet growth.
5. Conclusion The current investigations demonstrate a function of the adaptor protein SNTA in lipid droplet growth. SNTA does neither affect adipogenesis nor adipocyte function. Importantly, adipocytes with low SNTA are able to more effectively store surplus triglycerides suggesting a protective role under conditions of fatty acid excess.
Acknowledgments The study was supported by a Grant from the German Research Foundation (BU 1141/8-1). We thank Prof. Dr. Charalampos Aslanidis for helpful suggestions.
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