|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Sfrp5 expression and secretion in adipocytes are up-regulated during differentiation and are negatively correlated with insulin resistance
Ci Lv1, Youzhao Jiang1, Hui Wang and Bing Chen2
Department of Endocrinology, Southwest Hospital, Third Military Medical University, Chongqing, 400038, Peoples Republic of China
We have examined the patterns of Sfrp5 (secreted frizzled-related protein 5) mRNA expression and protein secretion during adipocyte differentiation, and investigated the potential role of Sfrp5 in IR (insulin resistance) in adipocytes. 3T3-L1 pre-adipocytes were induced for differentiation, and RT–PCR (reverse transcription–PCR) and ELISA assays were used to determine Sfrp5 mRNA expression and protein secretion. The results showed that with the differentiation and maturity of pre-adipocytes, transcription and protein secretion of Sfrp5 gradually increased, peaking on the 9th day of differentiation. Sfrp5 mRNA expression in mature adipocytes was decreased by 20, 22 and 32 upon treatment with dexamethasone, insulin and TNF (tumour necrosis factor) respectively, whereas Sfrp5 protein secretion was decreased by 15, 17 and 30%, correspondingly. In contrast, Sfrp5 mRNA expression in mature adipose was increased by 34 and 19% upon treatment with rosiglitazone and metformin respectively, whereas Sfrp5 protein secretion was increased by 10 and 6%, correspondingly. In conclusion, Sfrp5 mRNA expression and protein secretion depend on the differentiation of adipocytes. The dysregulation of Sfrp5 expression and secretion is directly correlated with IR. Up-regulation of Sfrp5 expression and secretion in adipocytes may be one crucial mechanism by which rosiglitazone and metformin improve insulin sensitivity.
Key words: adipogenesis, insulin resistance, obese, Sfrp5, 3T3-L1 pre-adipocytes
Abbreviations: Ct, threshold cycle, C/EBP, CCAAT/enhancer-binding protein, FBS, fetal bovine serum, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, IR, insulin resistance, JNK, c-Jun N-terminal kinase, PPARγ, peroxisome proliferator-activated receptor γ, RT–PCR, reverse transcription–PCR, Sfrp5, secreted frizzled-related protein-5, TNF, tumour necrosis factor, Wnt, wingless-type
1These authors have contributed equally to this work.
2To whom correspondence should be addressed (email email@example.com).
Increased incidences of obesity have unfortunately become concurrent with rapid economic development and improved living standards in modern society. Obesity, a common disease of nutritional disturbance, leads to metabolic diseases such as type 2 diabetes mellitus (Grundy et al., 2004). Obesity is also related to impairments in the endocrine functions of adipose tissues, such as low inflammatory conditions (Pajvani et al., 2005; Wellen and Hotamisligil, 2005; Scherer, 2006). The main cause of human obesity is the secretion imbalance of pro- and anti-inflammatory cytokines in adipose tissues (Heilbronn and Campbell, 2008).
Sfrp5 (secreted frizzled-related protein-5), a newly identified anti-inflammatory adipokine, has been shown to inhibit chronic inflammation and consequently improve insulin sensitivity. Sfrp5 expression is abundant in mouse adipocytes, but is decreased in various rodent models, such as obesity and type 2 diabetes mellitus Ouchi et al., 2010). Due to the high homology of CRD (cysteine-rich domain) of SFRPs to that of the Wnt (wingless-type) receptor frizzled, SFRPs could compete with the frizzled receptor to bind Wnt ligand and negatively regulate Wnt pathway (Suzuki et al., 2004; He et al., 2005). The anti-inflammatory function of Sfrp5 is enabled by its combination with Wnt5a expressed in adipose tissues, thereby inhibiting the activation of the downstream target JNK (c-Jun N-terminal kinase) of non-canonical Wnt signalling. Consequently, the secretion of pro-inflammatory cytokines is limited, and the phosphorylation of insulin receptor substrate-1 at Ser307 is antagonized (Hotamisligil, 2006; Ouchi et al., 2010). Adipocyte differentiation is closely related to glucose and lipid metabolism and play role in IR (insulin resistance), type 2 diabetes mellitus and hyperlipoidemia (Cho et al., 2004). Therefore, changes in Sfrp5 expression and secretion during the dynamic process of adipocyte differentiation need to be evaluated.
Rosiglitazone and metformin are the common drugs used for the treatment of type 2 diabetes mellitus in the clinic. However, it remains unclear whether these drugs improve insulin sensitivity by regulating the expression and function of Sfrp5. We have employed 3T3-L1 pre-adipocytes as a cell model of IR and investigated Sfrp5 expression and secretion during adipocyte differentiation. We also investigated the effects of rosiglitazone and metformin on Sfrp5 mRNA expression and protein secretion in differentiated adipocytes.
2. Materials and methods
2.1. Cell culture and differentiation of 3T3-L1 pre-adipocytes
3T3-L1 pre-adipocytes were purchased from Institute of Biochemical and Cell Biology, Shanghai branch of the Chinese Academy of Sciences. The cells were cultured and differentiated in accordance with the standard procedures (Kajimoto et al., 2005). Briefly, the cells were maintained in DMEM (Dulbecco's modified Eagle's medium; Thermo Scientific) supplemented with 10% FBS (fetal bovine serum) and 100 units/ml penicillin/streptomycin at 37°C (5% CO2, 95% air). The medium was changed every 2 days. Two days post-confluence (referred to as day 0), the cells were incubated with 10 mg/l insulin, 0.5 mM isobutylmethyl-xanthine and 1 mM dexamethasone. On day 2, the media were removed and fresh media supplemented with only insulin was added. On day 4, the media were removed and fresh media with 10% FBS, but with no additional hormones, were added. The media were changed every 2–3 days until the cells were collected. Cells were collected at different intervals (precursor, −2, 0, 3, 5, 6, and 9 days). For hormone treatments, 3T3-L1 adipocytes (used on day 6 of differentiation) were used. Cells were treated with an overnight incubation (24 h) with the indicated concentrations of treatment conditions. Cells were stored at −80°C until use.
2.2. Oil Red O dye staining
Cells (6 days) were collected and washed twice in PBS, fixed in 4% formaldehyde for 20 min, and washed twice in water. Oil Red O dye was dissolved in dimethyl carbinol and filtered. The cells were stained with the dye for 1 h, and washed with water until the background became transparent. Microscopic observations revealed that the tenuigenin overwhelmed the red. Images of the differentiation processes were recorded.
2.3. Real-time PCR
Total RNA was extracted from the cells using TRIzol® reagent (Invitrogen). cDNA was generated using the PrimeScriptTM RT reagent kit (Perfect Real Time, TaKaRa). The primers for Sfrp5 were 5-CTGTGCCTTGCCTCGCCTCTGT-3 and 5-GTAGGTGCGTGAAGCCATCC-3. The primers for GADPH were 5-GGGAAACTGTGGCGTGAT-3 and 5-AAAGGTGGAGGAGTGGGT-3. RT–PCR (reverse transcription–PCR) was done with an Ultra SYBR Mixture kit (with ROX). The signal and Ct (threshold cycle) value were examined using the iCycler software (Bio-Rad). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as internal control and relative level of Sfrp5 mRNA was calculated by 2−ΔΔ Ct.
Sfrp5 content in the conditioned medium was determined using ELISA kit (Shanghai Hushang). The average value was calculated as the final concentration, and the differentiation within the series was <5%.
2.5. Statistical analysis
SPSS 17 statistical software was used for statistical analyses. All results were expressed as means±S.E.M. One-way ANOVA was used to compare the averages of the various groups. P<0.05 was considered as significant.
3.1. Morphology of pre-adipocyte changes during the differentiation
We examined the morphology of the 3T3-L1 pre-adipocytes during the differentiation. Before differentiation, there were no red particles in the cells, and the cells outgrew the fibre cell state (Figure 1A). After culture in differentiation medium, the cells changed from their original shape of fusiform into circular forms. The sizes of the cells also dramatically grew. Red liquid droplets stained with Oil Red O increased (Figure 1B). On the 6th day, 70% of the cells differentiated into mature adipocytes. On the 9th day, the Oil Red liquid droplets accounted for >95% of the total mature adipocytes.
3.2. Changes in Sfrp5 mRNA expression and protein secretion during 3T3-L1 cells differentiation
Using RT–PCR and ELISA, changes in Sfrp5 expression and secretion were examined during the differentiation of 3T3-L1 cells from pre-adipocytes to adipocytes. Sfrp5 mRNA expression and protein secretion were not detected in 3T3-L1 pre-adipocytes. However, as the pre-adipocytes matured and differentiated, Sfrp5 mRNA transcription level gradually increased and peaked on the 9th day (10.02±0.28, P<0.01), except on the 5th day (Figure 2A). Similar pattern of changes was observed for Sfrp5 protein secretion in the conditioned medium of 3T3-L1 cells at different differentiated state (Figure 2B).
3.3. IR factors inhibit Sfrp5 mRNA expression and protein secretion in mature adipocytes
Sfrp5 mRNA expression in the mature adipocytes decreased by 20% (P<0.01), 22% (P<0.01), and 32% (P<0.01) after treatment with 1 μM dexamethasone, 100 nM insulin, and 10 ng/l TNF (tumour necrosis factor) respectively (Figure 3A). Sfrp5 protein secretion into conditioned medium of mature adipocytes was decreased by 15% (P<0.05), 17% (P<0.01) and 30% (P<0.01) after treatment with the corresponding agents (Figure 3B).
3.4. Rosiglitazone and metformin promote Sfrp5 mRNA expression and protein secretion in mature adipocytes
We finally examined the effects of rosiglitazone and metformin on Sfrp5 mRNA expression and protein secretion in mature adipocytes. RT–PCR analysis showed that Sfrp5 mRNA expression in the mature adipocytes decreased by 34% (P<0.01), and 19% (P<0.01) after treatment with 10 μM rosiglitazone and 1 mM metformin respectively (Figure 4A). ELISA analysis showed that Sfrp5 protein secretion into conditioned medium of mature adipocytes decreased by 10% (P<0.01) and 6% (P<0.05) after treatment with 10 μM rosiglitazone and 1 mM metformin respectively (Figure 4B).
We have shown that Sfrp5 mRNA expression and protein secretion were increased during the differentiation of 3T3-L1 pre-adipocytes. Sfrp5 mRNA was not detected in 3T3-L1 pre-adipocytes. However, as precursor adipocytes mature and differentiate, mRNA expression level of Sfrp5 tended to increase, and peaked on the 9th day. Sfrp5 mRNA expression increased at the start of the differentiation, whereas Sfrp5 protein secretion was not detected until the 2nd and 3rd day of differentiation.
One of the important metabolic adaptation for adipose tissues with energy overdose is the differentiation of precursor adipocytes into mature adipocytes. This process supports the overall energy storage capability in the body. During the adipogenesis, precursor cells initially undergo clonal expansion, followed by growth arrest and differentiation under the control of an array of sequentially induced transcription factors, such as the C/EBPs (CCAAT/enhancer-binding proteins) and PPARγ (peroxisome proliferator-activated receptor γ) (Ailhaud et al., 1992; Prusty et al., 2002). Interestingly, a recent study showed that the activation of Wnt signalling interfered with the normal differentiation of adipocytes via C/EBP resistance (Gustafson and Smith, 2006). Emerging evidence suggest the role of canonical Wnt signalling in adipogenesis and Wnts have been proposed as adipokines (Schinner et al., 2009). Based on our data on Sfrp5 expression during the differentiation, we propose that Sfrp5 functions to promote adipocyte differentiation by antagonizing canonical Wnt signalling. This speculation is supported by recent studies showing that shikonin and isorhamnetin inhibited the adipogenesis of 3T3-L1 pre-adipocytes by the activation of the WNT/β-catenin pathway (Lee et al., 2010, 2011).
To test the influence of IR on the expression and secretion of Sfrp5 in mature adipocytes, we treated 3T3-L1 adipocytes on day 6 of the differentiation with 1 μM dexamethasone, 100 nM insulin and 10 ng/l TNFα. The results showed that both Sfrp5 mRNA expression and protein secretion were decreased in the IR cell model.
TNFα is an adipocytokine generated by macrophages and adipocytes. TNFα promotes IR, and the circulating level of TNFα was elevated in obese subjects (Kern et al., 2001; Maury et al., 2009). In addition, TNFα could activate Wnt signalling to induce inflammation and terminate adipocyte differentiation (Gustafson et al., 2006). Our work shows that TNFα could decrease Sfrp5 gene expression and protein secretion in 3T3-L1 adipocytes. Together, the data suggest that TNFα stimulates Wnt signalling and inhibit adipocyte differentiation via the down-regulation of Sfrp5, a Wnt antagonist.
We have also shown that PPRP-r (phosphoribosylpyrophosphate) activators rosiglitazone and metformin could promote Sfrp5 mRNA expression and protein secretion in mature 3T3-L1 adipocytes. Rosiglitazone belongs to the group of thiazolidinediones. By activating PPARγ, rosiglitazone induces the expression of many proteins that control glucose and lipid metabolism, leading to decreased IR of peripheral tissues and increased glucose utilization of peripheral tissues. Consequently, glucose and lipid metabolism in type 2 diabetes mellitus patients are improved (Wagstaff et al., 2002; Boden et al., 2003). Rosiglitazone not only intensifies pre-adipocyte differentiation but also promotes the terminal differentiation of mature adipocytes by increasing β-catenin degradation and inhibiting Wnt signalling (Hammarstedt et al., 2007). In this aspect, our findings of the up-regulation of Sfrp5 expression and secretion by rosiglitazone provide new clues on the molecular mechanisms by which rosiglitazone inhibits Wnt signalling and promotes adipocytes to differentiate and mature.
Metformin is the most extensively used hypoglycaemic agent, and is effective in the treatment of type 2 diabetes mellitus. It inhibits liver gluconeogenesis and decreases the hepatic glucose output to promote the glucose uptake and utilization in skeletal muscles, fats and other tissues (Hammarstedt et al., 2007; He et al., 2009; Yoshida et al., 2009). Consequently, oxidative stress in adipose tissues is ameliorated, leading to increased liver and muscle insulin sensitivity and improved symptoms of diabetic mellitus (Anedda et al., 2008). Metformin can inhibit Wnt/β-catenin signalling by the activation of AMPK (AMP-activated protein kinase), providing molecular explanation for its action (Takatani et al., 2011). Our data showing the up-regulation of Sfrp5 by metformin suggest another important mechanism responsible for the inhibition of Wnt signalling by metformin.
Non-canonical Wnt signalling includes PCP (planar cell polarity) signalling and Wnt/calcium signalling, which are crucially involved in the regulation of cancer progression and inflammation (Pereira et al., 2008; Wang, 2009). Sfrp5 expressed in macrophages and adipocytes could bind non-canonical Wnt ligand Wnt5a in adipose tissues via paracrine and autocrine mechanisms (Solinas et al., 2007; Vallerie et al., 2008). Furthermore, JNK, the downstream target of non-canonical Wnt signalling, is a crucial mediator of obesity and IR (Hirosumi et al., 2002). Consequently, Sfrp5 could inhibit the activation of JNK and decrease the secretion of pro-inflammatory adipokines such as TNFα, IL-6 (interleukin 6) and MCP-1 (monocyte chemoattractant protein 1), leading to decreased IR (Ouchi et al., 2010). Therefore the regulatory axis of Sfrp5–Wnt in the adipose tissue could be exploited as the potential target to control obesity-linked abnormalities. Given our data showing that rosiglitazone and metformin up-regulated Sfrp5 expression and protein secretion in differentiated adipocytes, we speculate that these agents help sequester non-canonical Wnt ligands and restrain the chronic inflammatory state, contributing to improved insulin sensitivity.
In conclusion, Sfrp5 mRNA expression and protein secretion depend on the differentiation of adipocytes. Up-regulation of Sfrp5 expression and secretion in adipocytes is one crucial mechanism by which rosiglitazone and metformin improve IR. Sfrp5, an important anti-inflammatory adipokine, may play a protective role in obesity and related metabolic disorders. Further characterization of the mechanisms of Sfrp5 action will help reveal new therapeutic targets for obesity.
Bing Chen and Ci Lv conceived and designed the experiments. Ci Lv and Hui Wang performed the experiments. Ci Lv and Youzhao Jiang analysed the data. Ci Lv contributed reagents/materials/analysis tools. Ci Lv and Youzhao Jiang wrote the paper.
We thank the workers in the Southwest Hospital Experiment Center, Third Military Medical University.
This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Boden, G, Cheung, P, Mozzoli, M and Fried, SK (2003) Effect of thiazolidinediones on glucose and fatty acid metabolism in patients with type 2 diabetes. Metabolism 52, 753-9
Grundy, SM, Brewer, HB Jr, Cleeman, JI, Smith, SC Jr and Lenfant, C (2004) Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Arterioscler Thromb Vasc Biol 24, e13-8
Gustafson, B and Smith, U (2006) Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes. J Biol Chem 281, 9507-16
Hammarstedt, A, Isakson, P, Gustafson, B and Smith, U (2007) Wnt-signaling is maintained and adipogenesis inhibited by TNFα but not MCP-1 and resistin. Biochem Biophysl Res Commun 357, 700-6
He, B, Lee, AY, Dadfarmay, S, You, L, Xu, Z and Reguart, N (2005) Secreted frizzled-related protein 4 is silenced by hypermethylation and induces apoptosis in β-catenin-deficient human mesothelioma cells. Cancer Res 65, 743-8
He, L, Sabet, A, Djedjos, S, Miller, R, Sun, X and Hussain, MA (2009) Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein. Cell 137, 635-46
Kajimoto, K, Terada, H, Baba, Y and Shinohara, Y (2005) Essential role of citrate export from mitochondria at early differentiation stage of 3T3-L1 cells for their effective differentiation into fat cells, as revealed by studies using specific inhibitors of mitochondrial di-and tricarboxylate carriers. Mol Genet Metab 85, 46-53
Kern, PA, Ranganathan, S, Li, C, Wood, L and Ranganathan, G (2001) Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance. AM J Physiol Endocrinol Metab 280, E745-51
Lee, J, Lee, J, Jung, E, Hwang, W, Kim, YS and Park, D (2010) Isorhamnetin-induced anti-adipogenesis is mediated by stabilization of β-catenin protein. Life Sci 86, 416-23
Maury, E, Noel, L, Detry, R and Brichard, SM (2009) In vitro hyper-responsiveness to TNFα contributes to adipokine dysregulation in omental adipocytes of obese subjects. J Clin Endocrinol Metab 94, 1393-400
Ouchi, N, Higuchi, A, Ohashi, K, Oshima, Y, Gokce, N and Shibata, R (2010) Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity. Science 329, 454-7
Pajvani, UB, Trujillo, ME, Combs, TP, Iyengar, P, Jelicks, L and Roth, KA (2005) Fat apoptosis through targeted activation of caspase 8: a new mouse model of inducible and reversible lipoatrophy. Nat Med 11, 797-803
Pereira, C, Schaer, DJ, Bachli, EB, Kurrer, MO and Schoedon, G (2008) Wnt5A/CaMKII signaling contributes to the inflammatory response of macrophages and is a target for the antiinflammatory action of activated protein C and interleukin-10. Arterioscler Thromb Vasc Biol 28, 504-10
Prusty, D, Park, BH, Davis, KE and Farmer, SR (2002) Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor γ (PPARγ) and C/EBP alpha gene expression during the differentiation of 3T3-L1 preadipocytes. Biol Chem 277, 226-32
Solinas, G, Vilcu, C, Neels, JG, Bandyopadhyay, GK, Luo, JL and Naugler, W (2007) JNK1 in hematopoietically derived cells contributes to diet-induced inflammation and insulin resistance without affecting obesity. Cell Metab 6, 386-97
Suzuki, H, Watkins, DN, Jair, KW, Schuebel, KE, Markowitz, SD and Chen, WD (2004) Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat Genet 36, 417-22
Takatani, T, Minagawa, M, Takatani, R, Kinoshita, K and Kohno, Y (2011) AMP-activated protein kinase attenuates Wnt/β-catenin signaling in human ostesblastic Saos-2 cells. Mol Cell Endocrinol 339, 114-9
Vallerie, SN, Furuhashi, M, Fucho, R and Hotamisligil, GS (2008) A predominant role for parenchymal c-Jun amino terminal kinase (JNK) in the regulation of systemic insulin sensitivity. PLoS One 3, e3151
Yoshida, T, Okuno, A, Tanaka, J, Takahashi, K, Nakashima, R and Kanda, S (2009) Metformin primarily decreases plasma glucose not by gluconeogenesis suppression but by activating glucose utilization in a non-obese type 2 diabetes Goto-Kakizaki rats. Eur J Pharmacol 62 3, 141-7
Received 3 February 2012/17 April 2012; accepted 14 May 2012
Published as Cell Biology International Immediate Publication 14 May 2012, doi:10.1042/CBI20120054
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 2 Increased Sfrp5 expression and secretion during the differentiation of 3T3-L1 pre-adipocytes to adipocytes