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Secreted frizzled-related protein 4 (SFRP4) and fractalkine (CX3CL1) — Potential new biomarkers for β-cell dysfunction and diabetes
Clinical Biochemistry 47 (2014) 529–532
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Review
Secreted frizzled-related protein 4 (SFRP4) and fractalkine (CX3CL1) — Potential new biomarkers for β-cell dysfunction and diabetes Katarzyna Bergmann ⁎, Grazyna Sypniewska Department of Laboratory Medicine, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz, Poland
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 11 December 2013 Received in revised form 3 March 2014 Accepted 6 March 2014 Available online 24 March 2014
The discovery of new risk factors for diabetes is a major challenge for contemporary science. Pathogenesis of type 2 diabetes mellitus (T2DM) is closely related to adipose tissue dysfunction. The aim of this review was to describe recently discovered cytokines: fractalkine (CX3CL1, FKN) and secreted frizzled-related protein 4 (SFRP4) as potential biomarkers of early β cell dysfunction and diabetes. The association of CX3CL1 and SFRP4 with lowgrade inflammation in adipose tissue links obesity with disturbances in insulin secretion and impaired glucose metabolism, therefore it indicates new therapeutic and preventive targets in both healthy and diabetic subjects. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Keywords: Diabetes Insulin resistance Adipocytokines Secreted frizzled-related protein 4 Fractalkine
Contents Introduction . . . . . . . . . . . . . . Fractalkine (CX3CL1, FKN) . . . . . . . Secreted frizzled-related protein 4 (SFRP4) Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . .
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Introduction Diabetes mellitus and its cardiovascular complications are one of the leading causes of disability and deaths worldwide. According to the International Diabetes Federation (IDF) incidence of diabetes, especially type 2 (T2DM), will rise from 366 million in 2011 to 552 million by 2030, which means that the disease will affect one out of ten adults [1]. In Europe at least 131 billion dollars per year is spent on healthcare due to diabetes [2], which indicates that it is not only an important health, but also a socio-economic problem. Therefore the need for prevention as well as the discovery of new risk prediction factors for diabetes is a major challenge for contemporary laboratory medicine. Pathogenesis of T2DM is closely linked to adipose tissue dysfunction. Excessive body fat acts as an endocrine organ in which many adverse biochemical mechanisms occur. Cytokines produced by adipocytes such as ⁎ Corresponding author at: Department of Laboratory Medicine, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, M. Sklodowskiej-Curie 9, 85-094 Bydgoszcz, Poland. E-mail address: [email protected] (K. Bergmann).
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interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), resistin, retinol binding protein 4 (RBP-4), dipeptidyl peptidase 4 (DPP-4) and adipocyte fatty acid-binding protein (A-FABP) activate inflammation pathways, which decrease the quantity/activity of the insulin-dependent receptors, and affect gene expression and insulin production in human pancreatic islets, leading to insulin resistance and impaired glucose metabolism [3]. By the fact that elevated levels of various chemokines may predict the occurrence of disease even several years before its diagnosis they are considered to be potential risk factors in both apparently healthy, nondiabetic and overweight/obese subjects. Recent studies focused on newly discovered chemokines: fractalkine (CX3CL1, FKN) and secreted frizzled-related protein 4 (SFRP4) as promising biomarkers of early pancreatic β-cell dysfunction, however their diagnostic and clinical utility/relevance is not confirmed yet. Fractalkine (CX3CL1, FKN) Fractalkine is the only member of the CX3C chemokine subclass composed of 373 amino acids. It is synthesized as a transmembrane
http://dx.doi.org/10.1016/j.clinbiochem.2014.03.007 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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K. Bergmann, G. Sypniewska / Clinical Biochemistry 47 (2014) 529–532
molecule consisted of a soluble form — chemokine domain and the extracellular mucin-like stalk and the membrane-bound form (Fig. 1). The soluble CX3CL1 is generated by disintegrin-like metalloproteinases (ADAM) 10 and 17/TACE and has the chemoattractive activity for monocytes, natural killer (NK) cells, and T cells [4]. Membrane-bound form is produced in endothelial and epithelial cells, vascular smooth muscle cells, keratinocytes, dendritic cells, and neurons after stimulation by proinflammatory factors, such as TNF-α, interferon γ (IFN-γ), and IL-1, and supports integrin-independent leukocyte adhesion [5]. The presence of mRNA for FKN has been showed in various organs such as heart, kidneys, liver, adrenal gland and brain. The activity of CX3CL1 is related to its receptor called CX3CR1. This receptor occurs on the surface of monocytes, macrophages, NK cells, some T cells, neutrophils, mast cells, platelets, smooth muscle cells, and dendritic and microglia cells [4]. The discovery of fractalkine changed the existing opinions upon cell adhesion and migration through the endothelium. According to the classical pathway migration of leukocytes is dependent on the selectinmediated interactions between leukocytes and the endothelium and activation of integrins on leukocytes by chemokines presented on glycosaminoglycans. Therefore it was claimed that all chemokines are produced as soluble molecules associated with proteoglycans on the cell surface and tissue matrix. In contrast, membrane-bound form of FKN has a chemokine domain on the top of the mucin-like stalk, which acts as an adhesion molecule and is involved in all stages of the migration, while the soluble form shows strong chemotactic effect [6]. Leukocytes with CX3CR1 receptor connect selectively with a chemokine domain presented on endothelial cells which triggers rapid binding. Interaction between CX3CL1 and its receptor additionally can increase expression of integrins, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in leukocytes, which may result in stronger adhesion. Expression of fractalkine during the inflammation can also attract and activate NK cells, which are responsible for cytotoxic effect and cytolysis, therefore its contribution to inflammationdependent conditions seems to be very important [5]. Several studies confirmed the role of fractalkine in the pathogenesis of many human diseases, such as atherosclerosis and cardiovascular disease, rheumatoid arthritis, HIV and HCV infections and cancer in which activity of inflammatory factors is significant [7–10]. Type 2
diabetes is strictly associated with obesity and related to adipose tissue inflammation. In this process a recruitment of monocytes to fatty tissue plays a key role and initiates the first local production of cytokines and then leads to systemic inflammation and insulin resistance. FKN with its dual properties in the mechanism of leukocyte chemotaxis and adhesion might be a relevant pathogenetic factor in early adipocyte dysfunction and T2DM [11], although the data in this area are scarce. Study by Shah et al. [12] clearly confirmed that CX3CL1 is expressed and secreted by human adipocytes and stromal vascular cells, hence it can be considered as adipocytokine. This well-designed study was performed based on in vivo endotoxemia and adipose tissue biopsies in lean and obese subjects, in vitro study of primary human adipocytes and monocytes and case–control study performed in individuals with and without T2DM. Moderate dose (3 ng/kg) of endotoxin (standard lipopolysaccharide, LPS) increased significantly both adipose and blood FKN (33-fold, p b 0.001 and over 40-fold, p = 0.006 after 4 h, respectively). Subcutaneous adipose tissue levels of CX3CL1 were higher in obese compared with lean subjects (mean 0.420 vs. 0.228 ng/mL; p = 0.04) and in obese individuals, these levels were higher in visceral than in subcutaneous adipose tissue (mean 0.736 vs. 0.420 ng/mL; p = 0.01). Stimulation of CX3CL1 mRNA expression and protein secretion from adipocytes in vitro by LPS, TNF-α and IFN-γ was observed. Moreover, the use of CX3CL1 blocking antibody reduced monocyte adhesion to adipocytes by almost 50% (p b 0.001). Plasma FKN was significantly lower in non-diabetic subjects compared to T2DM patients (mean 0.422 vs. 0.506 ng/mL; p b 0.0001). A significant 2.77 odds ratio for diabetes for a 1 SD (0.21 ng/mL) increase in CX3CL1 concentration after adjusting for gender, BMI, ethnicity and other metabolic risk factors was indicated. The effect of controlled low-dose endotoxemia on fractalkine expression in human adipocytes in vitro was also observed by Mehta et al. [13]. In this study CX3CL1 mRNA increased 15-fold (p b 0.001) 4 h after 0.6 ng/kg LPS administration and it was the largest increase compared to the other cytokines (IL-6, TNF-α and MCP-1). Analysis of the impact of endotoxemia on impaired insulin activity showed 32% increase in HOMA-IR (p b 0.01) and 21% decrease in insulin sensitivity (p b 0.05) in LPS-treated subjects compared to placebo. Presented results broadly highlight the influence of adipose tissue inflammation including fractalkine-mediated pathway on T2DM development,
Fig. 1. Fractalkine (CX3CL1) structure.
K. Bergmann, G. Sypniewska / Clinical Biochemistry 47 (2014) 529–532
however do not explain the effect of FKN on β-cell function. In contrast, recent study by Lee et al. [14] demonstrates that fractalkine/CX3CR1 system is a regulatory mechanism for pancreatic islet β-cell function which improves insulin secretion and glucose uptake in vitro in mouse and human islets. These properties might be associated with three pathways: increased expression of Akt-1 kinase and its anti-apoptotic effect, suppression of inducible cAMP early repressor (ICER-1) and maintenance of β-cell gene expression, increased intracellular Ca2+ and stimulated insulin secretion. Expression of FKN in islets was decreased by high-fat diet, obesity and aging, therefore authors suggest that lower levels of CX3CL1/CX3CR1 may contribute to β-cell dysfunction in T2DM and emphasize its potential role as a new therapeutic target. However, these findings should be considered with caution if compared with data of Wan and Evans [15], which showed suppression of FKN expression and CX3CL1/CX3CR1 signaling by rosiglitazone, a drug for insulin resistance and type 2 diabetes. Interestingly, rosiglitazone acts by activation of an anti-inflammatory agent, peroxisome proliferator-activated receptor γ (PPAR-γ) and inhibits FKN-dependent migration and adhesion of macrophages to endothelial cells, which also may prevent the cardiovascular complications in diabetic patients. Not without significance several studies revealed increased fractalkine levels in T2DM subjects and its involvement in atherosclerosis and vascular disorders [16,17], as well as the fact that high glucose conditions might induce upregulation of fractalkine and monocyte chemotactic protein-1 [18] and that insulin has a well established anti-inflammatory activity [19]. Thus, it can be concluded that initial increase of insulin secretion by CX3CL1/CX3CR1 may not be a beneficial action, but a defensive reaction to inflammation, which can lead to chronic hyperinsulinemia and depletion of functional reserves of β-cells.
islets. A number of studies demonstrated that SFRP4 is a Wnt antagonist and inhibits Wnt signaling pathway, for example in human cancer cells and adipocytes [26,27]. Several studies exhibit different properties of SFRP4 in both, physiological and pathological states. It was shown to play a role in inhibition of angiogenesis, tumor suppression, promotion of epidermal differentiation, development of polycystic kidney disease and preeclampsia [28–32]. However, it should be noted that some authors suggest that activation of Wnt signaling in pancreatic cells is one of the major pathogenic factors in T2DM [33]. Enhanced Wnt signaling might play an adaptive role, for example, in promoting β-cell proliferation in the early stages of diabetes. On the other hand chronic pathway activation stimulates cell apoptosis and reduces insulin secretion. Therefore, increased expression of SFRP4 in β cells of diabetic subjects can be considered as a result of impaired Wnt signaling. Despite this interesting hypothesis, the role of SFRP4 in obesity and T2DM has not been sufficiently understood yet. Expression of SFRP4 mRNA and secretion of this protein from visceral adipose tissue are increased in obesity and correlate with decreased insulin secretion [34]. Taneera et al. [25] observed significant inverse correlation of SFRP4 expression in human pancreatic islets with insulin secretion (R = −0.28; p = 0.03) and positive relationship with glycated hemoglobin (HbA1c) levels (R = 0.45;
Secreted frizzled-related protein 4 (SFRP4) Secreted frizzled-related proteins (SFRPs) are a family of proteins which act as modulators of Wnt signaling. Wnt signaling is involved in many crucial mechanisms, like cell proliferation and migration. Several Wnt pathway components are associated with lipid and glucose metabolism, therefore they might play an important role in the pathogenesis of metabolic disorders. The relationship between Wnt signaling disturbances and type 2 diabetes is associated with adipogenesis and insulin signaling. Wnt has the ability to maintain preadipocytes in an undifferentiated state through inhibition of the pro-adipogenic factors [20]. Studies on animals showed 50% reduction of adipose mass in overexpression of Wnt-10b, hence Wnt signaling pathway negatively regulates adipogenesis [21]. Interestingly, in physiological state mature adipocytes produce Wnt ligands, like secreted frizzled-related protein 5 (SFRP5), which has anti-inflammatory and insulin-sensitizing properties [22]. In obesity and type 2 diabetes a low-grade inflammation in adipose tissue due to increased macrophage infiltration disturbs a proper Wnt action. In lean subjects the number of macrophages in adipose tissue is low. Production of Wnt antagonists, such as Wnt5a, by macrophages is negligible and additionally their activity is neutralized by SFRP5 produced by adipocytes. On the contrary, in obesity a large number of macrophages synthesize increased levels of Wnt5a and large mature adipocytes produce less SFRP5. Inflammation together with Wnt signaling impairment induces insulin resistance in adipocytes via c-Jun N-terminal kinase (JNK) interfering with insulin receptor substrate (IRS-1) [23]. In vitro study by Abiola et al. showed that activation of Wnt signaling increases insulin sensitivity in skeletal muscles due to inhibition of accumulation of intramyocellular lipids [24]. Secreted frizzled-related protein 4 (SFRP4) is a recently discovered cytokine which contains a cysteine-rich domain homologous to the putative Wnt-binding site of frizzled proteins — G protein-coupled receptor proteins related to Wnt signaling pathway [25]. Its expression is observed in several cells and tissues, including adipocytes, myocardium, endometrium, kidney and ovary, however its association with β cell dysfunction in diabetes is closely related to expression in pancreatic
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Fig. 2. Potential role of SFRP4 in insulin resistance, according to Mahdi et al. [35].
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p = 0.001). They also reported the previous independent results which showed that in human pancreatic islets recombinant SFRP4 inhibits in vitro insulin secretion by 30% and cell exocytosis by 50%, as well as that SFRP4 overexpression is associated with the occurrence of proinflammatory factors. Recently Mahdi et al. [35] noted that SFRP4 expression links inflammation and defective insulin secretion, therefore it indicates a novel purpose for SFRP4 investigation as a biomarker of the pancreatic islet dysfunction in T2DM. In this study, high overexpression of SFRP4 in islets of diabetic subjects and its relationship with inflammatory markers, particularly IL-1β stimulating its production, were observed. Reduced insulin secretion was explained by decreased expression of Ca2+ channels in the islets' cells and suppression of insulin exocytosis (Fig. 2). Serum SFRP4 concentration was significantly correlated with fasting glucose (β = 0.142; p = 0.004), reduced insulin sensitivity index (β = −0.176; p = 0.002) and lower disposition index (insulin secretion adjusted for insulin sensitivity; β = − 0.186; p = 0.029) in non-diabetic subjects and it was not affected by BMI, sex and patient age. Interestingly, the authors concluded that SFRP4 is elevated in the serum several years before clinical diagnosis of diabetes has been made and its presence increases the risk of diabetes up to three-fold. Therefore, SFRP4 might be used as an early risk predictor, especially in apparently healthy individuals. Conclusions Relationship between low-grade inflammation mediated by adipose tissue and metabolic disorders has been well understood in recent years, however the discoveries of new potential biomarkers and pathways are still an open question for researchers. Presented cytokines, secreted frizzled-related protein 4 and fractalkine, should meet general requirements for biomarkers prior to their application in routine clinical practice [36]. Firstly, there is a need for a standardized assay (preferably adapted for automatic analyzers) with proven analytical performance, instead of manual ELISA tests. Secondly, clinical performance and effectiveness evaluation are necessary in order to confirm their diagnostic/ therapeutic goals and prognostic value in assessing the risk of T2DM. Another important aspect is also a cost-effectiveness analysis which compares the changes in costs and in health effects of introducing a new test. Despite the presented promising results on the contribution of secreted frizzled-related protein 4 and fractalkine in the pathogenesis of T2DM, more detailed large population-based studies to evaluate their clinical and diagnostic utility are required. They may provide an opportunity for the development of new therapeutic targets. References [1] International Diabetes Federation (IDF). The global burden — diabetes. http://www. idf.org/diabetesatlas/5e/diabetes . [9.08.2013]. [2] International Diabetes Federation (IDF). Diabetes atlas. http://www.idf.org/ diabetesatlas/5e/europe?language=zh-hans . [9.03.2013]. [3] Bergmann K, Sypniewska G. Diabetes as a complication of adipose tissue dysfunction. Is there a role for potential new biomarkers? Clin Chem Lab Med 2013;51(1):177–85. [4] Jones BA, Beamer M, Ahmed S. Fractalkine/CX3CL1: a potential new target for inflammatory diseases. Mol Interv 2010;10(5):263–70. [5] Owlasiuk P, Zajkowska JM, Pietruczuk M, Pancewicz SA, Hermanowska-Szpakowicz T. Fractalkine — structure, functions and biological activity. Pol Merkur Lekarski 2009;26(153):253–7. [6] Umehara H, Bloom ET, Okazaki T, Nagano Y, Yoshie O, Imai T. Fractalkine in vascular biology: from basic research to clinical disease. Arterioscler Thromb Vasc Biol 2004;24(1):34–40. [7] Stolla M, Pelisek J, von Brühl ML, Schäfer A, Barocke V, Heider P, et al. Fractalkine is expressed in early and advanced atherosclerotic lesions and supports monocyte recruitment via CX3CR1. PLoS One 2012;7(8):e43572.
[8] Jones B, Koch AE, Ahmed S. Pathological role of fractalkine/CX3CL1 in rheumatic diseases: a unique chemokine with multiple functions. Front Immunol 2012;2:82. [9] García-Álvarez M, Berenguer J, Guzmán-Fulgencio M, Micheloud D, Catalán P, Muñoz-Fernandez MA, et al. High plasma fractalkine (CX3CL1) levels are associated with severe liver disease in HIV/HCV co-infected patients with HCV genotype 1. Cytokine 2011;54(3):244–8. [10] Tardáguila M, Mira E, García-Cabezas MA, Feijoo AM, Quintela-Fandino M, Azcoitia I, et al. CX3CL1 promotes breast cancer via transactivation of the EGF pathway. Cancer Res 2013;73(14):4461–73. [11] Cefalu WT. Fractalkine: a cellular link between adipose tissue inflammation and vascular pathologies. Diabetes 2011;60(5):1380–2. [12] Shah R, Hinkle CC, Ferguson JF, Mehta NN, Li M, Qu L, et al. Fractalkine is a novel human adipochemokine associated with type 2 diabetes. Diabetes 2011;60(5):1512–8. [13] Mehta NN, Heffron SP, Patel PN, Ferguson J, Shah RD, Hinkle CC, et al. A human model of inflammatory cardio-metabolic dysfunction; a double blind placebocontrolled crossover trial. J Transl Med 2012;10:124. [14] Lee YS, Morinaga H, Kim JJ, Lagakos W, Taylor S, Keshwani M, et al. The fractalkine/ CX3CR1 system regulates β cell function and insulin secretion. Cell 2013;153(2):413–25. [15] Wan Y, Evans RM. Rosiglitazone activation of PPAR-gamma suppresses fractalkine signaling. J Mol Endocrinol 2010;44(2):135–42. [16] Wong BW, Wong D, McManus BM. Characterization of fractalkine (CX3CL1) and CX3CR1 in human coronary arteries with native atherosclerosis, diabetes mellitus, and transplant vascular disease. Cardiovasc Pathol 2002;11(6):332–8. [17] Yao K, Lu H, Huang R, Zhang S, Hong X, Shi H, et al. Changes of dendritic cells and fractalkine in type 2 diabetic patients with unstable angina pectoris: a preliminary report. Cardiovasc Diabetol 2011;10:50. [18] Dragomir E, Manduteanu I, Calin M, Gan AM, Stan D, Koenen RR, et al. High glucose conditions induce upregulation of fractalkine and monocyte chemotactic protein-1 in human smooth muscle cells. Thromb Haemost 2008;100(6):1155–65. [19] Hyun E, Ramachandran R, Hollenberg MD, Vergnolle N. Mechanisms behind the anti-inflammatory actions of insulin. Crit Rev Immunol 2011;31(4):307–40. [20] Ip W, Chiang YT, Jin T. The involvement of the Wnt signaling pathway and TCF7L2 in diabetes mellitus: the current understanding, dispute, and perspective. Cell Biosci 2012;2(1):28. [21] Longo KA, Wright WS, Kang S, Gerin I, Chiang SH, Lucas PC, et al. Wnt10b inhibits development of white and brown adipose tissues. J Biol Chem 2004;279:35503–9. [22] Ouchi N, Higuchi A, Ohashi K, Oshima Y, Gokce N, Shibata R, et al. SFRP5 is an antiinflammatory adipokine that modulates metabolic dysfunction in obesity. Science 2010;329(5990):454–7. [23] Laudes M. Role of Wnt signalling in the determination of human mesenchymal stem cells into preadipocytes. J Mol Endocrinol 2011;46(2):R65–72. [24] Abiola M, Favier M, Christodoulou-Vafeiadou E, Pichard AL, Martelly I, Guillet-Deniau I. Activation of Wnt/beta-catenin signaling increases insulin sensitivity through a reciprocal regulation of Wnt10b and SREBP-1c in skeletal muscle cells. PLoS One 2009;4(12):e8509. [25] Taneera J, Lang S, Sharma A, Fadista J, Zhou Y, Ahlgvist E, et al. A systems genetics approach identifies genes and pathways for type 2 diabetes in human islets. Cell Metab 2012;16(1):122–34. [26] Carmon KS, Loose DS. Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells. Mol Cancer Res 2008;6(6):1017–28. [27] Park JR, Jung JW, Lee YS, Kang KS. The roles of Wnt antagonists Dkk1 and SFRP4 during adipogenesis of human adipose tissue-derived mesenchymal stem cells. Cell Prolif 2008;41(6):859–74. [28] Muley A, Majumder S, Kolluru GK, Parkinson S, Viola H, Hool L, et al. Secreted frizzledrelated protein 4: an angiogenesis inhibitor. Am J Pathol 2010;176(3):1505–16. [29] Jacob F, Ukegjini K, Nixdorf S, Ford CE, Olivier J, Caduff R, et al. Loss of secreted frizzled-related protein 4 correlates with an aggressive phenotype and predicts poor outcome in ovarian cancer patients. PLoS One 2012;7(2):e31885. [30] Maganga R, Giles N, Adcroft K, Unni A, Keeney D, Wood F, et al. Secreted frizzled related protein-4 (SFRP4) promotes epidermal differentiation and apoptosis. Biochem Biophys Res Commun 2008;377(2):606–11. [31] Romaker D, Puetz M, Teschner S, Donauer J, Geyer M, Gerke P, et al. Increased expression of secreted frizzled-related protein 4 in polycystic kidneys. J Am Soc Nephrol 2009;20(1):48–56. [32] Zhang Z, Zhang L, Zhang L, Jia L, Wang P, Gao Y. Association of Wnt2 and SFRP4 expression in the third trimester placenta in women with severe preeclampsia. Reprod Sci 2013;20(8):981–9. [33] Lee SH, Demeterco C, Geron I, Abrahamsson A, Levine F, Itkin-Ansari P. Islet specific Wnt activation in human type II diabetes. Exp Diabetes Res 2008;2008:728763. [34] Ehrlund A, Mejhert N, Lorente-Cebrián S, Aström G, Dahlman I, Laurencikiene J, et al. Characterization of the Wnt inhibitors secreted frizzled-related proteins (SFRPs) in human adipose tissue. J Clin Endocrinol Metab 2013;98(3):E503–8. [35] Mahdi T, Hänzelmann S, Salehi A, Muhammed SJ, Reinbothe TM, Tang Y, et al. Secreted frizzled-related protein 4 reduces insulin secretion and is overexpressed in type 2 diabetes. Cell Metab 2012;16(5):625–33. [36] Horvath AR, Lord SJ, StJohn A, Sandberg S, Cobbaert CM, Lorenz S, et al. From biomarkers to medical tests: the changing landscape of test evaluation. Clin Chim Acta 2014;427:49–57.
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Review
Secreted frizzled-related protein 4 (SFRP4) and fractalkine (CX3CL1) — Potential new biomarkers for β-cell dysfunction and diabetes Katarzyna Bergmann ⁎, Grazyna Sypniewska Department of Laboratory Medicine, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz, Poland
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 11 December 2013 Received in revised form 3 March 2014 Accepted 6 March 2014 Available online 24 March 2014
The discovery of new risk factors for diabetes is a major challenge for contemporary science. Pathogenesis of type 2 diabetes mellitus (T2DM) is closely related to adipose tissue dysfunction. The aim of this review was to describe recently discovered cytokines: fractalkine (CX3CL1, FKN) and secreted frizzled-related protein 4 (SFRP4) as potential biomarkers of early β cell dysfunction and diabetes. The association of CX3CL1 and SFRP4 with lowgrade inflammation in adipose tissue links obesity with disturbances in insulin secretion and impaired glucose metabolism, therefore it indicates new therapeutic and preventive targets in both healthy and diabetic subjects. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Keywords: Diabetes Insulin resistance Adipocytokines Secreted frizzled-related protein 4 Fractalkine
Contents Introduction . . . . . . . . . . . . . . Fractalkine (CX3CL1, FKN) . . . . . . . Secreted frizzled-related protein 4 (SFRP4) Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . .
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Introduction Diabetes mellitus and its cardiovascular complications are one of the leading causes of disability and deaths worldwide. According to the International Diabetes Federation (IDF) incidence of diabetes, especially type 2 (T2DM), will rise from 366 million in 2011 to 552 million by 2030, which means that the disease will affect one out of ten adults [1]. In Europe at least 131 billion dollars per year is spent on healthcare due to diabetes [2], which indicates that it is not only an important health, but also a socio-economic problem. Therefore the need for prevention as well as the discovery of new risk prediction factors for diabetes is a major challenge for contemporary laboratory medicine. Pathogenesis of T2DM is closely linked to adipose tissue dysfunction. Excessive body fat acts as an endocrine organ in which many adverse biochemical mechanisms occur. Cytokines produced by adipocytes such as ⁎ Corresponding author at: Department of Laboratory Medicine, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, M. Sklodowskiej-Curie 9, 85-094 Bydgoszcz, Poland. E-mail address: [email protected] (K. Bergmann).
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interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), resistin, retinol binding protein 4 (RBP-4), dipeptidyl peptidase 4 (DPP-4) and adipocyte fatty acid-binding protein (A-FABP) activate inflammation pathways, which decrease the quantity/activity of the insulin-dependent receptors, and affect gene expression and insulin production in human pancreatic islets, leading to insulin resistance and impaired glucose metabolism [3]. By the fact that elevated levels of various chemokines may predict the occurrence of disease even several years before its diagnosis they are considered to be potential risk factors in both apparently healthy, nondiabetic and overweight/obese subjects. Recent studies focused on newly discovered chemokines: fractalkine (CX3CL1, FKN) and secreted frizzled-related protein 4 (SFRP4) as promising biomarkers of early pancreatic β-cell dysfunction, however their diagnostic and clinical utility/relevance is not confirmed yet. Fractalkine (CX3CL1, FKN) Fractalkine is the only member of the CX3C chemokine subclass composed of 373 amino acids. It is synthesized as a transmembrane
http://dx.doi.org/10.1016/j.clinbiochem.2014.03.007 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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molecule consisted of a soluble form — chemokine domain and the extracellular mucin-like stalk and the membrane-bound form (Fig. 1). The soluble CX3CL1 is generated by disintegrin-like metalloproteinases (ADAM) 10 and 17/TACE and has the chemoattractive activity for monocytes, natural killer (NK) cells, and T cells [4]. Membrane-bound form is produced in endothelial and epithelial cells, vascular smooth muscle cells, keratinocytes, dendritic cells, and neurons after stimulation by proinflammatory factors, such as TNF-α, interferon γ (IFN-γ), and IL-1, and supports integrin-independent leukocyte adhesion [5]. The presence of mRNA for FKN has been showed in various organs such as heart, kidneys, liver, adrenal gland and brain. The activity of CX3CL1 is related to its receptor called CX3CR1. This receptor occurs on the surface of monocytes, macrophages, NK cells, some T cells, neutrophils, mast cells, platelets, smooth muscle cells, and dendritic and microglia cells [4]. The discovery of fractalkine changed the existing opinions upon cell adhesion and migration through the endothelium. According to the classical pathway migration of leukocytes is dependent on the selectinmediated interactions between leukocytes and the endothelium and activation of integrins on leukocytes by chemokines presented on glycosaminoglycans. Therefore it was claimed that all chemokines are produced as soluble molecules associated with proteoglycans on the cell surface and tissue matrix. In contrast, membrane-bound form of FKN has a chemokine domain on the top of the mucin-like stalk, which acts as an adhesion molecule and is involved in all stages of the migration, while the soluble form shows strong chemotactic effect [6]. Leukocytes with CX3CR1 receptor connect selectively with a chemokine domain presented on endothelial cells which triggers rapid binding. Interaction between CX3CL1 and its receptor additionally can increase expression of integrins, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in leukocytes, which may result in stronger adhesion. Expression of fractalkine during the inflammation can also attract and activate NK cells, which are responsible for cytotoxic effect and cytolysis, therefore its contribution to inflammationdependent conditions seems to be very important [5]. Several studies confirmed the role of fractalkine in the pathogenesis of many human diseases, such as atherosclerosis and cardiovascular disease, rheumatoid arthritis, HIV and HCV infections and cancer in which activity of inflammatory factors is significant [7–10]. Type 2
diabetes is strictly associated with obesity and related to adipose tissue inflammation. In this process a recruitment of monocytes to fatty tissue plays a key role and initiates the first local production of cytokines and then leads to systemic inflammation and insulin resistance. FKN with its dual properties in the mechanism of leukocyte chemotaxis and adhesion might be a relevant pathogenetic factor in early adipocyte dysfunction and T2DM [11], although the data in this area are scarce. Study by Shah et al. [12] clearly confirmed that CX3CL1 is expressed and secreted by human adipocytes and stromal vascular cells, hence it can be considered as adipocytokine. This well-designed study was performed based on in vivo endotoxemia and adipose tissue biopsies in lean and obese subjects, in vitro study of primary human adipocytes and monocytes and case–control study performed in individuals with and without T2DM. Moderate dose (3 ng/kg) of endotoxin (standard lipopolysaccharide, LPS) increased significantly both adipose and blood FKN (33-fold, p b 0.001 and over 40-fold, p = 0.006 after 4 h, respectively). Subcutaneous adipose tissue levels of CX3CL1 were higher in obese compared with lean subjects (mean 0.420 vs. 0.228 ng/mL; p = 0.04) and in obese individuals, these levels were higher in visceral than in subcutaneous adipose tissue (mean 0.736 vs. 0.420 ng/mL; p = 0.01). Stimulation of CX3CL1 mRNA expression and protein secretion from adipocytes in vitro by LPS, TNF-α and IFN-γ was observed. Moreover, the use of CX3CL1 blocking antibody reduced monocyte adhesion to adipocytes by almost 50% (p b 0.001). Plasma FKN was significantly lower in non-diabetic subjects compared to T2DM patients (mean 0.422 vs. 0.506 ng/mL; p b 0.0001). A significant 2.77 odds ratio for diabetes for a 1 SD (0.21 ng/mL) increase in CX3CL1 concentration after adjusting for gender, BMI, ethnicity and other metabolic risk factors was indicated. The effect of controlled low-dose endotoxemia on fractalkine expression in human adipocytes in vitro was also observed by Mehta et al. [13]. In this study CX3CL1 mRNA increased 15-fold (p b 0.001) 4 h after 0.6 ng/kg LPS administration and it was the largest increase compared to the other cytokines (IL-6, TNF-α and MCP-1). Analysis of the impact of endotoxemia on impaired insulin activity showed 32% increase in HOMA-IR (p b 0.01) and 21% decrease in insulin sensitivity (p b 0.05) in LPS-treated subjects compared to placebo. Presented results broadly highlight the influence of adipose tissue inflammation including fractalkine-mediated pathway on T2DM development,
Fig. 1. Fractalkine (CX3CL1) structure.
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however do not explain the effect of FKN on β-cell function. In contrast, recent study by Lee et al. [14] demonstrates that fractalkine/CX3CR1 system is a regulatory mechanism for pancreatic islet β-cell function which improves insulin secretion and glucose uptake in vitro in mouse and human islets. These properties might be associated with three pathways: increased expression of Akt-1 kinase and its anti-apoptotic effect, suppression of inducible cAMP early repressor (ICER-1) and maintenance of β-cell gene expression, increased intracellular Ca2+ and stimulated insulin secretion. Expression of FKN in islets was decreased by high-fat diet, obesity and aging, therefore authors suggest that lower levels of CX3CL1/CX3CR1 may contribute to β-cell dysfunction in T2DM and emphasize its potential role as a new therapeutic target. However, these findings should be considered with caution if compared with data of Wan and Evans [15], which showed suppression of FKN expression and CX3CL1/CX3CR1 signaling by rosiglitazone, a drug for insulin resistance and type 2 diabetes. Interestingly, rosiglitazone acts by activation of an anti-inflammatory agent, peroxisome proliferator-activated receptor γ (PPAR-γ) and inhibits FKN-dependent migration and adhesion of macrophages to endothelial cells, which also may prevent the cardiovascular complications in diabetic patients. Not without significance several studies revealed increased fractalkine levels in T2DM subjects and its involvement in atherosclerosis and vascular disorders [16,17], as well as the fact that high glucose conditions might induce upregulation of fractalkine and monocyte chemotactic protein-1 [18] and that insulin has a well established anti-inflammatory activity [19]. Thus, it can be concluded that initial increase of insulin secretion by CX3CL1/CX3CR1 may not be a beneficial action, but a defensive reaction to inflammation, which can lead to chronic hyperinsulinemia and depletion of functional reserves of β-cells.
islets. A number of studies demonstrated that SFRP4 is a Wnt antagonist and inhibits Wnt signaling pathway, for example in human cancer cells and adipocytes [26,27]. Several studies exhibit different properties of SFRP4 in both, physiological and pathological states. It was shown to play a role in inhibition of angiogenesis, tumor suppression, promotion of epidermal differentiation, development of polycystic kidney disease and preeclampsia [28–32]. However, it should be noted that some authors suggest that activation of Wnt signaling in pancreatic cells is one of the major pathogenic factors in T2DM [33]. Enhanced Wnt signaling might play an adaptive role, for example, in promoting β-cell proliferation in the early stages of diabetes. On the other hand chronic pathway activation stimulates cell apoptosis and reduces insulin secretion. Therefore, increased expression of SFRP4 in β cells of diabetic subjects can be considered as a result of impaired Wnt signaling. Despite this interesting hypothesis, the role of SFRP4 in obesity and T2DM has not been sufficiently understood yet. Expression of SFRP4 mRNA and secretion of this protein from visceral adipose tissue are increased in obesity and correlate with decreased insulin secretion [34]. Taneera et al. [25] observed significant inverse correlation of SFRP4 expression in human pancreatic islets with insulin secretion (R = −0.28; p = 0.03) and positive relationship with glycated hemoglobin (HbA1c) levels (R = 0.45;
Secreted frizzled-related protein 4 (SFRP4) Secreted frizzled-related proteins (SFRPs) are a family of proteins which act as modulators of Wnt signaling. Wnt signaling is involved in many crucial mechanisms, like cell proliferation and migration. Several Wnt pathway components are associated with lipid and glucose metabolism, therefore they might play an important role in the pathogenesis of metabolic disorders. The relationship between Wnt signaling disturbances and type 2 diabetes is associated with adipogenesis and insulin signaling. Wnt has the ability to maintain preadipocytes in an undifferentiated state through inhibition of the pro-adipogenic factors [20]. Studies on animals showed 50% reduction of adipose mass in overexpression of Wnt-10b, hence Wnt signaling pathway negatively regulates adipogenesis [21]. Interestingly, in physiological state mature adipocytes produce Wnt ligands, like secreted frizzled-related protein 5 (SFRP5), which has anti-inflammatory and insulin-sensitizing properties [22]. In obesity and type 2 diabetes a low-grade inflammation in adipose tissue due to increased macrophage infiltration disturbs a proper Wnt action. In lean subjects the number of macrophages in adipose tissue is low. Production of Wnt antagonists, such as Wnt5a, by macrophages is negligible and additionally their activity is neutralized by SFRP5 produced by adipocytes. On the contrary, in obesity a large number of macrophages synthesize increased levels of Wnt5a and large mature adipocytes produce less SFRP5. Inflammation together with Wnt signaling impairment induces insulin resistance in adipocytes via c-Jun N-terminal kinase (JNK) interfering with insulin receptor substrate (IRS-1) [23]. In vitro study by Abiola et al. showed that activation of Wnt signaling increases insulin sensitivity in skeletal muscles due to inhibition of accumulation of intramyocellular lipids [24]. Secreted frizzled-related protein 4 (SFRP4) is a recently discovered cytokine which contains a cysteine-rich domain homologous to the putative Wnt-binding site of frizzled proteins — G protein-coupled receptor proteins related to Wnt signaling pathway [25]. Its expression is observed in several cells and tissues, including adipocytes, myocardium, endometrium, kidney and ovary, however its association with β cell dysfunction in diabetes is closely related to expression in pancreatic
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Fig. 2. Potential role of SFRP4 in insulin resistance, according to Mahdi et al. [35].
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p = 0.001). They also reported the previous independent results which showed that in human pancreatic islets recombinant SFRP4 inhibits in vitro insulin secretion by 30% and cell exocytosis by 50%, as well as that SFRP4 overexpression is associated with the occurrence of proinflammatory factors. Recently Mahdi et al. [35] noted that SFRP4 expression links inflammation and defective insulin secretion, therefore it indicates a novel purpose for SFRP4 investigation as a biomarker of the pancreatic islet dysfunction in T2DM. In this study, high overexpression of SFRP4 in islets of diabetic subjects and its relationship with inflammatory markers, particularly IL-1β stimulating its production, were observed. Reduced insulin secretion was explained by decreased expression of Ca2+ channels in the islets' cells and suppression of insulin exocytosis (Fig. 2). Serum SFRP4 concentration was significantly correlated with fasting glucose (β = 0.142; p = 0.004), reduced insulin sensitivity index (β = −0.176; p = 0.002) and lower disposition index (insulin secretion adjusted for insulin sensitivity; β = − 0.186; p = 0.029) in non-diabetic subjects and it was not affected by BMI, sex and patient age. Interestingly, the authors concluded that SFRP4 is elevated in the serum several years before clinical diagnosis of diabetes has been made and its presence increases the risk of diabetes up to three-fold. Therefore, SFRP4 might be used as an early risk predictor, especially in apparently healthy individuals. Conclusions Relationship between low-grade inflammation mediated by adipose tissue and metabolic disorders has been well understood in recent years, however the discoveries of new potential biomarkers and pathways are still an open question for researchers. Presented cytokines, secreted frizzled-related protein 4 and fractalkine, should meet general requirements for biomarkers prior to their application in routine clinical practice [36]. Firstly, there is a need for a standardized assay (preferably adapted for automatic analyzers) with proven analytical performance, instead of manual ELISA tests. Secondly, clinical performance and effectiveness evaluation are necessary in order to confirm their diagnostic/ therapeutic goals and prognostic value in assessing the risk of T2DM. Another important aspect is also a cost-effectiveness analysis which compares the changes in costs and in health effects of introducing a new test. Despite the presented promising results on the contribution of secreted frizzled-related protein 4 and fractalkine in the pathogenesis of T2DM, more detailed large population-based studies to evaluate their clinical and diagnostic utility are required. They may provide an opportunity for the development of new therapeutic targets. References [1] International Diabetes Federation (IDF). The global burden — diabetes. http://www. idf.org/diabetesatlas/5e/diabetes . [9.08.2013]. [2] International Diabetes Federation (IDF). Diabetes atlas. http://www.idf.org/ diabetesatlas/5e/europe?language=zh-hans . [9.03.2013]. [3] Bergmann K, Sypniewska G. Diabetes as a complication of adipose tissue dysfunction. Is there a role for potential new biomarkers? Clin Chem Lab Med 2013;51(1):177–85. [4] Jones BA, Beamer M, Ahmed S. Fractalkine/CX3CL1: a potential new target for inflammatory diseases. Mol Interv 2010;10(5):263–70. [5] Owlasiuk P, Zajkowska JM, Pietruczuk M, Pancewicz SA, Hermanowska-Szpakowicz T. Fractalkine — structure, functions and biological activity. Pol Merkur Lekarski 2009;26(153):253–7. [6] Umehara H, Bloom ET, Okazaki T, Nagano Y, Yoshie O, Imai T. Fractalkine in vascular biology: from basic research to clinical disease. Arterioscler Thromb Vasc Biol 2004;24(1):34–40. [7] Stolla M, Pelisek J, von Brühl ML, Schäfer A, Barocke V, Heider P, et al. Fractalkine is expressed in early and advanced atherosclerotic lesions and supports monocyte recruitment via CX3CR1. PLoS One 2012;7(8):e43572.
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