Neuroscience Letters 558 (2014) 53–57
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S100B as a glial cell marker in diabetic peripheral neuropathy Asuman Celikbilek a,∗ , Lutfi Akyol b , Seda Sabah c , Nermin Tanik a , Mehmet Adam d , Mehmet Celikbilek e , Murat Korkmaz f , Neziha Yilmaz g a
Department of Neurology, Bozok University, Medical School, Yozgat, Turkey Department of Internal Medicine, Bozok University, Medical School, Yozgat, Turkey Department of Medical Biology, Bozok University, Medical School, Yozgat, Turkey d Department of Ophthalmology, Bozok University, Medical School, Yozgat, Turkey e Department of Gastroenterology, Bozok University, Medical School, Yozgat, Turkey f Department of Orthopaedics and Traumatology, Bozok University, Medical School, Yozgat, Turkey g Department of Infectious Diseases and Microbiology, Bozok University, Medical School, Yozgat, Turkey b
c
h i g h l i g h t s • We did not detect any concentration of GFAP in serum samples in both of the groups. • Markedly decreased serum levels of S100B were obtained in diabetic patients. • Serum S100B levels did not correlate with diabetic peripheral neuropathy in diabetics.
a r t i c l e
i n f o
Article history: Received 6 September 2013 Received in revised form 22 October 2013 Accepted 25 October 2013 Keywords: Diabetes Diabetic peripheral neuropathy GFAP S100B
a b s t r a c t Evidence suggests that acute and chronic hyperglycemia can cause oxidative stress in the peripheral nervous system which, in turn, can promote the development of diabetic neuropathy. Recent studies have found increased expression of glial fibrillary acidic protein (GFAP) and S100B, both of which are indicators of glial reactivity, in the neural and retinal tissues of diabetic rats. For the first time in the literature, the serum levels of GFAP and S100B were assessed in patients with diabetes to evaluate the potential of these factors to serve as peripheral glial biomarkers of diabetes and to investigate their relationship to diabetic peripheral neuropathy. This prospective clinical study included 72 patients with type 2 diabetes mellitus and 50 age- and sex-matched control subjects. All diabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy, and nephropathy. Serum samples were analyzed for human GFAP and S100B using a commercially available Enzyme-linked Immuno Sorbent Assay kit. GFAP was not detected in the serum samples of either diabetic or control patients (p > 0.05). However, we found a statistically significant decrease in S100B serum levels in patients with diabetes compared with control participants (p < 0.001). No associations between serum S100B levels and the presence of diabetic peripheral neuropathy or other microvascular complications were observed (p > 0.05). The findings of markedly decreased serum levels of S100B may possibly indicate a neuroprotective effect of S100B, whereas GFAP may be of no diagnostic value in human patients with diabetes. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes mellitus is the most common serious metabolic disorder in which microvascular complications, such as peripheral neuropathy, retinopathy, and nephropathy, regularly occur but may remain undetected in daily practice [12]. An easy and
∗ Corresponding author at: Bozok University, Department of Neurology, 66200 Yozgat, Turkey. Tel.: +90 505 653 26 15; fax: +90 354 217 10 72. E-mail address: [email protected] (A. Celikbilek). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.10.067
non-invasive screening test that can identify these types of complications would be of short-term and long-term benefit to patients with diabetes. This is particularly true in the case of peripheral neuropathy, which requires sensory and motor conduction studies on multiple nerves in the upper and lower limbs [17]. Needle examination and, rarely, sural and skin–punch biopsies are required for the diagnosis of a neuropathy when small fiber injury is the sole pathology and conduction studies are normal; however, these procedures are often painful to the patient [17]. Evidence suggests that acute and chronic hyperglycemia can cause oxidative stress in the peripheral nervous system (PNS)
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which, in turn, can promote the development of diabetic neuropathy [29]. Proteins damaged by oxidative stress exhibit decreased biological activity, which leads to reduced energy metabolism, decreased cell signaling and transport, and, ultimately, cell death [29]. Experimental animal and in vitro models of diabetes as well as clinical trials of antioxidants have strongly implicated hyperglycemia-induced oxidative stress in the manifestation of diabetic neuropathy [16,23,25]. Moreover, glial cells exhibit reactive gliosis, an early and obvious cellular response, following a variety of insults to the central nervous system (CNS; [4,6]). Glial cells play a vital role in the homeostatic regulation of the CNS as they are involved in neurotransmitter uptake, neuronal metabolic support, pH regulation, and neuronal survival against oxidative stress caused by free radicals [5,7]. Following hyperglycemiainduced oxidative or metabolic insults in the brain, astrocytes overexpress glial fibrillary acidic protein (GFAP) and S100B protein, both of which are indicators of glial reactivity [7]. Baydas et al. [5,8] recently found increased expression of GFAP and S100B in the neural and retinal tissues of diabetic rats. These authors suggested that the gliosis that accompanies diabetes occurs via free radical formation and that GFAP and S100B may be relevant markers of neurodegenerative changes in experimental models of diabetes [5,8]. Regarding clinical perspectives, these glial markers have been extensively studied in peripheral blood of patients with either traumatic brain injury [30] or cerebral infarcts [13] through the hypothesis of blood–brain barrier (BBB) permeability. Due primarily to the wide microcirculatory network, hyperglycemia-induced oxidative stress affects both central and peripheral organs, possibly resulting in overexpression of the glial biomarkers [29]. Then, we might expect that GFAP and S100B protein would be detected in the serum of individuals with diabetes, rather than being part of a mechanism regulating BBB permeability, in response to the excessive peripheral glial reactivity that accompanies diabetes. However, the level of glia in the serum samples of diabetic patients has yet to be investigated. Here, it is proposed that if hyperglycemiainduced oxidative stress were involved in the development of diabetic neuropathy, then peripheral glial markers, which represent diabetes-induced glial reactivity, may be a non-invasive assessment of peripheral neuropathy in diabetic patients. Thus, for the first time in the literature, the serum levels of GFAP and S100B were assessed in patients with diabetes to evaluate the potential of these factors to serve as peripheral glial biomarkers of diabetes and to investigate their relationship to diabetic peripheral neuropathy.
Dependent variables included systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI), which was calculated as weight in kilograms divided by the square of height in meters [26]. Furthermore, fasting venous blood samples were taken from all subjects between 8:30 and 10:00 am, and routine hematological and biochemical analyses were performed by standard methods in our laboratory.
2. Material and methods
Nephropathy was diagnosed according to the presence of microalbuminuria, macroalbuminuria, or creatinine clearance of 300 mg of albumin per gram of creatinine for macroalbuminuria. Three patients with diabetes were found to have nephropathy.
2.1. Study population This prospective clinical study included 72 patients with type 2 diabetes mellitus and 50 age- and sex-matched control subjects ranging from 25 to 75 years of age. Type 2 diabetes mellitus was identified by the presence of fasting glucose levels ≥126 mg/dl or postprandial glucose levels ≥200 mg/dl concomitant with symptoms of diabetes or by treatment with insulin and/or oral hypoglycemic agents of previously diagnosed diabetes [1]. Patients with malignancy; chronic renal, hepatic, cardiovascular, or connective tissue diseases; thyroid disease; chronic obstructive pulmonary disease; history of local trauma or surgery; infections (Lyme disease, hepatitis, etc.); a vitamin-B12 deficiency; or a family history of neuropathy or a disease known to cause neuropathy were excluded from the study. Additionally, those who were pregnant, morbidly obese, current smokers, current consumers of alcohol, or users of drugs that cause neuropathy were also excluded from the subject pool. The study protocol was approved by the Bozok University Research Ethics Committee, and written informed consent was obtained from all participants.
2.2. Assessment of peripheral neuropathy All diabetic patients underwent conventional sensory and motor nerve conduction studies performed by the same neurologist, who was blind to the results. All nerve stimulations, including of the median, ulnar, deep peroneal, and tibial motor nerves and median, ulnar, and of the sural sensory nerves in both limbs, were performed with a Meledec Synergy electromyography (EMG) machine (Meledec Synergy; Oxford Instruments; Surrey, UK). Filter settings included a 20–2000 Hz bandpass for sensory nerve studies and a 2–10,000 Hz bandpass for motor nerve studies. The limb temperature of all subjects was maintained above 31–32 ◦ C. Abnormal spontaneous activity, increased number of long-duration motor unit potentials, and decreased recruitment patterns were determined to be indicators of neuropathic changes. Based on EMG findings (nerve conduction velocity, amplitude, and distal latency) and a score of ≥4 on the Douleur Neuropathique 4 (DN4) questionnaire, peripheral neuropathy was confirmed or ruled out for each patient [9]. Of the diabetic patients, 37 (51.4%) were found to have peripheral neuropathy. 2.3. Assessment of retinopathy Retinopathy status was assessed by fundoscopic eye examination performed by the same ophthalmologist who was blind to the status of the patients. Non-proliferative retinopathy was diagnosed according to the presence of cotton wool spots, micro-aneurysms, and/or boat-shaped hemorrhages during direct ophthalmoscopic examination. Proliferative retinopathy was diagnosed according to the presence of neovascularization in the retina [10]. Of the diabetic patients, 18 (25%) were found to have retinopathy; four patients exhibited the proliferative type, and the remainder exhibited the non-proliferative type. 2.4. Assessment of nephropathy
2.5. Biochemical analysis Blood samples were collected in vacutainer tubes without anticoagulant supplements. All blood samples were centrifuged for 10 min at 3000 rpm, after which the supernatant was quickly removed and kept frozen at −80 ◦ C until the assays were performed by an investigator blind to patient status. Serum samples were analyzed for human GFAP and S100B using a commercially available Enzyme-linked Immuno Sorbent Assay (ELISA) kit (human GFAP and S100B, BioVendor Research and Diagnostic Products; Heidelberg; Germany). The limits of detection for human GFAP and S100B were 0.045 ng/mL and 15 pg/mL, respectively. Serum GFAP concentrations were expressed as ng/mL, whereas S100B concentrations were expressed as pg/mL. The S100B levels of 98% of all subjects
A. Celikbilek et al. / Neuroscience Letters 558 (2014) 53–57 Table 1 Demographic and laboratory data of control and diabetic patients. Variables
Control (n = 50)
Diabetic (n = 72)
p
Age (years) Gender (female/male) BMI (kg/m2 ) SBP (mmHg) DBP (mmHg) Fasting glucose (mg/dL) Creatinine (mg/dL) WBC (103 /mm3 ) Hemoglobin (mg/dL) Platelet (103 /mm3 ) AST (IU/L) ALT (IU/L) TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) TSH (uIU/mL) B12 vitamin (pg/mL) Folat (ng/mL)
58.9 ± 10.93 34 (68)/16 (32) 31.9 ± 5.46 120 (110–130) 70 (60–80) 89.5 (85.7–93.2) 0.7 (0.6–0.8) 7.84 ± 1.41 14.1 (13.2–15.3) 264.1 ± 66.96 20 ± 6.14 18 (12.7–25) 216.04 ± 36.74 146.5 (102–199) 42 (39.7–48) 137.66 ± 28.14 1.4 (0.9–2) 358.5 (297.2–457.2) 11 (10–13)
56.2 ± 9.34 50 (60.4)/22 (30.6) 31.4 ± 4.88 120 (110–130) 70 (60–80) 168 (130.7–214.7) 0.7 (0.6–0.87) 7.72 ± 1.25 13.9 (13.2–15.3) 261.1 ± 69.06 18.7 ± 5.44 20 (15–27) 210.78 ± 48.65 148 (107–206) 43 (37.2–46.7) 131.31 ± 34.09 1.5 (0.9–2.7) 355 (299–494.7) 12 (10–15)
0.153 0.865 0.572 0.895 0.895 0.05), and no significant associations among the BMI, SBP, and DBP parameters in patients with diabetes compared with controls were observed (p > 0.05). The two groups were also similar with respect to all laboratory results (p > 0.05), except for fasting glucose (p < 0.001). Additionally GFAP was not detected in the serum samples of either diabetic or control patients (p > 0.05). However, we found a statistically significant decrease in S100B serum levels in patients with diabetes compared with control participants (p < 0.001; Table 2). A detailed analysis of the associations Table 2 Serum levels of GFAP and S100B in control and diabetic patients. Variables
Control (n = 50)
Diabetic(n = 72)
p
GFAP (ng/mL) S100B (pg/mL)
0 (100) 14.878 ± 1.296
0 (100) 10.889 ± 2.787
0.895 0.05). 4. Discussion Two main findings emerged from the present study. First, we did not detect GFAP in either the diabetic or the control group, but markedly decreased serum levels of S100B were observed in the diabetic group. Second, we found no association between serum S100B levels and diabetic peripheral neuropathy in patients with diabetes. Diabetes causes a variety of functional and structural changes in the CNS and PNS [4,5]. For example, in patients with diabetes, chronic hyperglycemia causes oxidative stress in tissues prone to complications [22]; the most common peripheral neuropathy in diabetics is microvascular complications. As the testing procedures for this type of neuropathy are often painful for the patient, it is of great clinical interest to find diagnostic measures that can predict peripheral neuropathy using non-invasive methods. Experimental data strongly implicate hyperglycemia-induced oxidative stress in the manifestation of diabetic neuropathy [16,23,25]. Additionally, in a hyperglycemic environment, astrocytes in the brain overexpress GFAP and S100B, which are factors that represent glial reactivity in response to oxidative and metabolic insults [4–7]. Considered together, these data suggest that peripheral glial markers may represent a non-invasive approach with which to diagnose peripheral neuropathy in patients with diabetes. GFAP is a monomeric, intracellular, intermediate filament of cytoskeletal proteins within glial cells that has a molecular mass between 40 and 53 kD and is expressed almost exclusively by astrocytes in the CNS [15]. It has been suggested that GFAP is essential for the formation of stable astrocytic processes and, thus, increases in GFAP are commonly used to examine the distribution of glial cells following neuronal damage [6,15]. GFAP can be measured in the serum following head injury [28] and acute cerebral infarcts [13]. However, in this study, we did not detect any concentration of GFAP in the serum samples of patients with diabetes. This may be explained by the fact that GFAP is strictly specific to CNS-related astrocytic damage, whereas S100B may be detected in neural tissues as well as in non-neural tissues [2]. Therefore, it appears that GFAP is unsuitable as a non-invasive measure to diagnose symptoms in diabetic patients. Another intracellular glycoprotein that may be used as an astrocytic marker is S100B, an acidic calcium-binding protein that is localized primarily in the glial cells of the CNS and PNS [11]. Elevated serum levels of S100B have been reported by several clinical studies investigating traumatic brain injury [20],
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stroke [13], schizophrenia [14], systemic lupus erythematosus [19], and migraines [31] whereas no changes in this regard were observed in patients with Parkinson’s disease [24]. In this study, serum levels of S100B were significantly lower in patients with diabetes relative to controls. As an intracellular regulator, S100B is involved in the regulation of energy metabolism, transcription, protein phosphorylation, cell proliferation, survival, differentiation and motility, and calcium homeostasis via its interaction with a wide array of proteins in a restricted number of cell types [27]. As an extracellular signal, S100B engages the pattern recognition receptor and the receptor for advanced glycation end-products (RAGE) on immune cells, vascular smooth muscle cells, skeletal myoblasts, cardiomyocytes, and neuronal, astrocytic, and microglial cells [27]. Moreover, the extracellular effects of S100B vary depending on its local concentration. At nanomolar concentrations, such as under physiological conditions, S100B has neurotrophic and gliotrophic actions and may play an important role in normal CNS development and recovery following injury. At micromolar concentrations, S100B may be toxic and result in neuronal and glial cell death by apoptosis and, therefore, contribute to neuropathological changes during neurodegeneration and neuroinflammation. From this perspective, it is noteworthy that the markedly lower levels (pg/mL) of S100B in the diabetic group may indicate the pro-survival or neuroprotective effects of this protein on CNS neurons following chemical insults induced by hyperglycemia. The present hypothesis contrasts with previous data suggesting that higher levels of S100B represent a compensatory response by metabolically stressed cells [3]. Mohammadzadeh et al. [18] identified an enhancement of S100B levels in an experimental diabetes model, suggesting that the expression of this protein may serve to modulate cardiac remodeling and function. In contrast, no associations between serum levels of S100B and the presence of diabetic peripheral neuropathy or other microvascular complications were observed. S100B is found in Schwann cells in the PNS and is expressed by crushed nerves during the subsequent degeneration period [27]. Upon acute peripheral nerve injury, S100B is released from Schwann cells in damaged nerves, activates RAGE, and exerts beneficial effects by clearing cell debris and releasing cytokines and trophic factors, which have been shown to be crucial for the repair of injured nerves [21]. This indicates that S100B stimulates neuronal survival and differentiation via RAGE engagement in damaged nerves. Thus, according to the hypothetical protective role proposed here for S100B in patients with diabetes, S100B may be further decreased in patients with diabetes with peripheral neuropathy despite the lower levels in this population. However, this study failed to find any relationship between these variables, and it is likely that larger cohorts are needed to produce more definitive results. The present study has several potential limitations. First, we used a small sample, and it will be necessary to validate these findings with a larger cohort. Second, this study investigated only serum samples, and cerebrospinal fluid analysis would probably provide additional clarification. Third, data regarding the upper signaling pathways by which these proteins are regulated are lacking and may clarify the mechanisms underlying diabetes-induced glial reactivity. 5. Conclusions These are the first data to demonstrate that, unlike the markedly increased expression of GFAP and S100B observed in diabetic rats, significantly decreased serum levels of S100B are found in humans. It is possible that this indicates a neuroprotective effect of S100B, whereas GFAP may be of no diagnostic value in human patients with diabetes. Additional studies with a larger sample of participants are needed to confirm the present findings as well as to clarify
the contribution of markers of peripheral glial cell damage to the development of diabetic peripheral neuropathy. Acknowledgments This study was funded by the Bozok University Scientific Research Project Unit(2013 TF/A37) and was conducted at Bozok University Hospital. References [1] A. American Diabetes, Diagnosis and classification of diabetes mellitus, Diabetes Care 32 (Suppl. 1) (2009) S62–67. [2] R.E. Anderson, L.O. Hansson, O. Nilsson, J. Liska, G. Settergren, J. Vaage, Increase in serum S100A1-B and S100BB during cardiac surgery arises from extracerebral sources, The Annals of Thoracic Surgery 71 (2001) 1512–1517. [3] S.W. Barger, L.J. Van Eldik, M.P. Mattson, S100 beta protects hippocampal neurons from damage induced by glucose deprivation, Brain Research 677 (1995) 167–170. [4] G. Baydas, E. Donder, M. Kiliboz, E. Sonkaya, M. Tuzcu, A. Yasar, V.S. Nedzvetskii, Neuroprotection by alpha-lipoic acid in streptozotocin-induced diabetes, Biochemistry/Biokhimiia 69 (2004) 1001–1005. 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Tritschler, The roles of oxidative stress and antioxidant treatment in experimental diabetic neuropathy, Diabetes 46 (Suppl. 2) (1997) S38–S42. [17] A. Moghtaderi, A. Bakhshipour, H. Rashidi, Validation of Michigan neuropathy screening instrument for diabetic peripheral neuropathy, Clinical Neurology and Neurosurgery 108 (2006) 477–481. [18] F. Mohammadzadeh, J.F. Desjardins, J.N. Tsoporis, G. Proteau, H. Leong-Poi, T.G. Parker, S100B: role in cardiac remodeling and function following myocardial infarction in diabetes, Life Sciences 92 (2013) 639–647. [19] L.V. Portela, J.C. Brenol, R. Walz, M. Bianchin, A.B. Tort, U.P. Canabarro, S. Beheregaray, J.A. Marasca, R.M. Xavier, E.C. Neto, C.A. Goncalves, D.O. Souza, Serum S100B levels in patients with lupus erythematosus: preliminary observation, Clinical and Diagnostic Laboratory Immunology 9 (2002) 164–166. [20] A. Raabe, C. Grolms, O. Sorge, M. Zimmermann, V. 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[27] G. Sorci, F. Riuzzi, C. Arcuri, C. Tubaro, R. Bianchi, I. Giambanco, R. Donato, S100B protein in tissue development, repair and regeneration, World Journal of Biological Chemistry 4 (2013) 1–12. [28] W.J. van Geel, H.P. de Reus, H. Nijzing, M.M. Verbeek, P.E. Vos, K.J. Lamers, Measurement of glial fibrillary acidic protein in blood: an analytical method, Clinica Chimica Acta; International Journal of Clinical Chemistry 326 (2002) 151–154. [29] A.M. Vincent, J.W. Russell, P. Low, E.L. Feldman, Oxidative stress in the pathogenesis of diabetic neuropathy, Endocrine Reviews 25 (2004) 612–628. [30] P.E. Vos, B. Jacobs, T.M. Andriessen, K.J. Lamers, G.F. Borm, T. Beems, M. Edwards, C.F. Rosmalen, J.L. Vissers, GFAP and S100B are biomarkers of traumatic brain injury: an observational cohort study, Neurology 75 (2010) 1786–1793. [31] N. Yilmaz, K. Karaali, S. Ozdem, M. Turkay, A. Unal, B. Dora, Elevated S100B and neuron specific enolase levels in patients with migraine-without aura: evidence for neurodegeneration? Cellular and Molecular Neurobiology 31 (2011) 579–585.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
S100B as a glial cell marker in diabetic peripheral neuropathy Asuman Celikbilek a,∗ , Lutfi Akyol b , Seda Sabah c , Nermin Tanik a , Mehmet Adam d , Mehmet Celikbilek e , Murat Korkmaz f , Neziha Yilmaz g a
Department of Neurology, Bozok University, Medical School, Yozgat, Turkey Department of Internal Medicine, Bozok University, Medical School, Yozgat, Turkey Department of Medical Biology, Bozok University, Medical School, Yozgat, Turkey d Department of Ophthalmology, Bozok University, Medical School, Yozgat, Turkey e Department of Gastroenterology, Bozok University, Medical School, Yozgat, Turkey f Department of Orthopaedics and Traumatology, Bozok University, Medical School, Yozgat, Turkey g Department of Infectious Diseases and Microbiology, Bozok University, Medical School, Yozgat, Turkey b
c
h i g h l i g h t s • We did not detect any concentration of GFAP in serum samples in both of the groups. • Markedly decreased serum levels of S100B were obtained in diabetic patients. • Serum S100B levels did not correlate with diabetic peripheral neuropathy in diabetics.
a r t i c l e
i n f o
Article history: Received 6 September 2013 Received in revised form 22 October 2013 Accepted 25 October 2013 Keywords: Diabetes Diabetic peripheral neuropathy GFAP S100B
a b s t r a c t Evidence suggests that acute and chronic hyperglycemia can cause oxidative stress in the peripheral nervous system which, in turn, can promote the development of diabetic neuropathy. Recent studies have found increased expression of glial fibrillary acidic protein (GFAP) and S100B, both of which are indicators of glial reactivity, in the neural and retinal tissues of diabetic rats. For the first time in the literature, the serum levels of GFAP and S100B were assessed in patients with diabetes to evaluate the potential of these factors to serve as peripheral glial biomarkers of diabetes and to investigate their relationship to diabetic peripheral neuropathy. This prospective clinical study included 72 patients with type 2 diabetes mellitus and 50 age- and sex-matched control subjects. All diabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy, and nephropathy. Serum samples were analyzed for human GFAP and S100B using a commercially available Enzyme-linked Immuno Sorbent Assay kit. GFAP was not detected in the serum samples of either diabetic or control patients (p > 0.05). However, we found a statistically significant decrease in S100B serum levels in patients with diabetes compared with control participants (p < 0.001). No associations between serum S100B levels and the presence of diabetic peripheral neuropathy or other microvascular complications were observed (p > 0.05). The findings of markedly decreased serum levels of S100B may possibly indicate a neuroprotective effect of S100B, whereas GFAP may be of no diagnostic value in human patients with diabetes. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes mellitus is the most common serious metabolic disorder in which microvascular complications, such as peripheral neuropathy, retinopathy, and nephropathy, regularly occur but may remain undetected in daily practice [12]. An easy and
∗ Corresponding author at: Bozok University, Department of Neurology, 66200 Yozgat, Turkey. Tel.: +90 505 653 26 15; fax: +90 354 217 10 72. E-mail address: [email protected] (A. Celikbilek). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.10.067
non-invasive screening test that can identify these types of complications would be of short-term and long-term benefit to patients with diabetes. This is particularly true in the case of peripheral neuropathy, which requires sensory and motor conduction studies on multiple nerves in the upper and lower limbs [17]. Needle examination and, rarely, sural and skin–punch biopsies are required for the diagnosis of a neuropathy when small fiber injury is the sole pathology and conduction studies are normal; however, these procedures are often painful to the patient [17]. Evidence suggests that acute and chronic hyperglycemia can cause oxidative stress in the peripheral nervous system (PNS)
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which, in turn, can promote the development of diabetic neuropathy [29]. Proteins damaged by oxidative stress exhibit decreased biological activity, which leads to reduced energy metabolism, decreased cell signaling and transport, and, ultimately, cell death [29]. Experimental animal and in vitro models of diabetes as well as clinical trials of antioxidants have strongly implicated hyperglycemia-induced oxidative stress in the manifestation of diabetic neuropathy [16,23,25]. Moreover, glial cells exhibit reactive gliosis, an early and obvious cellular response, following a variety of insults to the central nervous system (CNS; [4,6]). Glial cells play a vital role in the homeostatic regulation of the CNS as they are involved in neurotransmitter uptake, neuronal metabolic support, pH regulation, and neuronal survival against oxidative stress caused by free radicals [5,7]. Following hyperglycemiainduced oxidative or metabolic insults in the brain, astrocytes overexpress glial fibrillary acidic protein (GFAP) and S100B protein, both of which are indicators of glial reactivity [7]. Baydas et al. [5,8] recently found increased expression of GFAP and S100B in the neural and retinal tissues of diabetic rats. These authors suggested that the gliosis that accompanies diabetes occurs via free radical formation and that GFAP and S100B may be relevant markers of neurodegenerative changes in experimental models of diabetes [5,8]. Regarding clinical perspectives, these glial markers have been extensively studied in peripheral blood of patients with either traumatic brain injury [30] or cerebral infarcts [13] through the hypothesis of blood–brain barrier (BBB) permeability. Due primarily to the wide microcirculatory network, hyperglycemia-induced oxidative stress affects both central and peripheral organs, possibly resulting in overexpression of the glial biomarkers [29]. Then, we might expect that GFAP and S100B protein would be detected in the serum of individuals with diabetes, rather than being part of a mechanism regulating BBB permeability, in response to the excessive peripheral glial reactivity that accompanies diabetes. However, the level of glia in the serum samples of diabetic patients has yet to be investigated. Here, it is proposed that if hyperglycemiainduced oxidative stress were involved in the development of diabetic neuropathy, then peripheral glial markers, which represent diabetes-induced glial reactivity, may be a non-invasive assessment of peripheral neuropathy in diabetic patients. Thus, for the first time in the literature, the serum levels of GFAP and S100B were assessed in patients with diabetes to evaluate the potential of these factors to serve as peripheral glial biomarkers of diabetes and to investigate their relationship to diabetic peripheral neuropathy.
Dependent variables included systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI), which was calculated as weight in kilograms divided by the square of height in meters [26]. Furthermore, fasting venous blood samples were taken from all subjects between 8:30 and 10:00 am, and routine hematological and biochemical analyses were performed by standard methods in our laboratory.
2. Material and methods
Nephropathy was diagnosed according to the presence of microalbuminuria, macroalbuminuria, or creatinine clearance of 300 mg of albumin per gram of creatinine for macroalbuminuria. Three patients with diabetes were found to have nephropathy.
2.1. Study population This prospective clinical study included 72 patients with type 2 diabetes mellitus and 50 age- and sex-matched control subjects ranging from 25 to 75 years of age. Type 2 diabetes mellitus was identified by the presence of fasting glucose levels ≥126 mg/dl or postprandial glucose levels ≥200 mg/dl concomitant with symptoms of diabetes or by treatment with insulin and/or oral hypoglycemic agents of previously diagnosed diabetes [1]. Patients with malignancy; chronic renal, hepatic, cardiovascular, or connective tissue diseases; thyroid disease; chronic obstructive pulmonary disease; history of local trauma or surgery; infections (Lyme disease, hepatitis, etc.); a vitamin-B12 deficiency; or a family history of neuropathy or a disease known to cause neuropathy were excluded from the study. Additionally, those who were pregnant, morbidly obese, current smokers, current consumers of alcohol, or users of drugs that cause neuropathy were also excluded from the subject pool. The study protocol was approved by the Bozok University Research Ethics Committee, and written informed consent was obtained from all participants.
2.2. Assessment of peripheral neuropathy All diabetic patients underwent conventional sensory and motor nerve conduction studies performed by the same neurologist, who was blind to the results. All nerve stimulations, including of the median, ulnar, deep peroneal, and tibial motor nerves and median, ulnar, and of the sural sensory nerves in both limbs, were performed with a Meledec Synergy electromyography (EMG) machine (Meledec Synergy; Oxford Instruments; Surrey, UK). Filter settings included a 20–2000 Hz bandpass for sensory nerve studies and a 2–10,000 Hz bandpass for motor nerve studies. The limb temperature of all subjects was maintained above 31–32 ◦ C. Abnormal spontaneous activity, increased number of long-duration motor unit potentials, and decreased recruitment patterns were determined to be indicators of neuropathic changes. Based on EMG findings (nerve conduction velocity, amplitude, and distal latency) and a score of ≥4 on the Douleur Neuropathique 4 (DN4) questionnaire, peripheral neuropathy was confirmed or ruled out for each patient [9]. Of the diabetic patients, 37 (51.4%) were found to have peripheral neuropathy. 2.3. Assessment of retinopathy Retinopathy status was assessed by fundoscopic eye examination performed by the same ophthalmologist who was blind to the status of the patients. Non-proliferative retinopathy was diagnosed according to the presence of cotton wool spots, micro-aneurysms, and/or boat-shaped hemorrhages during direct ophthalmoscopic examination. Proliferative retinopathy was diagnosed according to the presence of neovascularization in the retina [10]. Of the diabetic patients, 18 (25%) were found to have retinopathy; four patients exhibited the proliferative type, and the remainder exhibited the non-proliferative type. 2.4. Assessment of nephropathy
2.5. Biochemical analysis Blood samples were collected in vacutainer tubes without anticoagulant supplements. All blood samples were centrifuged for 10 min at 3000 rpm, after which the supernatant was quickly removed and kept frozen at −80 ◦ C until the assays were performed by an investigator blind to patient status. Serum samples were analyzed for human GFAP and S100B using a commercially available Enzyme-linked Immuno Sorbent Assay (ELISA) kit (human GFAP and S100B, BioVendor Research and Diagnostic Products; Heidelberg; Germany). The limits of detection for human GFAP and S100B were 0.045 ng/mL and 15 pg/mL, respectively. Serum GFAP concentrations were expressed as ng/mL, whereas S100B concentrations were expressed as pg/mL. The S100B levels of 98% of all subjects
A. Celikbilek et al. / Neuroscience Letters 558 (2014) 53–57 Table 1 Demographic and laboratory data of control and diabetic patients. Variables
Control (n = 50)
Diabetic (n = 72)
p
Age (years) Gender (female/male) BMI (kg/m2 ) SBP (mmHg) DBP (mmHg) Fasting glucose (mg/dL) Creatinine (mg/dL) WBC (103 /mm3 ) Hemoglobin (mg/dL) Platelet (103 /mm3 ) AST (IU/L) ALT (IU/L) TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) TSH (uIU/mL) B12 vitamin (pg/mL) Folat (ng/mL)
58.9 ± 10.93 34 (68)/16 (32) 31.9 ± 5.46 120 (110–130) 70 (60–80) 89.5 (85.7–93.2) 0.7 (0.6–0.8) 7.84 ± 1.41 14.1 (13.2–15.3) 264.1 ± 66.96 20 ± 6.14 18 (12.7–25) 216.04 ± 36.74 146.5 (102–199) 42 (39.7–48) 137.66 ± 28.14 1.4 (0.9–2) 358.5 (297.2–457.2) 11 (10–13)
56.2 ± 9.34 50 (60.4)/22 (30.6) 31.4 ± 4.88 120 (110–130) 70 (60–80) 168 (130.7–214.7) 0.7 (0.6–0.87) 7.72 ± 1.25 13.9 (13.2–15.3) 261.1 ± 69.06 18.7 ± 5.44 20 (15–27) 210.78 ± 48.65 148 (107–206) 43 (37.2–46.7) 131.31 ± 34.09 1.5 (0.9–2.7) 355 (299–494.7) 12 (10–15)
0.153 0.865 0.572 0.895 0.895 0.05), and no significant associations among the BMI, SBP, and DBP parameters in patients with diabetes compared with controls were observed (p > 0.05). The two groups were also similar with respect to all laboratory results (p > 0.05), except for fasting glucose (p < 0.001). Additionally GFAP was not detected in the serum samples of either diabetic or control patients (p > 0.05). However, we found a statistically significant decrease in S100B serum levels in patients with diabetes compared with control participants (p < 0.001; Table 2). A detailed analysis of the associations Table 2 Serum levels of GFAP and S100B in control and diabetic patients. Variables
Control (n = 50)
Diabetic(n = 72)
p
GFAP (ng/mL) S100B (pg/mL)
0 (100) 14.878 ± 1.296
0 (100) 10.889 ± 2.787
0.895 0.05). 4. Discussion Two main findings emerged from the present study. First, we did not detect GFAP in either the diabetic or the control group, but markedly decreased serum levels of S100B were observed in the diabetic group. Second, we found no association between serum S100B levels and diabetic peripheral neuropathy in patients with diabetes. Diabetes causes a variety of functional and structural changes in the CNS and PNS [4,5]. For example, in patients with diabetes, chronic hyperglycemia causes oxidative stress in tissues prone to complications [22]; the most common peripheral neuropathy in diabetics is microvascular complications. As the testing procedures for this type of neuropathy are often painful for the patient, it is of great clinical interest to find diagnostic measures that can predict peripheral neuropathy using non-invasive methods. Experimental data strongly implicate hyperglycemia-induced oxidative stress in the manifestation of diabetic neuropathy [16,23,25]. Additionally, in a hyperglycemic environment, astrocytes in the brain overexpress GFAP and S100B, which are factors that represent glial reactivity in response to oxidative and metabolic insults [4–7]. Considered together, these data suggest that peripheral glial markers may represent a non-invasive approach with which to diagnose peripheral neuropathy in patients with diabetes. GFAP is a monomeric, intracellular, intermediate filament of cytoskeletal proteins within glial cells that has a molecular mass between 40 and 53 kD and is expressed almost exclusively by astrocytes in the CNS [15]. It has been suggested that GFAP is essential for the formation of stable astrocytic processes and, thus, increases in GFAP are commonly used to examine the distribution of glial cells following neuronal damage [6,15]. GFAP can be measured in the serum following head injury [28] and acute cerebral infarcts [13]. However, in this study, we did not detect any concentration of GFAP in the serum samples of patients with diabetes. This may be explained by the fact that GFAP is strictly specific to CNS-related astrocytic damage, whereas S100B may be detected in neural tissues as well as in non-neural tissues [2]. Therefore, it appears that GFAP is unsuitable as a non-invasive measure to diagnose symptoms in diabetic patients. Another intracellular glycoprotein that may be used as an astrocytic marker is S100B, an acidic calcium-binding protein that is localized primarily in the glial cells of the CNS and PNS [11]. Elevated serum levels of S100B have been reported by several clinical studies investigating traumatic brain injury [20],
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stroke [13], schizophrenia [14], systemic lupus erythematosus [19], and migraines [31] whereas no changes in this regard were observed in patients with Parkinson’s disease [24]. In this study, serum levels of S100B were significantly lower in patients with diabetes relative to controls. As an intracellular regulator, S100B is involved in the regulation of energy metabolism, transcription, protein phosphorylation, cell proliferation, survival, differentiation and motility, and calcium homeostasis via its interaction with a wide array of proteins in a restricted number of cell types [27]. As an extracellular signal, S100B engages the pattern recognition receptor and the receptor for advanced glycation end-products (RAGE) on immune cells, vascular smooth muscle cells, skeletal myoblasts, cardiomyocytes, and neuronal, astrocytic, and microglial cells [27]. Moreover, the extracellular effects of S100B vary depending on its local concentration. At nanomolar concentrations, such as under physiological conditions, S100B has neurotrophic and gliotrophic actions and may play an important role in normal CNS development and recovery following injury. At micromolar concentrations, S100B may be toxic and result in neuronal and glial cell death by apoptosis and, therefore, contribute to neuropathological changes during neurodegeneration and neuroinflammation. From this perspective, it is noteworthy that the markedly lower levels (pg/mL) of S100B in the diabetic group may indicate the pro-survival or neuroprotective effects of this protein on CNS neurons following chemical insults induced by hyperglycemia. The present hypothesis contrasts with previous data suggesting that higher levels of S100B represent a compensatory response by metabolically stressed cells [3]. Mohammadzadeh et al. [18] identified an enhancement of S100B levels in an experimental diabetes model, suggesting that the expression of this protein may serve to modulate cardiac remodeling and function. In contrast, no associations between serum levels of S100B and the presence of diabetic peripheral neuropathy or other microvascular complications were observed. S100B is found in Schwann cells in the PNS and is expressed by crushed nerves during the subsequent degeneration period [27]. Upon acute peripheral nerve injury, S100B is released from Schwann cells in damaged nerves, activates RAGE, and exerts beneficial effects by clearing cell debris and releasing cytokines and trophic factors, which have been shown to be crucial for the repair of injured nerves [21]. This indicates that S100B stimulates neuronal survival and differentiation via RAGE engagement in damaged nerves. Thus, according to the hypothetical protective role proposed here for S100B in patients with diabetes, S100B may be further decreased in patients with diabetes with peripheral neuropathy despite the lower levels in this population. However, this study failed to find any relationship between these variables, and it is likely that larger cohorts are needed to produce more definitive results. The present study has several potential limitations. First, we used a small sample, and it will be necessary to validate these findings with a larger cohort. Second, this study investigated only serum samples, and cerebrospinal fluid analysis would probably provide additional clarification. Third, data regarding the upper signaling pathways by which these proteins are regulated are lacking and may clarify the mechanisms underlying diabetes-induced glial reactivity. 5. Conclusions These are the first data to demonstrate that, unlike the markedly increased expression of GFAP and S100B observed in diabetic rats, significantly decreased serum levels of S100B are found in humans. It is possible that this indicates a neuroprotective effect of S100B, whereas GFAP may be of no diagnostic value in human patients with diabetes. Additional studies with a larger sample of participants are needed to confirm the present findings as well as to clarify
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