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Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance
ORCP-594; No. of Pages 6
ARTICLE IN PRESS
Obesity Research & Clinical Practice (2016) xxx, xxx—xxx
SHORT COMMUNICATION
Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance Miaoxin Chen a,b,c, Bo Liu b, David Wilkinson b,d, Amy T. Hutchison b, Campbell H. Thompson b, Gary A. Wittert b, Leonie K. Heilbronn b,c,∗ a
Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, China b Discipline of Medicine, University of Adelaide, Australia c Research Centre for Reproductive Health, Robinson Research Institute, University of Adelaide, Australia d Discipline of Acute Care Medicine, University of Adelaide, Australia Received 22 June 2016 ; received in revised form 19 July 2016; accepted 22 July 2016
KEYWORDS Selenoprotein P; Obesity; Insulin resistance
∗
Summary Selenoprotein P (SeP) is secreted primarily by the liver and postulated to cause insulin resistance. The aim of this study was to measure plasma SeP in individuals who are lean (N = 29) or overweight/obese (N = 34), and examine relationships between circulating SeP, SEPP1 (SeP, plasma 1) expression in subcutaneous adipose tissue, and markers of insulin resistance. SeP was higher in individuals who were overweight/obese (P < 0.001), and associated with insulin resistance by HOMA-IR and by clamp, but not independently of BMI. SEPP1 mRNA was correlated negatively with BMI, suggesting there may be tissue specific regulation. This study suggests that obesity, rather than insulin resistance, is central to the increase in SeP. Crown Copyright © 2016 Published by Elsevier Ltd on behalf of Asia Oceania Association for the Study of Obesity. All rights reserved.
Correspondence to: SAHMRI, North Terrace, PO Box 11060, Adelaide, SA 5000, Australia. E-mail address: [email protected] (L.K. Heilbronn).
http://dx.doi.org/10.1016/j.orcp.2016.07.004 1871-403X/Crown Copyright © 2016 Published by Elsevier Ltd on behalf of Asia Oceania Association for the Study of Obesity. All rights reserved.
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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2
M. Chen et al.
Introduction
Methods
The liver plays a key role in maintaining glucose and lipid homeostasis [1]. Accumulation of fat in the liver relates more strongly with metabolic syndrome, insulin resistance and type 2 diabetes than increased body fat and visceral fat mass [2]. Accumulating evidence suggests that liver derived cytokines or ‘‘hepatokines’’ that are altered in obesity may directly contribute to development of insulin resistance and metabolic disease [1]. Selenoprotein P (SeP) is encoded by the SeP, plasma 1 (SEPP1) gene. It is an abundant extracellular glycoprotein, the circulating form of which is predominantly secreted by the liver, although all tissues express the protein [3]. SeP comprises ten selenocysteine residues [4], serves as a major circulating selenium transporter, and plays an important role in protecting cells against oxidative stress due to its phospholipid hydroperoxide thiol peroxidase activity [3]. Circulating SeP levels and hepatic expression of SeP are elevated in patients with type 2 diabetes [5]. Serum SeP levels are also increased in patients who are overweight and obese, have prediabetes, or non-alcoholic fatty liver disease (NAFLD) versus lean healthy individuals, and correlate with insulin resistance, inflammation and atherosclerosis [5—7]. However, it is unclear whether serum SeP is causal in development of insulin resistance, and whether this occurs independently of obesity in humans. Administration of purified human SeP at physiological levels inhibits insulin signalling in hepatocytes and myocytes, and impairs glucose metabolism in C57BL/6J mice [5]. Treatment with metformin reduces hepatic Sepp1 gene expression and secretion in both rats and mice, by activating AMPK and subsequently inactivating FoxO3a [8,9]. Furthermore, knockout and RNA interference-mediated knockdown of SeP improved glucose tolerance and insulin sensitivity in KKAy mice with type 2 diabetes [5]. Paradoxically however, emerging reports are that adipose tissue expression of Sepp1 is reduced in obese rodents [10]. Whether this dichotomy between circulating SeP and Sepp1 adipose tissue expression exists in humans is unknown. The aim of this study was to measure plasma SeP levels in individuals who are lean or overweight/obese, and examine the relationships between plasma SeP and fasting glucose, fasting insulin, insulin sensitivity, and in subcutaneous adipose tissue, the gene expression of SEPP1 and markers of oxidative stress, inflammation and angiogenesis.
Subjects Fasting blood samples were obtained from 63 individuals who participated in three separate studies that were conducted in our clinical research facility between 2011—2015. One study recruited young lean and obese individuals aged >17—26 years (N = 34) [11], one study recruited overweight and obese males aged >18 years (N = 9) and one study recruited overweight and obese females aged >35 years (N = 20). In all studies, individuals were excluded if they reported any significant medical conditions, or took medications known to affect glucose metabolism, energy metabolism, body weight, or appetite, if they smoked cigarettes, or drank >140 g of alcohol/week. All female participants were tested in the follicular phase of their menstrual cycle. The Royal Adelaide Hospital Research Ethics Committee approved the study protocols, and subjects provided written, informed consent. Studies are registered with clinicaltrials.gov (NCT01230632, NCT02009813, NCT01769976, respectively).
Metabolic tests Participants attended the clinical research facility at 8am after a 12-h overnight fast at baseline. Weight and height were measured in a hospital gown after voiding and fasting blood samples were obtained. In a subset of 25 subjects (14 men and 11 women; 15 leans and 10 subjects with overweight/obese), a needle biopsy of periumbilical subcutaneous adipose tissue was performed by previously described methods [12] to obtain ∼150 mg of tissue which was snap frozen for later analysis. Insulin sensitivity was measured using a 2-h hyperinsulinaemic—euglycaemic clamp (at 60—80 mU/m2 body surface/min). Glucose was infused at a variable rate to maintain glucose at 5.0 mmol/l and the steady-state glucose infusion rate (GIR) was normalized to fat free mass (FFM) and plasma insulin levels as described previously [13]. The formula is GIR × [average group steady state insulin/individual steady state insulin] and adjusted for metabolic size (FFM + 17.7 kg) [13]. The plasma steady state insulin levels were not statistically different between BMI groups (P = 0.49). Homeostasis model of assessment—–insulin resistance (HOMA-IR) was calculated as fasting glucose (mmol/l) × fasting insulin (mU/L)/22.5.
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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Selenoprotein P is elevated in individuals with obesity
Quantitative real-time PCR Total RNA were extracted from adipose tissue (100—150 mg) using Trizol (Invitrogen, USA) following manufacturer’s instructions. The concentration and purity of RNA were determined by Nanodrop (Thermo Fisher Scientific, California, USA). cDNA was synthesized from 800 ng of each RNA sample in 20 ul reactions using the QuantiTect reverse transcription kit (Qiagen, Valencia, CA) consistent with the manufacturer’s protocol. Standard control samples (25 ng/l) pooled from each cDNA were diluted to create a standard curve. Quantitative real-time PCR was performed with the ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA) using TaqMan primers and probes for CD40 (Hs01002913 g1), CD68 (Hs02836816 g1), CD163 (Hs00174705 m1), MCP-1 (chemokine (C-C motif) ligand 2, Hs00234140 m1), SOD1 (superoxide dismutase 1, Hs00533490 m1), SOD2 (superoxide dismutase 2, Hs00167309 m1), GPX1 (glutathione peroxidase, Hs00829989 gH), CAT (catalase, Hs00156308 m1), VEGF-A (vascular endothelial growth factor A, Hs00900055 m1), SEPP1 (Hs01032845 m1), and reference gene ACTB (beta-actin, Hs99999903-ml) (Applied Biosystems) according to manufacturer’s instructions. The samples were run in duplicate with internal negative controls and a standard curve. Data were analysed using the 2−(CT) method and expressed as the fold change relative to a calibrator sample, which was included in each run.
Biochemical analysis Plasma glucose concentrations were measured using commercial enzymatic kits (Beckman Coulter Inc, Brea, CA, USA) on a Beckman AU480 clinical analyser (Beckman Coulter Inc, Brea, CA, USA). Plasma insulin was assayed by radioimmunoassay (Millipore, St Charles, MO, USA). Blood lipids were examined by photometric assays in the laboratory of SA Pathology, South Australia. Plasma SeP levels were measured by an enzyme-linked immunosorbent assay (ELISA) (Cat. No.SEB809Hu, Uscn Life Science Inc., Wuhan, China) with intra- and interassay coefficients of variation (CVs) <10% and <12% respectively.
Statistics Data are shown as mean ± standard deviation (SD), unless otherwise stated. Statistics were analysed with SPSS 23 (SPSS, Chicago, IL, USA). Data were lntransformed for analysis if they were not normally distributed. Baseline differences were analysed by
3 two-tailed Student’s t-test. Mann—Whitney U tests were used for the statistical analysis of fasting insulin and SeP. Correlations were calculated using Pearson Correlation Coefficients or Partial Correlations controlling for body mass index, sex and age. Differences were considered statistically significant at P < 0.05. Lean is defined as BMI <25, whereas overweight and obese are defined as having a BMI >25—< 30 or a BMI> = 30 respectively.
Results As shown in Table 1, individuals who were overweight/obese were older (P < 0.001), and had increased LDL cholesterol, triglycerides, fasting insulin levels and HOMA-IR, and lower peripheral insulin sensitivity expressed as GIR/FFM adjusted for steady-state insulin (P < 0.04). SeP levels were significantly higher in individuals who were overweight and obese (Table 1, P < 0.001). SeP was correlated with BMI (Fig. 1A, r = 0.69, P < 0.0001). After adjustment for sex and age, the correlation was still significant (r = 0.55, P < 0.0001). SeP levels were positively correlated with fasting insulin (r = 0.36, P = 0.006) and HOMA-IR (r = 0.37, P = 0.005), and inversely correlated with peripheral insulin sensitivity (r = −0.39, P = 0.005) (Fig. 1B,C), but this relationship was lost after adjustment with BMI. SeP levels were positively correlated with total cholesterol (r = 0.46, P = 0.001), LDL cholesterol (r = 0.35, P = 0.02) and triglycerides (r = 0.48, P = 0.001), but this correlation was only significant with total cholesterols after adjustment with BMI, sex and age (r = 0.37, P = 0.01). SEPP1 gene expression in subcutaneous adipose tissue was not statistically different between individuals who were lean or overweight/obese (Table 1), but was correlated negatively with BMI (r = −0.44, P = 0.03). SEPP1 mRNA was also associated negatively with fasting insulin (r = −0.49 P = 0.014), HOMA-IR (r = −0.46, P = 0.02), and positively with peripheral insulin sensitivity (r = 0.49, P = 0.01), but this was not independent of BMI, sex and age. There was no correlation between plasma SeP levels and SEPP1 gene expression in subcutaneous adipose tissue. Gene expression of GPX1, CD68 and CD163 was increased, whereas VEGF-A mRNA levels were decreased in subcutaneous adipose tissue in individuals who were overweight and obese compared with lean (P < 0.01, Table 1). Plasma SeP levels were negatively correlated with gene expression of VEGF-A (r = −0.68, P = 0.0002) in subcutaneous adipose tissue after adjustment of BMI, sex and age (Fig. 1D). SeP levels
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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M. Chen et al. Table 1
Subject’s characteristics.
Variable
Whole cohort
Lean
Overweight/obese
P Value
N (M/F) Age (Years) Weight (Kg) Height (Cm) Body mass index (Kg/m2 ) Total cholesterol (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) Triglycerides (mmol/l) Fasting glucose (mmol/l) Fasting insulin (U/ml) HOMA-IR GIR*SSIR/FFM (mol/kg*U/ml/min) SeP (g/ml) SEPP1 mRNA expression# VEGFa mRNA expression# GPX1 mRNA expression# CD68 mRNA expression# CD163 mRNA expression#
63 23.2 ± 15.5 75.7 ± 16.8 169 ± 8 26.4 ± 5.4 4.6 ± 0.7 1.5 ± 0.4 2.6 ± 0.6 1.0 ± 0.4 5.0 ± 0.5 14.4 ± 5.5 3.2 ± 1.5 64 ± 28 32.7 ± 34.2 1.1 ± 0.2 1.0 ± 0.2 1.1 ± 0.3 1.1 ± 0.2 1.3 ± 0.3
29 (9/20) 20.9 ± 2.5 62.1 ± 9.4 168 ± 9 21.3 ± 2.2 4.4 ± 0.8 1.5 ± 0.4 2.5 ± 0.6 0.9 ± 0.4 4.9 ± 0.3 11.2 ± 2.6 2.4 ± 0.6 73 ± 27 14.5 ± 12.8 1.1 ± 0.2 1.1 ± 0.2 0.9 ± 0.2 1.0 ± 0.2 1.1 ± 0.3
34 (10/24) 43.8 ± 14.2 87.3 ± 12.6 170 ± 8 30.8 ± 2.9 4.8 ± 0.6 1.4 ± 0.3 2.8 ± 0.5 1.1 ± 0.5 5.1 ± 0.6 17.1 ± 6.0 3.9 ± 1.6 55 ± 25 52.3 ± 39.1 1.0 ± 0.3 0.8 ± 0.2 1.4 ± 0.3 1.3 ± 0.2 1.5 ± 0.2
<0.001 <0.001 0.31 0.001 0.12 0.24 0.03 0.04 0.12 0.001 0.001 0.01 0.001 0.15 0.001 0.001 0.001 0.005
Data are presented as mean ± SD. HOMA-IR: homeostasis model of assessment—–insulin resistance; GIR: steady-state glucose infusion rate; FFM: fat free mass SSIR: steady-state insulin ratio (average group steady state insulin/individual steady state insulin). Mann—Whitney U tests were used for the statistical analysis of fasting insulin and SeP. # N = 25. Lean is defined as BMI <25, whereas overweight and obese are defined as having a BMI >25—< 30 or a BMI> = 30 respectively.
Figure 1 Correlations between SeP levels and BMI (A), HOMA-IR (B), peripheral insulin sensitivity GIR*SSIR/FFM (C), and gene expression of VEGF-A (D) in subcutaneous adipose tissue. Significance in the correlations between SeP and measures of insulin sensitivity were lost after adjustment with BMI (1B, 1C).
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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Selenoprotein P is elevated in individuals with obesity were also positively correlated with GPX1 expression (r = 0.42, P = 0.03), CD68 expression (r = 0.54, P = 0.005) and CD163 expression (r = 0.41, P = 0.04). However, these associations were lost after adjustment with BMI.
Discussion Obesity is associated with fatty liver, and changes in the secretion of liver derived ‘‘hepatokines’’, that are increasingly being recognised to contribute towards insulin resistance and metabolic disease. Elevations in hepatic Sepp1 mRNA and serum SeP levels are observed in rodent models of T2DM as well as in patients with T2DM [5]. Serum SeP levels have also been correlated positively with BMI, fasting glucose and fasting insulin in individuals who are overweight or obese, display impaired glucose tolerance or NAFLD vs lean individuals [5—7]. Consistent with this, we observed that SeP levels were increased in overweight and obese individuals, and that this correlated with both HOMA-IR and peripheral insulin sensitivity by clamp. The age difference between groups remains a limitation to interpretation of this data despite adjustment. Of note, this association was lost after adjustment with BMI, suggesting this relationship may be mediated primarily by adiposity. This is supported by a Korean study in which circulating SeP concentrations were independently associated with visceral fat area and hepatic fat content in subjects with visceral obesity [7]. We did not obtain a measure of central or hepatic adiposity, and cannot perform this same analysis, which is another limitation of this study. Also, pregnant women with and without gestational diabetes displayed similar levels of SeP, but SeP was correlated with BMI [14]. During weight gain, significant adipose tissue expansion and remodelling occurs. This requires the expansion of new vasculature through angiogenesis, adipogenesis and changes to the extracellular matrix [15]. Impairments in angiogenesis in adipose tissue has been linked with development of insulin resistance and metabolic disease [15—17]. Vascular endothelial growth factor (VEGF) is a master regulator of angiogenesis, and likely plays an important role in promoting appropriate adipose tissue remodelling during weight gain [18], although its role in humans is controversial with reports of increases [17,19], and decreases in VEGF in obesity [20]. SeP has been shown to inhibit VEGF signalling and cell proliferation, tubule formation and migration in cultured human umbilical vein endothelial cells and impair wound closure [21]. In the present
5 study, we observed reduced gene expression of VEGF-A in subcutaneous adipose tissue in individuals who are overweight and obese, and an inverse correlation between circulating SeP levels and VEGF-A gene expression, and it is tempting to speculate that SeP could contribute to impaired adipose tissue remodelling and the development of insulin resistance in individuals who are obese. A possible role for SeP in adipogenesis has recently been identified [10]. In this study, SeP knockdown in pre-adipocytes decreased glutathione peroxidase activity, and increased the expression of inflammatory cytokines MCP-1 and IL-6. This was associated with a reduction in expression of the key regulator of adipogenesis, peroxisome proliferator-activated receptor gamma (PPAR-␥), and inhibition of adipogenesis in 3T3L1 cells [10]. Treatment with TNF alpha and hydrogen peroxide also downregulated Sepp1 gene expression in 3T3-L1 adipocytes [10]. This study showed that Sepp1 mRNA expression and SeP content in adipose tissue was decreased in diet induced obese mice, and administration of the anti-diabetic agent rosiglitazone increased SeP levels in adipose tissue [10]. In the present study, we did not observe statistical difference between BMI groups, however, this may be a reflection of the small sample size. We did note that the expression of SEPP1 in subcutaneous adipose tissue was negatively associated with BMI, insulin resistance by HOMA-IR and by hyperinsulinemic-euglycaemic clamp. Of note, there was no correlation between circulating levels of SeP and SEPP1 expression in adipose tissue. Together these studies suggest that in obesity, reductions in local SEPP1 expression may occur and could contribute to adipose tissue inflammation, oxidative stress and impaired adipogenesis, or vice versa. In conclusion, we observed that individuals who are overweight and obese have high circulating levels of SeP and tended to have lower levels of expression of SEPP1 in adipose tissue. The associations that were observed between SeP and markers of insulin resistance were lost after adjustment with BMI, suggesting that adiposity may be the main driver in this relationship, although this requires further study in larger, carefully controlled cohorts.
References [1] Stefan N, Haring HU. The role of hepatokines in metabolism. Nat Rev Endocrinol 2013;9:144—52. [2] Fabbrini E, Magkos F, Mohammed BS, Pietka T, Abumrad NA, Patterson BW, et al. Intrahepatic fat, not visceral fat, is
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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linked with metabolic complications of obesity. Proc Natl Acad Sci U S A 2009;106:15430—5. Burk RF, Hill KE. Selenoprotein P: an extracellular protein with unique physical characteristics and a role in selenium homeostasis. Annu Rev Nutr 2005;25:215—35. Burk RF, Hill KE, Motley AK. Plasma selenium in specific and non-specific forms. BioFactors 2001;14:107—14. Misu H, Takamura T, Takayama H, Hayashi H, MatsuzawaNagata N, Kurita S, et al. A liver-derived secretory protein, selenoprotein P, causes insulin resistance. Cell Metab 2010;12:483—95. Yang SJ, Hwang SY, Choi HY, Yoo HJ, Seo JA, Kim SG, et al. Serum selenoprotein P levels in patients with type 2 diabetes and prediabetes: implications for insulin resistance, inflammation, and atherosclerosis. J Clin Endocrinol Metab 2011;96:E1325—9. Choi HY, Hwang SY, Lee CH, Hong HC, Yang SJ, Yoo HJ, et al. Increased selenoprotein p levels in subjects with visceral obesity and nonalcoholic Fatty liver disease. Diabetes Metab J 2013;37:63—71. Takayama H, Misu H, Iwama H, Chikamoto K, Saito Y, Murao K, et al. Metformin suppresses expression of the selenoprotein P gene via an AMP-activated kinase (AMPK)/FoxO3a pathway in H4IIEC3 hepatocytes. J Biol Chem 2014;289:335—45. Speckmann B, Sies H, Steinbrenner H. Attenuation of hepatic expression and secretion of selenoprotein P by metformin. Biochem Biophys Res Commun 2009;387:158—63. Zhang Y, Chen X. Reducing selenoprotein P expression suppresses adipocyte differentiation as a result of increased preadipocyte inflammation. Am J Physiol Endocrinol Metab 2011;300:E77—85. Chen M, Wu L, Zhao J, Wu F, Davies MJ, Wittert GA, et al. Altered glucose metabolism in mouse and humans conceived by IVF. Diabetes 2014;63:3189—98. Heilbronn LK, Gan SK, Turner N, Campbell LV, Chisholm DJ. Markers of mitochondrial biogenesis and metabolism are
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lower in overweight and obese insulin-resistant subjects. J Clin Endocrinol Metab 2007;92:1467—73. Tam CS, Xie W, Johnson WD, Cefalu WT, Redman LM, Ravussin E. Defining insulin resistance from hyperinsulinemic-euglycemic clamps. Diabetes Care 2012;35:1605—10. Altinova AE, Iyidir OT, Ozkan C, Ors D, Ozturk M, Gulbahar O, et al. Selenoprotein P is not elevated in gestational diabetes mellitus. Gynecol Endocrinol 2015;31:874—6. Heilbronn LK, Liu B. Do adipose tissue macrophages promote insulin resistance or adipose tissue remodelling in humans? Horm Mol Biol Clin Investig 2014;20:3—13. Corvera S, Gealekman O. Adipose tissue angiogenesis: impact on obesity and type-2 diabetes. Biochim Biophys Acta 1842;2014:463—72. Gealekman O, Guseva N, Hartigan C, Apotheker S, Gorgoglione M, Gurav K, et al. Depot-specific differences and insufficient subcutaneous adipose tissue angiogenesis in human obesity. Circulation 2011;123:186—94. Sun K, Kusminski CM, Scherer PE. Adipose tissue remodeling and obesity. J Clin Invest 2011;121:2094—101. Tinahones FJ, Coin-Araguez L, Mayas MD, Garcia-Fuentes E, Hurtado-Del-Pozo C, Vendrell J, et al. Obesity-associated insulin resistance is correlated to adipose tissue vascular endothelial growth factors and metalloproteinase levels. BMC Physiol 2012;12:4. Pasarica M, Sereda OR, Redman LM, Albarado DC, Hymel DT, Roan LE, et al. Reduced adipose tissue oxygenation in human obesity: evidence for rarefaction, macrophage chemotaxis, and inflammation without an angiogenic response. Diabetes 2009;58:718—25. Ishikura K, Misu H, Kumazaki M, Takayama H, MatsuzawaNagata N, Tajima N, et al. Selenoprotein P as a diabetes-associated hepatokine that impairs angiogenesis by inducing VEGF resistance in vascular endothelial cells. Diabetologia 2014;57:1968—76.
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Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
ARTICLE IN PRESS
Obesity Research & Clinical Practice (2016) xxx, xxx—xxx
SHORT COMMUNICATION
Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance Miaoxin Chen a,b,c, Bo Liu b, David Wilkinson b,d, Amy T. Hutchison b, Campbell H. Thompson b, Gary A. Wittert b, Leonie K. Heilbronn b,c,∗ a
Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, China b Discipline of Medicine, University of Adelaide, Australia c Research Centre for Reproductive Health, Robinson Research Institute, University of Adelaide, Australia d Discipline of Acute Care Medicine, University of Adelaide, Australia Received 22 June 2016 ; received in revised form 19 July 2016; accepted 22 July 2016
KEYWORDS Selenoprotein P; Obesity; Insulin resistance
∗
Summary Selenoprotein P (SeP) is secreted primarily by the liver and postulated to cause insulin resistance. The aim of this study was to measure plasma SeP in individuals who are lean (N = 29) or overweight/obese (N = 34), and examine relationships between circulating SeP, SEPP1 (SeP, plasma 1) expression in subcutaneous adipose tissue, and markers of insulin resistance. SeP was higher in individuals who were overweight/obese (P < 0.001), and associated with insulin resistance by HOMA-IR and by clamp, but not independently of BMI. SEPP1 mRNA was correlated negatively with BMI, suggesting there may be tissue specific regulation. This study suggests that obesity, rather than insulin resistance, is central to the increase in SeP. Crown Copyright © 2016 Published by Elsevier Ltd on behalf of Asia Oceania Association for the Study of Obesity. All rights reserved.
Correspondence to: SAHMRI, North Terrace, PO Box 11060, Adelaide, SA 5000, Australia. E-mail address: [email protected] (L.K. Heilbronn).
http://dx.doi.org/10.1016/j.orcp.2016.07.004 1871-403X/Crown Copyright © 2016 Published by Elsevier Ltd on behalf of Asia Oceania Association for the Study of Obesity. All rights reserved.
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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M. Chen et al.
Introduction
Methods
The liver plays a key role in maintaining glucose and lipid homeostasis [1]. Accumulation of fat in the liver relates more strongly with metabolic syndrome, insulin resistance and type 2 diabetes than increased body fat and visceral fat mass [2]. Accumulating evidence suggests that liver derived cytokines or ‘‘hepatokines’’ that are altered in obesity may directly contribute to development of insulin resistance and metabolic disease [1]. Selenoprotein P (SeP) is encoded by the SeP, plasma 1 (SEPP1) gene. It is an abundant extracellular glycoprotein, the circulating form of which is predominantly secreted by the liver, although all tissues express the protein [3]. SeP comprises ten selenocysteine residues [4], serves as a major circulating selenium transporter, and plays an important role in protecting cells against oxidative stress due to its phospholipid hydroperoxide thiol peroxidase activity [3]. Circulating SeP levels and hepatic expression of SeP are elevated in patients with type 2 diabetes [5]. Serum SeP levels are also increased in patients who are overweight and obese, have prediabetes, or non-alcoholic fatty liver disease (NAFLD) versus lean healthy individuals, and correlate with insulin resistance, inflammation and atherosclerosis [5—7]. However, it is unclear whether serum SeP is causal in development of insulin resistance, and whether this occurs independently of obesity in humans. Administration of purified human SeP at physiological levels inhibits insulin signalling in hepatocytes and myocytes, and impairs glucose metabolism in C57BL/6J mice [5]. Treatment with metformin reduces hepatic Sepp1 gene expression and secretion in both rats and mice, by activating AMPK and subsequently inactivating FoxO3a [8,9]. Furthermore, knockout and RNA interference-mediated knockdown of SeP improved glucose tolerance and insulin sensitivity in KKAy mice with type 2 diabetes [5]. Paradoxically however, emerging reports are that adipose tissue expression of Sepp1 is reduced in obese rodents [10]. Whether this dichotomy between circulating SeP and Sepp1 adipose tissue expression exists in humans is unknown. The aim of this study was to measure plasma SeP levels in individuals who are lean or overweight/obese, and examine the relationships between plasma SeP and fasting glucose, fasting insulin, insulin sensitivity, and in subcutaneous adipose tissue, the gene expression of SEPP1 and markers of oxidative stress, inflammation and angiogenesis.
Subjects Fasting blood samples were obtained from 63 individuals who participated in three separate studies that were conducted in our clinical research facility between 2011—2015. One study recruited young lean and obese individuals aged >17—26 years (N = 34) [11], one study recruited overweight and obese males aged >18 years (N = 9) and one study recruited overweight and obese females aged >35 years (N = 20). In all studies, individuals were excluded if they reported any significant medical conditions, or took medications known to affect glucose metabolism, energy metabolism, body weight, or appetite, if they smoked cigarettes, or drank >140 g of alcohol/week. All female participants were tested in the follicular phase of their menstrual cycle. The Royal Adelaide Hospital Research Ethics Committee approved the study protocols, and subjects provided written, informed consent. Studies are registered with clinicaltrials.gov (NCT01230632, NCT02009813, NCT01769976, respectively).
Metabolic tests Participants attended the clinical research facility at 8am after a 12-h overnight fast at baseline. Weight and height were measured in a hospital gown after voiding and fasting blood samples were obtained. In a subset of 25 subjects (14 men and 11 women; 15 leans and 10 subjects with overweight/obese), a needle biopsy of periumbilical subcutaneous adipose tissue was performed by previously described methods [12] to obtain ∼150 mg of tissue which was snap frozen for later analysis. Insulin sensitivity was measured using a 2-h hyperinsulinaemic—euglycaemic clamp (at 60—80 mU/m2 body surface/min). Glucose was infused at a variable rate to maintain glucose at 5.0 mmol/l and the steady-state glucose infusion rate (GIR) was normalized to fat free mass (FFM) and plasma insulin levels as described previously [13]. The formula is GIR × [average group steady state insulin/individual steady state insulin] and adjusted for metabolic size (FFM + 17.7 kg) [13]. The plasma steady state insulin levels were not statistically different between BMI groups (P = 0.49). Homeostasis model of assessment—–insulin resistance (HOMA-IR) was calculated as fasting glucose (mmol/l) × fasting insulin (mU/L)/22.5.
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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Selenoprotein P is elevated in individuals with obesity
Quantitative real-time PCR Total RNA were extracted from adipose tissue (100—150 mg) using Trizol (Invitrogen, USA) following manufacturer’s instructions. The concentration and purity of RNA were determined by Nanodrop (Thermo Fisher Scientific, California, USA). cDNA was synthesized from 800 ng of each RNA sample in 20 ul reactions using the QuantiTect reverse transcription kit (Qiagen, Valencia, CA) consistent with the manufacturer’s protocol. Standard control samples (25 ng/l) pooled from each cDNA were diluted to create a standard curve. Quantitative real-time PCR was performed with the ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA) using TaqMan primers and probes for CD40 (Hs01002913 g1), CD68 (Hs02836816 g1), CD163 (Hs00174705 m1), MCP-1 (chemokine (C-C motif) ligand 2, Hs00234140 m1), SOD1 (superoxide dismutase 1, Hs00533490 m1), SOD2 (superoxide dismutase 2, Hs00167309 m1), GPX1 (glutathione peroxidase, Hs00829989 gH), CAT (catalase, Hs00156308 m1), VEGF-A (vascular endothelial growth factor A, Hs00900055 m1), SEPP1 (Hs01032845 m1), and reference gene ACTB (beta-actin, Hs99999903-ml) (Applied Biosystems) according to manufacturer’s instructions. The samples were run in duplicate with internal negative controls and a standard curve. Data were analysed using the 2−(CT) method and expressed as the fold change relative to a calibrator sample, which was included in each run.
Biochemical analysis Plasma glucose concentrations were measured using commercial enzymatic kits (Beckman Coulter Inc, Brea, CA, USA) on a Beckman AU480 clinical analyser (Beckman Coulter Inc, Brea, CA, USA). Plasma insulin was assayed by radioimmunoassay (Millipore, St Charles, MO, USA). Blood lipids were examined by photometric assays in the laboratory of SA Pathology, South Australia. Plasma SeP levels were measured by an enzyme-linked immunosorbent assay (ELISA) (Cat. No.SEB809Hu, Uscn Life Science Inc., Wuhan, China) with intra- and interassay coefficients of variation (CVs) <10% and <12% respectively.
Statistics Data are shown as mean ± standard deviation (SD), unless otherwise stated. Statistics were analysed with SPSS 23 (SPSS, Chicago, IL, USA). Data were lntransformed for analysis if they were not normally distributed. Baseline differences were analysed by
3 two-tailed Student’s t-test. Mann—Whitney U tests were used for the statistical analysis of fasting insulin and SeP. Correlations were calculated using Pearson Correlation Coefficients or Partial Correlations controlling for body mass index, sex and age. Differences were considered statistically significant at P < 0.05. Lean is defined as BMI <25, whereas overweight and obese are defined as having a BMI >25—< 30 or a BMI> = 30 respectively.
Results As shown in Table 1, individuals who were overweight/obese were older (P < 0.001), and had increased LDL cholesterol, triglycerides, fasting insulin levels and HOMA-IR, and lower peripheral insulin sensitivity expressed as GIR/FFM adjusted for steady-state insulin (P < 0.04). SeP levels were significantly higher in individuals who were overweight and obese (Table 1, P < 0.001). SeP was correlated with BMI (Fig. 1A, r = 0.69, P < 0.0001). After adjustment for sex and age, the correlation was still significant (r = 0.55, P < 0.0001). SeP levels were positively correlated with fasting insulin (r = 0.36, P = 0.006) and HOMA-IR (r = 0.37, P = 0.005), and inversely correlated with peripheral insulin sensitivity (r = −0.39, P = 0.005) (Fig. 1B,C), but this relationship was lost after adjustment with BMI. SeP levels were positively correlated with total cholesterol (r = 0.46, P = 0.001), LDL cholesterol (r = 0.35, P = 0.02) and triglycerides (r = 0.48, P = 0.001), but this correlation was only significant with total cholesterols after adjustment with BMI, sex and age (r = 0.37, P = 0.01). SEPP1 gene expression in subcutaneous adipose tissue was not statistically different between individuals who were lean or overweight/obese (Table 1), but was correlated negatively with BMI (r = −0.44, P = 0.03). SEPP1 mRNA was also associated negatively with fasting insulin (r = −0.49 P = 0.014), HOMA-IR (r = −0.46, P = 0.02), and positively with peripheral insulin sensitivity (r = 0.49, P = 0.01), but this was not independent of BMI, sex and age. There was no correlation between plasma SeP levels and SEPP1 gene expression in subcutaneous adipose tissue. Gene expression of GPX1, CD68 and CD163 was increased, whereas VEGF-A mRNA levels were decreased in subcutaneous adipose tissue in individuals who were overweight and obese compared with lean (P < 0.01, Table 1). Plasma SeP levels were negatively correlated with gene expression of VEGF-A (r = −0.68, P = 0.0002) in subcutaneous adipose tissue after adjustment of BMI, sex and age (Fig. 1D). SeP levels
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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M. Chen et al. Table 1
Subject’s characteristics.
Variable
Whole cohort
Lean
Overweight/obese
P Value
N (M/F) Age (Years) Weight (Kg) Height (Cm) Body mass index (Kg/m2 ) Total cholesterol (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) Triglycerides (mmol/l) Fasting glucose (mmol/l) Fasting insulin (U/ml) HOMA-IR GIR*SSIR/FFM (mol/kg*U/ml/min) SeP (g/ml) SEPP1 mRNA expression# VEGFa mRNA expression# GPX1 mRNA expression# CD68 mRNA expression# CD163 mRNA expression#
63 23.2 ± 15.5 75.7 ± 16.8 169 ± 8 26.4 ± 5.4 4.6 ± 0.7 1.5 ± 0.4 2.6 ± 0.6 1.0 ± 0.4 5.0 ± 0.5 14.4 ± 5.5 3.2 ± 1.5 64 ± 28 32.7 ± 34.2 1.1 ± 0.2 1.0 ± 0.2 1.1 ± 0.3 1.1 ± 0.2 1.3 ± 0.3
29 (9/20) 20.9 ± 2.5 62.1 ± 9.4 168 ± 9 21.3 ± 2.2 4.4 ± 0.8 1.5 ± 0.4 2.5 ± 0.6 0.9 ± 0.4 4.9 ± 0.3 11.2 ± 2.6 2.4 ± 0.6 73 ± 27 14.5 ± 12.8 1.1 ± 0.2 1.1 ± 0.2 0.9 ± 0.2 1.0 ± 0.2 1.1 ± 0.3
34 (10/24) 43.8 ± 14.2 87.3 ± 12.6 170 ± 8 30.8 ± 2.9 4.8 ± 0.6 1.4 ± 0.3 2.8 ± 0.5 1.1 ± 0.5 5.1 ± 0.6 17.1 ± 6.0 3.9 ± 1.6 55 ± 25 52.3 ± 39.1 1.0 ± 0.3 0.8 ± 0.2 1.4 ± 0.3 1.3 ± 0.2 1.5 ± 0.2
<0.001 <0.001 0.31 0.001 0.12 0.24 0.03 0.04 0.12 0.001 0.001 0.01 0.001 0.15 0.001 0.001 0.001 0.005
Data are presented as mean ± SD. HOMA-IR: homeostasis model of assessment—–insulin resistance; GIR: steady-state glucose infusion rate; FFM: fat free mass SSIR: steady-state insulin ratio (average group steady state insulin/individual steady state insulin). Mann—Whitney U tests were used for the statistical analysis of fasting insulin and SeP. # N = 25. Lean is defined as BMI <25, whereas overweight and obese are defined as having a BMI >25—< 30 or a BMI> = 30 respectively.
Figure 1 Correlations between SeP levels and BMI (A), HOMA-IR (B), peripheral insulin sensitivity GIR*SSIR/FFM (C), and gene expression of VEGF-A (D) in subcutaneous adipose tissue. Significance in the correlations between SeP and measures of insulin sensitivity were lost after adjustment with BMI (1B, 1C).
Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004
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Selenoprotein P is elevated in individuals with obesity were also positively correlated with GPX1 expression (r = 0.42, P = 0.03), CD68 expression (r = 0.54, P = 0.005) and CD163 expression (r = 0.41, P = 0.04). However, these associations were lost after adjustment with BMI.
Discussion Obesity is associated with fatty liver, and changes in the secretion of liver derived ‘‘hepatokines’’, that are increasingly being recognised to contribute towards insulin resistance and metabolic disease. Elevations in hepatic Sepp1 mRNA and serum SeP levels are observed in rodent models of T2DM as well as in patients with T2DM [5]. Serum SeP levels have also been correlated positively with BMI, fasting glucose and fasting insulin in individuals who are overweight or obese, display impaired glucose tolerance or NAFLD vs lean individuals [5—7]. Consistent with this, we observed that SeP levels were increased in overweight and obese individuals, and that this correlated with both HOMA-IR and peripheral insulin sensitivity by clamp. The age difference between groups remains a limitation to interpretation of this data despite adjustment. Of note, this association was lost after adjustment with BMI, suggesting this relationship may be mediated primarily by adiposity. This is supported by a Korean study in which circulating SeP concentrations were independently associated with visceral fat area and hepatic fat content in subjects with visceral obesity [7]. We did not obtain a measure of central or hepatic adiposity, and cannot perform this same analysis, which is another limitation of this study. Also, pregnant women with and without gestational diabetes displayed similar levels of SeP, but SeP was correlated with BMI [14]. During weight gain, significant adipose tissue expansion and remodelling occurs. This requires the expansion of new vasculature through angiogenesis, adipogenesis and changes to the extracellular matrix [15]. Impairments in angiogenesis in adipose tissue has been linked with development of insulin resistance and metabolic disease [15—17]. Vascular endothelial growth factor (VEGF) is a master regulator of angiogenesis, and likely plays an important role in promoting appropriate adipose tissue remodelling during weight gain [18], although its role in humans is controversial with reports of increases [17,19], and decreases in VEGF in obesity [20]. SeP has been shown to inhibit VEGF signalling and cell proliferation, tubule formation and migration in cultured human umbilical vein endothelial cells and impair wound closure [21]. In the present
5 study, we observed reduced gene expression of VEGF-A in subcutaneous adipose tissue in individuals who are overweight and obese, and an inverse correlation between circulating SeP levels and VEGF-A gene expression, and it is tempting to speculate that SeP could contribute to impaired adipose tissue remodelling and the development of insulin resistance in individuals who are obese. A possible role for SeP in adipogenesis has recently been identified [10]. In this study, SeP knockdown in pre-adipocytes decreased glutathione peroxidase activity, and increased the expression of inflammatory cytokines MCP-1 and IL-6. This was associated with a reduction in expression of the key regulator of adipogenesis, peroxisome proliferator-activated receptor gamma (PPAR-␥), and inhibition of adipogenesis in 3T3L1 cells [10]. Treatment with TNF alpha and hydrogen peroxide also downregulated Sepp1 gene expression in 3T3-L1 adipocytes [10]. This study showed that Sepp1 mRNA expression and SeP content in adipose tissue was decreased in diet induced obese mice, and administration of the anti-diabetic agent rosiglitazone increased SeP levels in adipose tissue [10]. In the present study, we did not observe statistical difference between BMI groups, however, this may be a reflection of the small sample size. We did note that the expression of SEPP1 in subcutaneous adipose tissue was negatively associated with BMI, insulin resistance by HOMA-IR and by hyperinsulinemic-euglycaemic clamp. Of note, there was no correlation between circulating levels of SeP and SEPP1 expression in adipose tissue. Together these studies suggest that in obesity, reductions in local SEPP1 expression may occur and could contribute to adipose tissue inflammation, oxidative stress and impaired adipogenesis, or vice versa. In conclusion, we observed that individuals who are overweight and obese have high circulating levels of SeP and tended to have lower levels of expression of SEPP1 in adipose tissue. The associations that were observed between SeP and markers of insulin resistance were lost after adjustment with BMI, suggesting that adiposity may be the main driver in this relationship, although this requires further study in larger, carefully controlled cohorts.
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Please cite this article in press as: Chen M, et al. Selenoprotein P is elevated in individuals with obesity, but is not independently associated with insulin resistance. Obes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.orcp.2016.07.004