METABOLIC SYNDROME AND RELATED DISORDERS Volume 13, Number 8, 2015 Mary Ann Liebert, Inc. Pp. 336–342 DOI: 10.1089/met.2014.0164
Relationship Between Serum IL-12 and p40 Subunit Concentrations and Lipid Parameters in Overweight and Obese Women Agnieszka Nikołajuk, PhD,1 Monika Karczewska-Kupczewska, MD, PhD,1,2 and Marek Straczkowski, MD, PhD1,2
Abstract
Background: Obesity is a chronic low-grade inflammatory state associated with the development of insulin resistance, type 2 diabetes mellitus, and atherosclerosis. Interleukin-12 (IL-12) is a proinflammatory cytokine composed of a 40-kD (p40) subunit and a 35-kD (p35) subunit. p40 might have an independent role in initiating the immune response. Recent findings indicate that IL-12 could be involved in the development of obesityassociated co-morbidities, especially atherosclerosis. It is unclear if there are alternations in circulating concentrations of total IL-12 and its subunit p40 in young subjects with increased adiposity without overt metabolic disturbances. The aim of the present study was to estimate serum total IL-12 together with its p40 subunit in young overweight and obese women and to investigate the associations of these parameters with insulin sensitivity and serum lipids. Methods: We studied 77 healthy women (37 lean and 40 obese or overweight). Anthropometric measurements, blood biochemical analyses, determination of serum IL-12, IL-12p40 concentrations, and euglycemic– hyperinsulinemic clamp were performed. Results: We demonstrated an increase in serum p40 in obese subjects (P = 0.029). We found positive correlations between p40 and fat mass (r = 0.24, P = 0.04) and significant negative associations with high-density lipoprotein cholesterol (HDL-C) (r = - 0.27, P = 0.002). Detectable concentrations of serum IL-12 were observed in 55% of subjects. Individuals with detectable serum concentrations of IL-12 had significantly higher levels of serum triglycerides (P = 0.049). A significant association between IL-12 and serum total cholesterol (r = 0.32, P = 0.042) was observed in this subgroup. No association between p40 or IL-12 and insulin sensitivity was found. Conclusions: Our data indicate that the IL-12/IL-12p40 system may be associated with lipid abnormalities in obese subjects.
Introduction
A
relationship between obesity and chronic lowgrade inflammation has been demonstrated in many studies, suggesting that inflammation may be a potential mechanism in which obesity leads to diseases associated with insulin resistance, like type 2 diabetes mellitus (T2DM) and atherosclerosis.1–3 It seems that adipose tissue (AT) may play an important role as a site and a source of inflammation. Intensive rodent and human studies have shown that macrophages accumulate along with increasing adiposity.4–6 Although these data indicated that macrophages in AT rep-
resent a major source of proinflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), and interferong (IFN-g).4 Moreover, the macrophage proinflammatory phenotype has been well described in pathogenesis of obesityassociated insulin resistance.7 Recent studies suggest that the accumulation various types of T lymphocytes and T cell– derived cytokines may play a crucial role in adipose inflammation and insulin resistance.8–10 These authors observed an increase in the number of T helper 1 (Th1) polarized T cells and effector T cells and a decrease in number of T helper 2 (Th2) polarized T cells in obese AT.9–11 Moreover, it seems
1 Department of Prophylaxis of Metabolic Diseases, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Bialystok, Poland. 2 Department of Metabolic Diseases, Medical University of Bialystok, Bialystok Poland.
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plausible that T cells and Th1/Th2 cytokines effect macrophage responses during the development of obesity-associated inflammation and insulin resistance. Furthermore, these authors proposed that early lymphocyte infiltration of AT occurs before the accumulation of macrophages.9,10 However, despite significant progress in identifying mechanisms involved in obesity-associated inflammation, it is really not known what initiates the development of inflammation. IL-12 is a heterodimeric protein (p70) composed of two disulfide-linked subunits designated p35 (light-chain 35-kD) and p40 (heavy-chain 40-kD). Notably, while p40 is expressed and secreted as monomer and homodimer, p35 is never found by itself,12 and the p40 subunit is expressed in greater quantities than required for only p35p40 formation.13 Recent publications have suggested that IL-12p40 might have an independent role in initiating the immune response, and both pro- and anti-inflammatory functions have been ascribed.14 IL-12 is produced by monocytes, macrophages, dendritic cells, and natural killer cells. Importantly, IL-12 induces polarization of naı¨ve T cells to the Th1 phenotype and favors Th1 responses, including IFN-g and TNF-a production.15 Taking into account these data, it was hypothesized that IL-12 potentially plays an important role in systemic low-grade inflammation and development of obesity-related insulin resistance. Plasma IL-12 levels were found to be elevated in obesity16 and in patients with metabolic syndrome17 and T2DM with and without cardiovascular complications.18,19 In addition, IL-12 is expressed in atherosclerotic plaque and involved in atherogenesis; it probably accelerates the progress of macrovascular complications in T2DM.20–23 Furthermore, increased mRNA expression of IL-12p40 and IL-12p35 was reported in insulin-responsive tissues under conditions of obesity24 and AT associated proinflammatory macrophages. It is not clear whether the changes in the concentrations of IL-12 and its subunits are present in young people with an excess of AT without clear metabolic complications. Therefore, the aim of this study was to estimate circulating levels of IL-12 and IL-12p40 and their correlation with obesity, serum lipids, and insulin sensitivity evaluated by the euglycemic–hyperinsulinemic clamp in normal weight and overweight/obese women.
Materials and Methods The study group consisted of 77 normally menstruating young women; 40 women were overweight or obese [mean age 24.73 – 5.60 years with a mean body mass index (BMI) of 30.72 – 3.94 kg/m2] and 37 were lean, young women (mean age 24.35 – 4.39 years with BMI < 25 kg/m2). Before entering the study, a physical examination and appropriate laboratory tests were performed. All study participants had no cardiovascular disease, hypertension, hyperandrogenism, infections, or other serious medical problems; all were nonsmokers and were not taking any anti-inflammatory drugs or drugs known to affect glucose and lipid metabolism. All analyses were performed after an overnight fast. A standard 75-gram oral glucose tolerance test (OGTT) was performed, and all subjects had normal glucose tolerance according to the World Health Organization criteria. The study protocol was approved by Ethical Committee of Human Studies of the Medical University of Bialystok, Poland. All participants gave written informed consent before entering the study.
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Anthropometric measurements The BMI was calculated as body weight in kilograms divided by height in meters squared (kg/m2). The waist circumference was measured at the smallest circumference between the waist and thighs. The percent of body fat was assessed by bioelectric impedance analysis using a Tanita TBF-511 Body Fat Analyzer (Tanita Corp., Tokyo, Japan), and fat-free mass was calculated.
Insulin sensitivity Insulin sensitivity was evaluated by the euglycemic– hyperinsulinemic clamp, as previously described.25,26 The rate of whole-body glucose uptake (M value) was calculated as the mean glucose infusion rate from 80 to 120 min, corrected for glucose space and normalized per kilogram of fatfree mass (FFM).
Biochemical analyses Fasting blood samples were also taken from the antecubital vein before the beginning of the clamp. Before the determination of serum insulin, IL-12, and IL-12p40 concentrations, samples were frozen at -70C until analyses. Plasma glucose was measured immediately by the enzymatic method using a glucose analyzer (YSI 2300 STAT Plus, YSI, Inc., Yellow Springs, OH) for the OGTT and the clamp study. Serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs) were measured by an enzymatic method (Cormay, Warsaw, Poland). The concentration of low-density lipoprotein cholesterol (LDL-C) was calculated from the Friedewald formula. Serum insulin was measured with an immunoradiometric assay (IRMA; BioSource Europe, Nivelles, Belgium). Serum high-sensitivity IL-12 was measured with a quantitative sandwich enzyme immunoassay kit (R&D Systems, Minneapolis, MN) with a lowest detectable limit of 0.5 pg/ mL and with intra-assay and interassay coefficients of variation (CV) below 4.9% and 12.6% respectively. Serum IL-12p40 concentrations were determined with an enzymelinked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). The minimum detectable concentration was 15 pg/mL, and intra- and interassay CV values were, respectively, below 6.6% and 6.9%.
Statistical analysis The statistics were performed with STATISTICA 8.0 software. The variables that did not have normal distribution (IL-12p40, insulin, TGs) were log-transformed prior to analyses. For the purpose of presenting the data, these variables were transformed to absolute values (see Results section). The differences between the groups were estimated with an unpaired Student t-test. The relationships between variables were studied with the Pearson product-moment correlation analysis and with multiple regression analysis. The level of significance was accepted at P < 0.05.
Results The clinical characteristic of the studied groups is shown in Table 1. We found that obese women had higher serum postload glucose (P = 0.014), fasting insulin, and postload insulin (P = 0.009 and P = 0.003, respectively) than lean
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Table 1.
Age (years) Weight (kg) BMI (kg/m2) Waist (cm) Body fat (%) Fasting glucose (mg/dL) Postload glucose (mg/dL) Fasting insulin (mIU/mL) Postload insulin (mIU/mL) Cholesterol (mg/dL) Serum TGs (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) IL-12 (pg/mL) IL-12p40 (pg/mL) M (mg/kgFFM/min)
Clinical and Biochemical Characteristics of the Groups Studied Overweight/obese (n = 40)
Lean (n = 37)
P value
24.73 – 5.60 84.62 – 13.23 30.72 – 3.93 93.71 – 12.70 40.15 – 7.58 82.65 – 6.35 92.29 – 21.20 16.79 – 8.69 63.59 – 43.60 174.23 – 30.18 90.78 – 40.02 56.40 – 8.49 99.39 – 31.16 0.51 – 0.68 96.85 – 40.50 8.47 – 3.28
24.35 – 4.39 58.94 – 6.81 21.29 – 1.89 71.90 – 6.62 25.89 – 6.68 80.18 – 7.56 81.20 – 17.17 12.10 – 5.35 38.05 – 21.21 185.22 – 32.63 72.12 – 31.34 62.89 – 12.56 104.72 – 36.28 0.77 – 0.70 79.13 – 38.39 10.69 – 3.16
0.75 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.12 0.014 0.009 0.003 0.13 0.028 0.010 0.51 0.23 0.029 0.003
Data are presented as mean – standard deviation (SD). BMI, body mass index; TGs, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; IL12, interleukin 12; IL-12p40, subunit p40 of IL-12; M value, whole-body glucose uptake normalized per kilogram of fat-free mass (FFM), postload glucose, and insulin refer to value at 120 min of the oral glucose tolerance test.
women. We observed significantly higher serum TGs concentrations in the obese women in comparison with the lean women (P = 0.028). Additionally, obese women had significantly lower serum HDL-C and insulin sensitivity (P = 0.009 and P = 0.003, respectively).
We showed an increase in serum IL-12p40 in obese subjects (P = 0.029), but serum IL-12 did not differ between obese and control (lean) subjects (P = 0.23). We found positive correlations between IL-12p40 and fat mass (r = 0.24, P = 0.04) and significant negative associations with HDL-C (r = - 0.27,
FIG. 1. Correlations between serum concentrations of interleukin-12 (IL-12)p40 and high-density lipoprotein cholesterol (HDL-C) in the entire group (n = 77). The relationships between variables were studied with the Pearson product-moment correlation analysis. The level of significance was accepted at P < 0.05. Values of IL-12p40 are shown on a log-transformed scale. (Filled circle) overweight/obese group; (open circle) normal weight group; (solid line) slope of the correlation. The dotted lines indicate 95% confidence intervals.
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Table 2. Clinical and Biochemical Characteristics of the Studied Subgroups Subjects with nondetectable IL-12 (n = 34)
Subjects with detectable IL-12 (n = 43)
Weight (kg) 73.27 – 15.90 71.49 – 17.43 BMI (kg/m2) 26.50 – 5.13 25.95 – 6.11 Waist (cm) 83.76 – 13.88 82.80 – 15.91 Body fat (%) 32.50 – 10.39 33.63 – 9.95 Fasting glucose (mg/dL) 82.79 – 5.11 80.40 – 8.13 Fasting insulin (mIU/mL) 14.71 – 6.21 14.15 – 8.56 Cholesterol (mg/dL) 177.85 – 31.91 180.72 – 31.74 Serum TGs (mg/dL) 72.68 – 28.65 89.43 – 41.64* HDL-C (mg/dL) 58.61 – 8.45 60.10 – 12.81 LDL-C (mg/dL) 104.82 – 33.52 99.31 – 33.86 IL-12p40 87.46 – 35.73 89.03 – 43.89 Log IL12p40 1.90 – 0.20 1.90 – 0.20 M (mg/kgFFM/min) 9.27 – 3.23 9.74 – 3.53 Data are presented as mean – standard deviation (SD). The differences between the groups were estimated with an unpaired Student t-test and level of significance was accepted at *P < 0.05. IL-12, interleukin 12; BMI, body mass index; TGs, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; IL-12p40, subunit p40 of IL-12; M value, whole-body glucose uptake normalized per kilogram of fat-free mass (FFM).
P = 0.002) (Fig. 1) in the entire studied group. In multiple regression analysis, correlation between IL-12p40 and HDL-C was independent of fat mass (b = - 0.39, P = 0.024). We reported detectable concentration of serum IL-12 in 43 participants (55% of the entire group). Clinical and biochemical parameters in subjects with and without detectable IL-12 are shown in Table 2. Subjects with detectable and nondetectable IL-12 (ND IL-12) were similar in BMI, waist circumference, percent body fat, fasting glucose and insulin, serum cholesterol, HDL-C, and LDL-C. The serum IL-12p40 concentration and insulin sensitivity did not differ between these subgroups (P = 0.87 and P = 0.57, respectively). Individuals with detectable serum concentrations of IL-12 had slightly higher levels of serum TGs (detectable IL-12, serum TGs were 89.43 – 41.64 mg/dL; ND IL-12, serum TGs were 72.68 – 28.65 mg/dL; P = 0.049). In the subgroup analysis, we found a positive correlation between serum IL-12 and cholesterol in the subjects with detectable IL-12 (r = 0.32, P = 0.042). No association between p40 or IL-12 and insulin sensitivity was found.
Discussion In the present study, we demonstrated that the concentrations of IL-12 subunit p40 were increased in obese women and that about 55% of our group have detectable levels of IL-12. Our observations may be indirectly confirmed by the results of other authors. Higher concentrations of IL-12 in patients with metabolic syndrome were observed.17 Data showed that IL-12 plasma concentrations were elevated in T2DM,18 both in patients with antidiabetic therapy and newly diagnosed T2DM.19,27 Additionally, the authors also found that level of IL-12 was associated with obesity and insulin resistance estimated by homeostasis model assessment of insulin resistance (HOMA-IR).19,27
Our study did not reveal the significant difference between serum IL-12 concentrations in obese and lean women and the relation of IL-12 to anthropometric parameters, but we found positive correlations between IL-12p40 and fat mass in the entire group. Even though we observed significantly higher levels of postload glucose, fasting and postload insulin, serum p40, and lower insulin sensitivity in obese women in comparison to healthy controls, we did not prove any significant correlations between insulin sensitivity and serum level IL-12 or subunit p40 in the entire studied group as well as in the subgroups. Studies on the relationship between the concentration of IL-12 and insulin resistance are contradictory. As mentioned, some investigators have observed the relationship of this cytokine to insulin sensitivity.19,27 In contrast, in the study conducted by Suarez-Alvarez et al.,16 no significant associations were observed among serum level of IL-12 and fasting insulin and insulin resistance denoted as HOMA-IR. These differences may come from the heterogeneity of the studied groups and different degrees of insulin resistance as well as differences arising from the methods of measuring insulin action. In our study, we used the euglycemic– hyperinsulinemic clamp technique, which is appropriate and important to use as the reference method in measuring insulin sensitivity. In the above-cited studies, the authors assessed insulin resistance HOMA.16,17,19 In addition, HOMA reflects mainly hepatic insulin resistance, whereas the whole-body glucose uptake normalized per kilogram of FFM estimated by euglycemic clamp reflects peripheral (predominantly skeletal muscle) insulin sensitivity. Additionally, our obese group exhibited only mild insulin resistance, which may underestimate the direct association between concentrations of IL-12 and p40 and parameters of glucose and lipid metabolism. However, we performed only indirect estimations of fat free mass by bioelectric impedance, which may be a limitation of our study. It has been shown that hyperglycemia may increase levels of IL-12 mRNA in mouse macrophages28 and IL-12 production from peripheral blood mononuclear cells (PBMCs) was increased in hyperglycemic conditions.29,30 Furthermore, our entire study group was much younger than in the cited study and had normal glucose tolerance.18,19,27,29 It is generally accepted that dyslipidemia is the most common complication of metabolic syndrome and T2DM. Low HDL levels and hypertriglyceridemia are lipid and lipoprotein abnormalities often observed in insulin resistance.31,32 Consistent with these findings, in our study, the individuals with detectable serum concentrations of IL-12 had significantly higher levels of serum TGs. The subjects with and without detectable IL-12 had comparable anthropometric parameters, indicating that this association was independent of obesity. Furthermore, the analysis of the subgroup with detectable IL-12 indicated a positive association between serum IL-12 and TC. Similar results were observed by Suarez-Alvarez et al.16 It is worth pointing out that these investigators grouped their study population into two clusters according to high and low producers of IL-12. They observed that more than 70% of the high producers of IL-12 showed increased levels of TGs and increased production of IL-12 was related to body weight gain. In contrast to these studies in our subgroups, we did not observe any differences in anthropometric and biochemical parameters. The association between the acute and chronic
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inflammation with alterations in TGs and cholesterol metabolism was described.33–35 On the other hand, it is well known that production of this cytokine is tightly regulated by other factors, like IFN-g, TNF-a, and vice versa. Cross-regulatory actions between these cytokines may be responsible for enhanced lipolysis,36,37 de novo fatty acid synthesis,38 or inhibition of lipase lipoproteins activity.37 IL-12 may impair reverse cholesterol transport (RCT) and cholesterol efflux, alone or through an effect on other cytokines39 in the manner promoting atherosclerosis. An important limitation of the present study is that it does not show any cause-and-effect relationships. Our data did not show whether the high levels of IL-12 were a cause or a consequence of high TGs concentration. Finally, also we have to consider that in previous studies, concentrations of IL-12 were detected in all subjects in the general population,17,19,27 whereas we have shown the presence of this cytokine in only nearly half of our study population. The inconsistent results may be attributable to differences in the methods of assay and in subjects’ characteristics. Our study group was younger and without apparent metabolic complications; additionally, overweight women outnumbered the obese women. On the other hand, IL-12 occurs in circulation in small, usually undetectable, concentrations, and the production of the p35 subunit is rate limited and tightly regulated and it requires co-expression of p40. Moreover, the p40 subunit is expressed in greater quantities than required for only p35p40 formation14 and occurs in the circulation in higher concentrations than IL-12. IL-12p40 acts as a chemoattractant for macrophages and promotes the migration of bacterially stimulated dendritic cells. It has been suggested that IL-12p40 might have a pivotal and independent role in initiating the immune response.14 Taking these data into account, we hypothesized that IL-12p40 may reflect the effects of IL-12 in cells. We demonstrated the increase in serum concentrations of IL-12p40 in obese women. These observations suggest that IL-12p40 may contribute to the induction of the immune response in obesity-associated inflammation. As mentioned, we observed the significant association between p40 and the fat mass in the entire group. There is an evidence demonstrating that AT is an important source of proinflammatory mediators.1–4 On the other hand, it is well known that AT is infiltrated by macrophages5,6 and different cells of the immune system (regulatory T cells, Th1 and Th2 polarized T cells, effector T cells, and B cells) that may be a source of proinflammatory cytokines.9–11 Only a few studies have addressed the role played by T cell–derived cytokines in AT inflammation and insulin resistance.9,17 So far, the data regarding the relationship between IL12p40 and inflammation, adiposity, and insulin resistance are limited. Strissel et al.9 observed the recruitment of macrophages robustly expressing IL-12p40 mRNA during diet-induced obesity in mice. The increased IL-12p40 mRNA and Th1 priming with progression of AT inflammation and insulin resistance in this model was also reported.9,10 In addition, increased IL-12 secretion has also been observed in human macrophages treated with resistin, a proinflammatory cytokine elevated in obese individuals and T2DM patients.40 In a recently published study, the increased levels of both Th1 and Th2 cytokines as well as their negative association with adiponectin and a positive association with the HOMA-IR and high-sensitivity
C-reactive protein were observed in subjects with metabolic syndrome.17 Furthermore, in other studies, it was reported that IL-12 family cytokines are regulated in white AT in a manner that it is dependent on the developmental stage of obesity as well as the inflammatory progression associated with obesity.24 The association of the cytokine with adipose inflammation in humans was observed in overweight and obese individuals and T2DM patients.16,19,27 However, the exact mechanism of the association between IL-12 and insulin resistance and inflammation is not clearly understood. The imbalance between release of proinflammatory and anti-inflammatory cytokines from these cells within AT enhances local inflammation and might contribute to local and peripheral insulin resistance, which might account for the increased levels of serum cytokines. These data indicated that IL-12/IL12p40 may play a part of inflammation associated with obesity. Further studies are needed to test these assumptions. Our results also demonstrate negative correlations between HDL and serum IL-12p40, an association suggesting that changes in p40 serum concentrations might be involved in the pathogenesis of atherosclerosis. These findings are in accordance with those reported by other researchers.17,19,22 We observed that our correlation between IL-12p40 and HDL-C was independent of fat mass. Wang et al. revealed that macrophages releasing IL-12 overexpressed endothelial lipase, which is responsible for local HDL hydrolysis.41 Previous studies have shown that inadequate levels of HDL in conjunction with more atherogenic forms of LDL disturb the natural homeostatic process and contribute to atherogenesis.42 In conclusion, the present study demonstrated the significantly higher serum p40 concentration in overweight and obese individuals than in normal weight subjects. Additionally, the levels of p40 showed a relationship with fat mass and a negative association with HDL-C. We hypothesize that high production of IL-12/IL-12p40 may be a marker related to body weight gain, associated with lipid abnormalities. The possible limitation of our study is that this report is a small cross-sectional study. In addition, we note that the demonstrated relationship between IL-12/IL12p40 and metabolic parameters is weak to modest and it does not show any cause-and-effect relationships. Further studies are required to explore the possible role of the IL-12/ IL-12p40 system in developing insulin resistance, dyslipidemias, and other obesity-associated changes in humans.
Acknowledgments This study was supported by the statutory funding of the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, and grant number 3-50690 from the Medical University of Bia1ystok, Poland. The study protocol was approved by the Ethics Committee of Medical University of Bialystok, Poland.
Author Disclosure Statement No competing financial interests exist.
References 1. Bastard JP, Maachi M, Lagathu C, et al. Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur Cytokine Netw 2006;17:4–12.
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2. Fernandez-Real JM, Pickup JC. Innate immunity, insulin resistance and type 2 diabetes. Trends Endocrinol Metab 2008;19:10–16. 3. Gustafson B. Adipose tissue, inflammation and atherosclerosis. J Atheroscler Thromb 2010;17:332–341. 4. Weisberg SP, McCann D, Desai M, et al. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003;112:1796–1808. 5. Curat CA, Wegner V, Sengenes C, et al. Macrophages in human visceral adipose tissue: Increased accumulation in obesity and a source of resistin and visfatin. Diabetologia 2006;49:744–747. 6. Cancello R, Tordjman J, Poitou C, et al. Increased infiltration of macrophages in omental adipose tissue is associated with marked hepatic lesions in morbid human obesity. Diabetes 2006;55:1554–1561. 7. Xu H, Barnes GT, Yang Q, et al. Chronic inflammation in fat plays a crucial role in the development of obesityrelated insulin resistance. J Clin Invest 2003;112:1821– 1830. 8. Duffaut C, Galitzky J, Lafontan M, Bouloumie A. Unexpected trafficking of immune cells within the adipose tissue during the onset of obesity. Biochem Biophys Res Commun 2009;384:482–485. 9. Strissel KJ, DeFuria J, Shaul ME, et al. T cell recruitment and Th1 polarization in adipose tissue during diet-induced obesity in C57BL/6 mice. Obesity 2010;18:1918–1925. 10. Kintscher U, Hartge M, Hess K, et al. T-lymphocyte infiltration in visceral adipose tissue, a primary event in adipose tissue inflammation and the development of obesity-mediated insulin resistance. Arterioscler Thromb Vasc Biol 2008;28:1304–1310. 11. Feuerer M, Herrero L, Cipolletta D, et al. Lean but not obese fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat Med 2009;15: 930–939. 12. Gillessen S, Carvajal D, Ling P, et al. Mouse interleukin-12 (IL-12) p40 homodimer: A potent IL-12 antagonist. Eur J Immunol 1995;25:200–206. 13. Trinchieri G, Wysocka M, D’Andrea A, et al. Natural killer cell stimulatory factor (NKSF) or interleukin-12 is a key regulator of immune response and inflammation. Prog Growth Factor Res 1992;4:355–368. 14. Cooper AM, Khader SA. IL-12p40: an inherently agonistic cytokine. Trends Immunol 2006;28:33–38. 15. Schulz EG, Mariani L, Radbruch A, Hofer T. Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-gamma and interleukin 12. Immunity 2009; 30:673–683. 16. Suarez-Alvarez K, Solis-Lozano L, Leon-Cabrera S, et al. Serum IL-12 is increased in Mexican obese subjects and associated with low-grade inflammation and obesity-related parameters. Mediators Inflamm 2013:967067. 17. Surendar J, Mohan V, Rao MM, et al. Increased levels of both Th1 and Th2 cytokines in subjects with metabolic syndrome (CURES-103). Diabetes Technol Ther 2011;13: 477–482. 18. Winkler G, Dworak O, Salamon F, et al. Increased interleukin-12 plasma concentrations in both, insulin-depended and non insulin-depended diabetes mellitus. Diabetologia 1998;41:488. 19. Wegner M, Winiarska H, Bobkiewicz-Koz1owska T, Dworacka M. IL-12 serum levels in patients with type 2 diabetes treated with sulphonylureas. Cytokine 2008;42: 312–316.
341
20. Fernandes JL, Mamoni RL, Orford JL, et al. Increased Th1 activity in patients with coronary artery disease. Cytokine 2004;26:131–137. 21. Yamashita H, Shimada K, Seki E, et al. Concentrations of interleukins, interferon, and C-reactive protein in stable and unstable angina pectoris. Am J Cardiol 2003;91:133–136. 22. Uyemura K, Demer LL, Castle SC, et al. Corss-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis. J Clin Invest 1996;97:2130–2138. 23. Hauer AD, Vyttenhore C, De Vos P, et al. Blockade of interleukin-12 function by protein vaccination attenuates atherosclerosis. Circulation 2005;112:1054–1062. 24. Nam H, Ferguson BS, Stephens JM, et al. Impact of obesity on family gene expression in insulin responsive tissues. Biochim Biophys Acta 2013;1832:11–19. 25. Straczkowski M, Kowalska I, Nikolajuk A, et al. Plasma levels of soluble tumor necrosis factor-alpha receptors are related to total and LDL-cholesterol in lean, but not in obese subjects. Cardiovasc Diabetol 2006;5:14. 26. Karczewska-Kupczewska M, Straczkowski M, Adamska A, et al. Insulin sensitivity, metabolic flexibility, and serum adiponectin concentration in women with anorexia nervosa. Metabolism 2010;59:473–477. 27. Mishra M, Kumar H, Bajpai S, et al. Level of serum IL-12 and its correlation with endothelial dysfunction, insulin resistance, proinflammatory cytokine and lipids profile in newly diagnosed T2DM. Diabetes Res Clin Pract 2011;94: 255–261. 28. Wen Y, Gu J, Li SL, et al. Elevated glucose and diabetes promote interleukin-12 cytokine gene expression in mouse macrophages. Endocrinology 2006;147:2518–2525. 29. Wu HP, Kuo SF, Wu SY, et al. High interleukin-12 production from stimulated peripheral blood mononuclear cells of type 2 diabetes patients. Cytokine 2010;51:298– 304. 30. Wu HP, Chen CH, Hsieh HC, et al. Effects of insulin and glucose on cytokine production from peripheral blood mononuclear cells. Chang Gung Med J 2008;31:253–258. 31. Ginsberg HN, Zhang Y, Hernandez-Ono A. Regulation of plasma triglycerides in insulin resistance and diabetes. Arch Med Res 2005;36:232–240. 32. Adiels M., Olofsson S., Taskinen M., Boren J. Overproduction of very low-density lipoproteins is the hallmark of the dyslipdemia in the metabolic syndrome. Arterioscler Thromb Vasc Biol 2008;28:1225–1236. 33. Wolfe RR, Shaw JH, Durkot MJ. Energy metabolism in trauma and sepsis: The role of fat. Prog Clin Biol Res 1983;111:89–109. 34. Park YB, Lee SK, Lee WK, et al. Lipid profiles in untreated patients with rheumatoid arthritis. J Rheumatol 1999;26: 1701–1704. 35. Svenungsson E, Gunnarsson I, Fei GZ, et al. Elevated triglycerides and low levels of high-density lipoprotein as markers of disease activity in association with up-regulation of the tumor necrosis factor alpha/tumor necrosis factor receptor system in systemic lupus erythematosus. Arthritis Rheum 2003;48:2533–2540. 36. Popa C, Netea MC, Riel PLCM, et al. The role of TNFa in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. J Lipid Res 2007;48:751– 762. 37. Patton JS, Shepard HM., Wilking H, et al. Interferons and tumor necrosis factors have similar catabolic effects on 3T3 L1 cells. Proc Natl Acad Sci USA 1986;83:8313– 8317.
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38. Grunfeld C, Soued M, Adi S, et al. Interleukin 4 inhibits stimulation of hepatic lipogenesis by tumor necrosis factor, interleukin 1, and interleukin 6 but not by interferon-alpha. Cancer Res 1991;51:2803–2807. 39. Ohashi R, Mu H, Wang X, et al. Reverse cholesterol transport and cholesterol efflux in atherosclerosis. Q J Med 2005;98:845–856. 40. Silswal N, Singh AK, Aruna B, et al. Human resistin stimulates the proinflammatory cytokines TNFa and IL-12 in macrophages by NF-kB-dependent pathway. Biochem Biophys Res Commun 2005;334:1092–1101. 41. Wang X, Jin W, Rader DJ Upregulation of macrophage endothelial lipase by toll-like receptors 4 and 3 modulates macrophage interleukin-10 and -12 production. Circ Res 2007;100:1008–1015.
42. Van der Velde AE. Reverse cholesterol transport: From classical view to new insights. World J Gastroenterol 2010; 47:5908–5915.
Address correspondence to: Agnieszka Niko1ajuk, MD, PhD Department of Prophylaxis of Metabolic Diseases Institute of Animal Reproduction and Food Research Polish Academy of Sciences M.C. Sk1odowskiej 24A 15-276 Bialystok Poland E-mail: [email protected]
Relationship Between Serum IL-12 and p40 Subunit Concentrations and Lipid Parameters in Overweight and Obese Women Agnieszka Nikołajuk, PhD,1 Monika Karczewska-Kupczewska, MD, PhD,1,2 and Marek Straczkowski, MD, PhD1,2
Abstract
Background: Obesity is a chronic low-grade inflammatory state associated with the development of insulin resistance, type 2 diabetes mellitus, and atherosclerosis. Interleukin-12 (IL-12) is a proinflammatory cytokine composed of a 40-kD (p40) subunit and a 35-kD (p35) subunit. p40 might have an independent role in initiating the immune response. Recent findings indicate that IL-12 could be involved in the development of obesityassociated co-morbidities, especially atherosclerosis. It is unclear if there are alternations in circulating concentrations of total IL-12 and its subunit p40 in young subjects with increased adiposity without overt metabolic disturbances. The aim of the present study was to estimate serum total IL-12 together with its p40 subunit in young overweight and obese women and to investigate the associations of these parameters with insulin sensitivity and serum lipids. Methods: We studied 77 healthy women (37 lean and 40 obese or overweight). Anthropometric measurements, blood biochemical analyses, determination of serum IL-12, IL-12p40 concentrations, and euglycemic– hyperinsulinemic clamp were performed. Results: We demonstrated an increase in serum p40 in obese subjects (P = 0.029). We found positive correlations between p40 and fat mass (r = 0.24, P = 0.04) and significant negative associations with high-density lipoprotein cholesterol (HDL-C) (r = - 0.27, P = 0.002). Detectable concentrations of serum IL-12 were observed in 55% of subjects. Individuals with detectable serum concentrations of IL-12 had significantly higher levels of serum triglycerides (P = 0.049). A significant association between IL-12 and serum total cholesterol (r = 0.32, P = 0.042) was observed in this subgroup. No association between p40 or IL-12 and insulin sensitivity was found. Conclusions: Our data indicate that the IL-12/IL-12p40 system may be associated with lipid abnormalities in obese subjects.
Introduction
A
relationship between obesity and chronic lowgrade inflammation has been demonstrated in many studies, suggesting that inflammation may be a potential mechanism in which obesity leads to diseases associated with insulin resistance, like type 2 diabetes mellitus (T2DM) and atherosclerosis.1–3 It seems that adipose tissue (AT) may play an important role as a site and a source of inflammation. Intensive rodent and human studies have shown that macrophages accumulate along with increasing adiposity.4–6 Although these data indicated that macrophages in AT rep-
resent a major source of proinflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), and interferong (IFN-g).4 Moreover, the macrophage proinflammatory phenotype has been well described in pathogenesis of obesityassociated insulin resistance.7 Recent studies suggest that the accumulation various types of T lymphocytes and T cell– derived cytokines may play a crucial role in adipose inflammation and insulin resistance.8–10 These authors observed an increase in the number of T helper 1 (Th1) polarized T cells and effector T cells and a decrease in number of T helper 2 (Th2) polarized T cells in obese AT.9–11 Moreover, it seems
1 Department of Prophylaxis of Metabolic Diseases, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Bialystok, Poland. 2 Department of Metabolic Diseases, Medical University of Bialystok, Bialystok Poland.
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plausible that T cells and Th1/Th2 cytokines effect macrophage responses during the development of obesity-associated inflammation and insulin resistance. Furthermore, these authors proposed that early lymphocyte infiltration of AT occurs before the accumulation of macrophages.9,10 However, despite significant progress in identifying mechanisms involved in obesity-associated inflammation, it is really not known what initiates the development of inflammation. IL-12 is a heterodimeric protein (p70) composed of two disulfide-linked subunits designated p35 (light-chain 35-kD) and p40 (heavy-chain 40-kD). Notably, while p40 is expressed and secreted as monomer and homodimer, p35 is never found by itself,12 and the p40 subunit is expressed in greater quantities than required for only p35p40 formation.13 Recent publications have suggested that IL-12p40 might have an independent role in initiating the immune response, and both pro- and anti-inflammatory functions have been ascribed.14 IL-12 is produced by monocytes, macrophages, dendritic cells, and natural killer cells. Importantly, IL-12 induces polarization of naı¨ve T cells to the Th1 phenotype and favors Th1 responses, including IFN-g and TNF-a production.15 Taking into account these data, it was hypothesized that IL-12 potentially plays an important role in systemic low-grade inflammation and development of obesity-related insulin resistance. Plasma IL-12 levels were found to be elevated in obesity16 and in patients with metabolic syndrome17 and T2DM with and without cardiovascular complications.18,19 In addition, IL-12 is expressed in atherosclerotic plaque and involved in atherogenesis; it probably accelerates the progress of macrovascular complications in T2DM.20–23 Furthermore, increased mRNA expression of IL-12p40 and IL-12p35 was reported in insulin-responsive tissues under conditions of obesity24 and AT associated proinflammatory macrophages. It is not clear whether the changes in the concentrations of IL-12 and its subunits are present in young people with an excess of AT without clear metabolic complications. Therefore, the aim of this study was to estimate circulating levels of IL-12 and IL-12p40 and their correlation with obesity, serum lipids, and insulin sensitivity evaluated by the euglycemic–hyperinsulinemic clamp in normal weight and overweight/obese women.
Materials and Methods The study group consisted of 77 normally menstruating young women; 40 women were overweight or obese [mean age 24.73 – 5.60 years with a mean body mass index (BMI) of 30.72 – 3.94 kg/m2] and 37 were lean, young women (mean age 24.35 – 4.39 years with BMI < 25 kg/m2). Before entering the study, a physical examination and appropriate laboratory tests were performed. All study participants had no cardiovascular disease, hypertension, hyperandrogenism, infections, or other serious medical problems; all were nonsmokers and were not taking any anti-inflammatory drugs or drugs known to affect glucose and lipid metabolism. All analyses were performed after an overnight fast. A standard 75-gram oral glucose tolerance test (OGTT) was performed, and all subjects had normal glucose tolerance according to the World Health Organization criteria. The study protocol was approved by Ethical Committee of Human Studies of the Medical University of Bialystok, Poland. All participants gave written informed consent before entering the study.
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Anthropometric measurements The BMI was calculated as body weight in kilograms divided by height in meters squared (kg/m2). The waist circumference was measured at the smallest circumference between the waist and thighs. The percent of body fat was assessed by bioelectric impedance analysis using a Tanita TBF-511 Body Fat Analyzer (Tanita Corp., Tokyo, Japan), and fat-free mass was calculated.
Insulin sensitivity Insulin sensitivity was evaluated by the euglycemic– hyperinsulinemic clamp, as previously described.25,26 The rate of whole-body glucose uptake (M value) was calculated as the mean glucose infusion rate from 80 to 120 min, corrected for glucose space and normalized per kilogram of fatfree mass (FFM).
Biochemical analyses Fasting blood samples were also taken from the antecubital vein before the beginning of the clamp. Before the determination of serum insulin, IL-12, and IL-12p40 concentrations, samples were frozen at -70C until analyses. Plasma glucose was measured immediately by the enzymatic method using a glucose analyzer (YSI 2300 STAT Plus, YSI, Inc., Yellow Springs, OH) for the OGTT and the clamp study. Serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs) were measured by an enzymatic method (Cormay, Warsaw, Poland). The concentration of low-density lipoprotein cholesterol (LDL-C) was calculated from the Friedewald formula. Serum insulin was measured with an immunoradiometric assay (IRMA; BioSource Europe, Nivelles, Belgium). Serum high-sensitivity IL-12 was measured with a quantitative sandwich enzyme immunoassay kit (R&D Systems, Minneapolis, MN) with a lowest detectable limit of 0.5 pg/ mL and with intra-assay and interassay coefficients of variation (CV) below 4.9% and 12.6% respectively. Serum IL-12p40 concentrations were determined with an enzymelinked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). The minimum detectable concentration was 15 pg/mL, and intra- and interassay CV values were, respectively, below 6.6% and 6.9%.
Statistical analysis The statistics were performed with STATISTICA 8.0 software. The variables that did not have normal distribution (IL-12p40, insulin, TGs) were log-transformed prior to analyses. For the purpose of presenting the data, these variables were transformed to absolute values (see Results section). The differences between the groups were estimated with an unpaired Student t-test. The relationships between variables were studied with the Pearson product-moment correlation analysis and with multiple regression analysis. The level of significance was accepted at P < 0.05.
Results The clinical characteristic of the studied groups is shown in Table 1. We found that obese women had higher serum postload glucose (P = 0.014), fasting insulin, and postload insulin (P = 0.009 and P = 0.003, respectively) than lean
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Table 1.
Age (years) Weight (kg) BMI (kg/m2) Waist (cm) Body fat (%) Fasting glucose (mg/dL) Postload glucose (mg/dL) Fasting insulin (mIU/mL) Postload insulin (mIU/mL) Cholesterol (mg/dL) Serum TGs (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) IL-12 (pg/mL) IL-12p40 (pg/mL) M (mg/kgFFM/min)
Clinical and Biochemical Characteristics of the Groups Studied Overweight/obese (n = 40)
Lean (n = 37)
P value
24.73 – 5.60 84.62 – 13.23 30.72 – 3.93 93.71 – 12.70 40.15 – 7.58 82.65 – 6.35 92.29 – 21.20 16.79 – 8.69 63.59 – 43.60 174.23 – 30.18 90.78 – 40.02 56.40 – 8.49 99.39 – 31.16 0.51 – 0.68 96.85 – 40.50 8.47 – 3.28
24.35 – 4.39 58.94 – 6.81 21.29 – 1.89 71.90 – 6.62 25.89 – 6.68 80.18 – 7.56 81.20 – 17.17 12.10 – 5.35 38.05 – 21.21 185.22 – 32.63 72.12 – 31.34 62.89 – 12.56 104.72 – 36.28 0.77 – 0.70 79.13 – 38.39 10.69 – 3.16
0.75 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.12 0.014 0.009 0.003 0.13 0.028 0.010 0.51 0.23 0.029 0.003
Data are presented as mean – standard deviation (SD). BMI, body mass index; TGs, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; IL12, interleukin 12; IL-12p40, subunit p40 of IL-12; M value, whole-body glucose uptake normalized per kilogram of fat-free mass (FFM), postload glucose, and insulin refer to value at 120 min of the oral glucose tolerance test.
women. We observed significantly higher serum TGs concentrations in the obese women in comparison with the lean women (P = 0.028). Additionally, obese women had significantly lower serum HDL-C and insulin sensitivity (P = 0.009 and P = 0.003, respectively).
We showed an increase in serum IL-12p40 in obese subjects (P = 0.029), but serum IL-12 did not differ between obese and control (lean) subjects (P = 0.23). We found positive correlations between IL-12p40 and fat mass (r = 0.24, P = 0.04) and significant negative associations with HDL-C (r = - 0.27,
FIG. 1. Correlations between serum concentrations of interleukin-12 (IL-12)p40 and high-density lipoprotein cholesterol (HDL-C) in the entire group (n = 77). The relationships between variables were studied with the Pearson product-moment correlation analysis. The level of significance was accepted at P < 0.05. Values of IL-12p40 are shown on a log-transformed scale. (Filled circle) overweight/obese group; (open circle) normal weight group; (solid line) slope of the correlation. The dotted lines indicate 95% confidence intervals.
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Table 2. Clinical and Biochemical Characteristics of the Studied Subgroups Subjects with nondetectable IL-12 (n = 34)
Subjects with detectable IL-12 (n = 43)
Weight (kg) 73.27 – 15.90 71.49 – 17.43 BMI (kg/m2) 26.50 – 5.13 25.95 – 6.11 Waist (cm) 83.76 – 13.88 82.80 – 15.91 Body fat (%) 32.50 – 10.39 33.63 – 9.95 Fasting glucose (mg/dL) 82.79 – 5.11 80.40 – 8.13 Fasting insulin (mIU/mL) 14.71 – 6.21 14.15 – 8.56 Cholesterol (mg/dL) 177.85 – 31.91 180.72 – 31.74 Serum TGs (mg/dL) 72.68 – 28.65 89.43 – 41.64* HDL-C (mg/dL) 58.61 – 8.45 60.10 – 12.81 LDL-C (mg/dL) 104.82 – 33.52 99.31 – 33.86 IL-12p40 87.46 – 35.73 89.03 – 43.89 Log IL12p40 1.90 – 0.20 1.90 – 0.20 M (mg/kgFFM/min) 9.27 – 3.23 9.74 – 3.53 Data are presented as mean – standard deviation (SD). The differences between the groups were estimated with an unpaired Student t-test and level of significance was accepted at *P < 0.05. IL-12, interleukin 12; BMI, body mass index; TGs, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; IL-12p40, subunit p40 of IL-12; M value, whole-body glucose uptake normalized per kilogram of fat-free mass (FFM).
P = 0.002) (Fig. 1) in the entire studied group. In multiple regression analysis, correlation between IL-12p40 and HDL-C was independent of fat mass (b = - 0.39, P = 0.024). We reported detectable concentration of serum IL-12 in 43 participants (55% of the entire group). Clinical and biochemical parameters in subjects with and without detectable IL-12 are shown in Table 2. Subjects with detectable and nondetectable IL-12 (ND IL-12) were similar in BMI, waist circumference, percent body fat, fasting glucose and insulin, serum cholesterol, HDL-C, and LDL-C. The serum IL-12p40 concentration and insulin sensitivity did not differ between these subgroups (P = 0.87 and P = 0.57, respectively). Individuals with detectable serum concentrations of IL-12 had slightly higher levels of serum TGs (detectable IL-12, serum TGs were 89.43 – 41.64 mg/dL; ND IL-12, serum TGs were 72.68 – 28.65 mg/dL; P = 0.049). In the subgroup analysis, we found a positive correlation between serum IL-12 and cholesterol in the subjects with detectable IL-12 (r = 0.32, P = 0.042). No association between p40 or IL-12 and insulin sensitivity was found.
Discussion In the present study, we demonstrated that the concentrations of IL-12 subunit p40 were increased in obese women and that about 55% of our group have detectable levels of IL-12. Our observations may be indirectly confirmed by the results of other authors. Higher concentrations of IL-12 in patients with metabolic syndrome were observed.17 Data showed that IL-12 plasma concentrations were elevated in T2DM,18 both in patients with antidiabetic therapy and newly diagnosed T2DM.19,27 Additionally, the authors also found that level of IL-12 was associated with obesity and insulin resistance estimated by homeostasis model assessment of insulin resistance (HOMA-IR).19,27
Our study did not reveal the significant difference between serum IL-12 concentrations in obese and lean women and the relation of IL-12 to anthropometric parameters, but we found positive correlations between IL-12p40 and fat mass in the entire group. Even though we observed significantly higher levels of postload glucose, fasting and postload insulin, serum p40, and lower insulin sensitivity in obese women in comparison to healthy controls, we did not prove any significant correlations between insulin sensitivity and serum level IL-12 or subunit p40 in the entire studied group as well as in the subgroups. Studies on the relationship between the concentration of IL-12 and insulin resistance are contradictory. As mentioned, some investigators have observed the relationship of this cytokine to insulin sensitivity.19,27 In contrast, in the study conducted by Suarez-Alvarez et al.,16 no significant associations were observed among serum level of IL-12 and fasting insulin and insulin resistance denoted as HOMA-IR. These differences may come from the heterogeneity of the studied groups and different degrees of insulin resistance as well as differences arising from the methods of measuring insulin action. In our study, we used the euglycemic– hyperinsulinemic clamp technique, which is appropriate and important to use as the reference method in measuring insulin sensitivity. In the above-cited studies, the authors assessed insulin resistance HOMA.16,17,19 In addition, HOMA reflects mainly hepatic insulin resistance, whereas the whole-body glucose uptake normalized per kilogram of FFM estimated by euglycemic clamp reflects peripheral (predominantly skeletal muscle) insulin sensitivity. Additionally, our obese group exhibited only mild insulin resistance, which may underestimate the direct association between concentrations of IL-12 and p40 and parameters of glucose and lipid metabolism. However, we performed only indirect estimations of fat free mass by bioelectric impedance, which may be a limitation of our study. It has been shown that hyperglycemia may increase levels of IL-12 mRNA in mouse macrophages28 and IL-12 production from peripheral blood mononuclear cells (PBMCs) was increased in hyperglycemic conditions.29,30 Furthermore, our entire study group was much younger than in the cited study and had normal glucose tolerance.18,19,27,29 It is generally accepted that dyslipidemia is the most common complication of metabolic syndrome and T2DM. Low HDL levels and hypertriglyceridemia are lipid and lipoprotein abnormalities often observed in insulin resistance.31,32 Consistent with these findings, in our study, the individuals with detectable serum concentrations of IL-12 had significantly higher levels of serum TGs. The subjects with and without detectable IL-12 had comparable anthropometric parameters, indicating that this association was independent of obesity. Furthermore, the analysis of the subgroup with detectable IL-12 indicated a positive association between serum IL-12 and TC. Similar results were observed by Suarez-Alvarez et al.16 It is worth pointing out that these investigators grouped their study population into two clusters according to high and low producers of IL-12. They observed that more than 70% of the high producers of IL-12 showed increased levels of TGs and increased production of IL-12 was related to body weight gain. In contrast to these studies in our subgroups, we did not observe any differences in anthropometric and biochemical parameters. The association between the acute and chronic
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inflammation with alterations in TGs and cholesterol metabolism was described.33–35 On the other hand, it is well known that production of this cytokine is tightly regulated by other factors, like IFN-g, TNF-a, and vice versa. Cross-regulatory actions between these cytokines may be responsible for enhanced lipolysis,36,37 de novo fatty acid synthesis,38 or inhibition of lipase lipoproteins activity.37 IL-12 may impair reverse cholesterol transport (RCT) and cholesterol efflux, alone or through an effect on other cytokines39 in the manner promoting atherosclerosis. An important limitation of the present study is that it does not show any cause-and-effect relationships. Our data did not show whether the high levels of IL-12 were a cause or a consequence of high TGs concentration. Finally, also we have to consider that in previous studies, concentrations of IL-12 were detected in all subjects in the general population,17,19,27 whereas we have shown the presence of this cytokine in only nearly half of our study population. The inconsistent results may be attributable to differences in the methods of assay and in subjects’ characteristics. Our study group was younger and without apparent metabolic complications; additionally, overweight women outnumbered the obese women. On the other hand, IL-12 occurs in circulation in small, usually undetectable, concentrations, and the production of the p35 subunit is rate limited and tightly regulated and it requires co-expression of p40. Moreover, the p40 subunit is expressed in greater quantities than required for only p35p40 formation14 and occurs in the circulation in higher concentrations than IL-12. IL-12p40 acts as a chemoattractant for macrophages and promotes the migration of bacterially stimulated dendritic cells. It has been suggested that IL-12p40 might have a pivotal and independent role in initiating the immune response.14 Taking these data into account, we hypothesized that IL-12p40 may reflect the effects of IL-12 in cells. We demonstrated the increase in serum concentrations of IL-12p40 in obese women. These observations suggest that IL-12p40 may contribute to the induction of the immune response in obesity-associated inflammation. As mentioned, we observed the significant association between p40 and the fat mass in the entire group. There is an evidence demonstrating that AT is an important source of proinflammatory mediators.1–4 On the other hand, it is well known that AT is infiltrated by macrophages5,6 and different cells of the immune system (regulatory T cells, Th1 and Th2 polarized T cells, effector T cells, and B cells) that may be a source of proinflammatory cytokines.9–11 Only a few studies have addressed the role played by T cell–derived cytokines in AT inflammation and insulin resistance.9,17 So far, the data regarding the relationship between IL12p40 and inflammation, adiposity, and insulin resistance are limited. Strissel et al.9 observed the recruitment of macrophages robustly expressing IL-12p40 mRNA during diet-induced obesity in mice. The increased IL-12p40 mRNA and Th1 priming with progression of AT inflammation and insulin resistance in this model was also reported.9,10 In addition, increased IL-12 secretion has also been observed in human macrophages treated with resistin, a proinflammatory cytokine elevated in obese individuals and T2DM patients.40 In a recently published study, the increased levels of both Th1 and Th2 cytokines as well as their negative association with adiponectin and a positive association with the HOMA-IR and high-sensitivity
C-reactive protein were observed in subjects with metabolic syndrome.17 Furthermore, in other studies, it was reported that IL-12 family cytokines are regulated in white AT in a manner that it is dependent on the developmental stage of obesity as well as the inflammatory progression associated with obesity.24 The association of the cytokine with adipose inflammation in humans was observed in overweight and obese individuals and T2DM patients.16,19,27 However, the exact mechanism of the association between IL-12 and insulin resistance and inflammation is not clearly understood. The imbalance between release of proinflammatory and anti-inflammatory cytokines from these cells within AT enhances local inflammation and might contribute to local and peripheral insulin resistance, which might account for the increased levels of serum cytokines. These data indicated that IL-12/IL12p40 may play a part of inflammation associated with obesity. Further studies are needed to test these assumptions. Our results also demonstrate negative correlations between HDL and serum IL-12p40, an association suggesting that changes in p40 serum concentrations might be involved in the pathogenesis of atherosclerosis. These findings are in accordance with those reported by other researchers.17,19,22 We observed that our correlation between IL-12p40 and HDL-C was independent of fat mass. Wang et al. revealed that macrophages releasing IL-12 overexpressed endothelial lipase, which is responsible for local HDL hydrolysis.41 Previous studies have shown that inadequate levels of HDL in conjunction with more atherogenic forms of LDL disturb the natural homeostatic process and contribute to atherogenesis.42 In conclusion, the present study demonstrated the significantly higher serum p40 concentration in overweight and obese individuals than in normal weight subjects. Additionally, the levels of p40 showed a relationship with fat mass and a negative association with HDL-C. We hypothesize that high production of IL-12/IL-12p40 may be a marker related to body weight gain, associated with lipid abnormalities. The possible limitation of our study is that this report is a small cross-sectional study. In addition, we note that the demonstrated relationship between IL-12/IL12p40 and metabolic parameters is weak to modest and it does not show any cause-and-effect relationships. Further studies are required to explore the possible role of the IL-12/ IL-12p40 system in developing insulin resistance, dyslipidemias, and other obesity-associated changes in humans.
Acknowledgments This study was supported by the statutory funding of the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, and grant number 3-50690 from the Medical University of Bia1ystok, Poland. The study protocol was approved by the Ethics Committee of Medical University of Bialystok, Poland.
Author Disclosure Statement No competing financial interests exist.
References 1. Bastard JP, Maachi M, Lagathu C, et al. Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur Cytokine Netw 2006;17:4–12.
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2. Fernandez-Real JM, Pickup JC. Innate immunity, insulin resistance and type 2 diabetes. Trends Endocrinol Metab 2008;19:10–16. 3. Gustafson B. Adipose tissue, inflammation and atherosclerosis. J Atheroscler Thromb 2010;17:332–341. 4. Weisberg SP, McCann D, Desai M, et al. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003;112:1796–1808. 5. Curat CA, Wegner V, Sengenes C, et al. Macrophages in human visceral adipose tissue: Increased accumulation in obesity and a source of resistin and visfatin. Diabetologia 2006;49:744–747. 6. Cancello R, Tordjman J, Poitou C, et al. Increased infiltration of macrophages in omental adipose tissue is associated with marked hepatic lesions in morbid human obesity. Diabetes 2006;55:1554–1561. 7. Xu H, Barnes GT, Yang Q, et al. Chronic inflammation in fat plays a crucial role in the development of obesityrelated insulin resistance. J Clin Invest 2003;112:1821– 1830. 8. Duffaut C, Galitzky J, Lafontan M, Bouloumie A. Unexpected trafficking of immune cells within the adipose tissue during the onset of obesity. Biochem Biophys Res Commun 2009;384:482–485. 9. Strissel KJ, DeFuria J, Shaul ME, et al. T cell recruitment and Th1 polarization in adipose tissue during diet-induced obesity in C57BL/6 mice. Obesity 2010;18:1918–1925. 10. Kintscher U, Hartge M, Hess K, et al. T-lymphocyte infiltration in visceral adipose tissue, a primary event in adipose tissue inflammation and the development of obesity-mediated insulin resistance. Arterioscler Thromb Vasc Biol 2008;28:1304–1310. 11. Feuerer M, Herrero L, Cipolletta D, et al. Lean but not obese fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat Med 2009;15: 930–939. 12. Gillessen S, Carvajal D, Ling P, et al. Mouse interleukin-12 (IL-12) p40 homodimer: A potent IL-12 antagonist. Eur J Immunol 1995;25:200–206. 13. Trinchieri G, Wysocka M, D’Andrea A, et al. Natural killer cell stimulatory factor (NKSF) or interleukin-12 is a key regulator of immune response and inflammation. Prog Growth Factor Res 1992;4:355–368. 14. Cooper AM, Khader SA. IL-12p40: an inherently agonistic cytokine. Trends Immunol 2006;28:33–38. 15. Schulz EG, Mariani L, Radbruch A, Hofer T. Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-gamma and interleukin 12. Immunity 2009; 30:673–683. 16. Suarez-Alvarez K, Solis-Lozano L, Leon-Cabrera S, et al. Serum IL-12 is increased in Mexican obese subjects and associated with low-grade inflammation and obesity-related parameters. Mediators Inflamm 2013:967067. 17. Surendar J, Mohan V, Rao MM, et al. Increased levels of both Th1 and Th2 cytokines in subjects with metabolic syndrome (CURES-103). Diabetes Technol Ther 2011;13: 477–482. 18. Winkler G, Dworak O, Salamon F, et al. Increased interleukin-12 plasma concentrations in both, insulin-depended and non insulin-depended diabetes mellitus. Diabetologia 1998;41:488. 19. Wegner M, Winiarska H, Bobkiewicz-Koz1owska T, Dworacka M. IL-12 serum levels in patients with type 2 diabetes treated with sulphonylureas. Cytokine 2008;42: 312–316.
341
20. Fernandes JL, Mamoni RL, Orford JL, et al. Increased Th1 activity in patients with coronary artery disease. Cytokine 2004;26:131–137. 21. Yamashita H, Shimada K, Seki E, et al. Concentrations of interleukins, interferon, and C-reactive protein in stable and unstable angina pectoris. Am J Cardiol 2003;91:133–136. 22. Uyemura K, Demer LL, Castle SC, et al. Corss-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis. J Clin Invest 1996;97:2130–2138. 23. Hauer AD, Vyttenhore C, De Vos P, et al. Blockade of interleukin-12 function by protein vaccination attenuates atherosclerosis. Circulation 2005;112:1054–1062. 24. Nam H, Ferguson BS, Stephens JM, et al. Impact of obesity on family gene expression in insulin responsive tissues. Biochim Biophys Acta 2013;1832:11–19. 25. Straczkowski M, Kowalska I, Nikolajuk A, et al. Plasma levels of soluble tumor necrosis factor-alpha receptors are related to total and LDL-cholesterol in lean, but not in obese subjects. Cardiovasc Diabetol 2006;5:14. 26. Karczewska-Kupczewska M, Straczkowski M, Adamska A, et al. Insulin sensitivity, metabolic flexibility, and serum adiponectin concentration in women with anorexia nervosa. Metabolism 2010;59:473–477. 27. Mishra M, Kumar H, Bajpai S, et al. Level of serum IL-12 and its correlation with endothelial dysfunction, insulin resistance, proinflammatory cytokine and lipids profile in newly diagnosed T2DM. Diabetes Res Clin Pract 2011;94: 255–261. 28. Wen Y, Gu J, Li SL, et al. Elevated glucose and diabetes promote interleukin-12 cytokine gene expression in mouse macrophages. Endocrinology 2006;147:2518–2525. 29. Wu HP, Kuo SF, Wu SY, et al. High interleukin-12 production from stimulated peripheral blood mononuclear cells of type 2 diabetes patients. Cytokine 2010;51:298– 304. 30. Wu HP, Chen CH, Hsieh HC, et al. Effects of insulin and glucose on cytokine production from peripheral blood mononuclear cells. Chang Gung Med J 2008;31:253–258. 31. Ginsberg HN, Zhang Y, Hernandez-Ono A. Regulation of plasma triglycerides in insulin resistance and diabetes. Arch Med Res 2005;36:232–240. 32. Adiels M., Olofsson S., Taskinen M., Boren J. Overproduction of very low-density lipoproteins is the hallmark of the dyslipdemia in the metabolic syndrome. Arterioscler Thromb Vasc Biol 2008;28:1225–1236. 33. Wolfe RR, Shaw JH, Durkot MJ. Energy metabolism in trauma and sepsis: The role of fat. Prog Clin Biol Res 1983;111:89–109. 34. Park YB, Lee SK, Lee WK, et al. Lipid profiles in untreated patients with rheumatoid arthritis. J Rheumatol 1999;26: 1701–1704. 35. Svenungsson E, Gunnarsson I, Fei GZ, et al. Elevated triglycerides and low levels of high-density lipoprotein as markers of disease activity in association with up-regulation of the tumor necrosis factor alpha/tumor necrosis factor receptor system in systemic lupus erythematosus. Arthritis Rheum 2003;48:2533–2540. 36. Popa C, Netea MC, Riel PLCM, et al. The role of TNFa in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. J Lipid Res 2007;48:751– 762. 37. Patton JS, Shepard HM., Wilking H, et al. Interferons and tumor necrosis factors have similar catabolic effects on 3T3 L1 cells. Proc Natl Acad Sci USA 1986;83:8313– 8317.
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38. Grunfeld C, Soued M, Adi S, et al. Interleukin 4 inhibits stimulation of hepatic lipogenesis by tumor necrosis factor, interleukin 1, and interleukin 6 but not by interferon-alpha. Cancer Res 1991;51:2803–2807. 39. Ohashi R, Mu H, Wang X, et al. Reverse cholesterol transport and cholesterol efflux in atherosclerosis. Q J Med 2005;98:845–856. 40. Silswal N, Singh AK, Aruna B, et al. Human resistin stimulates the proinflammatory cytokines TNFa and IL-12 in macrophages by NF-kB-dependent pathway. Biochem Biophys Res Commun 2005;334:1092–1101. 41. Wang X, Jin W, Rader DJ Upregulation of macrophage endothelial lipase by toll-like receptors 4 and 3 modulates macrophage interleukin-10 and -12 production. Circ Res 2007;100:1008–1015.
42. Van der Velde AE. Reverse cholesterol transport: From classical view to new insights. World J Gastroenterol 2010; 47:5908–5915.
Address correspondence to: Agnieszka Niko1ajuk, MD, PhD Department of Prophylaxis of Metabolic Diseases Institute of Animal Reproduction and Food Research Polish Academy of Sciences M.C. Sk1odowskiej 24A 15-276 Bialystok Poland E-mail: [email protected]
