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Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content
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Original Article
Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content Renata Vargas a, Elena Ryder a, Marı´a Diez-Ewald a, Jesu´s Mosquera a,*, Anyelo Dura´n a, ˜ ez b, Caterina Pen ˜ a c, Erika Ferna´ndez a Nereida Valero a, Adriana Pedrean a
Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela Ca´tedra de Inmunologı´a. Escuela de Bionanalisis, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela c Ca´tedra de Gene´tica, Escuela de Bionanalisis, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela b
A R T I C L E I N F O
A B S T R A C T
Keywords: Obesity Inflammation Cytokines Insulin
Aims: Chronic inflammation in obesity is associated with co-morbidities such as, hyperglycemia, hypertension and hyperlipidemia. Leukocytes play an important role in this inflammation and C-reactive protein (CRP) and Interleukin-2 (IL-2) can be important effectors during the immune response in obesity; however, the initial inflammatory events in obesity remain unclear. The aim of this study was to determine the circulating levels of CRP, IL-2, insulin and adiponectin, their association and the association with leukocyte count in obese individuals without co-morbidities and with or without insulin resistance (IR). Materials and methods: Nineteen obese non-diabetic and 9 lean subjects were studied for serum levels of CRP, IL-2, insulin, adiponectin, lipids, glycated hemoglobin, glycemia, for homeostasis model assessment of insulin resistance (HOMA-IR), arterial pressure and anthropometric parameters, and for leukocyte counts. Neutrophil/lymphocyte ratio (N/L) was calculated using the loge of leukocyte counts. Associations were determined by Pearson’s correlation. Results: None of the studied groups presented co-morbidities and two groups of obese individuals with normal or high levels of insulin (IR) were found. Increased CRP concentration and decreased IL-2 and adiponectin concentrations in obese were observed. Positive correlation between leukocyte type counts with CRP in obese with IR was found; however, no correlations with IL-2 in obese were observed. Insulin in obese were positively correlated with CRP and negatively correlated with IL-2 in IR obese individuals. Adiponectin in obese was negatively correlated with CRP. Conclusion: CRP and IL-2 may represent two important effectors in the early inflammatory events in obese individuals without co-morbidities. Adiponectin and insulin may be involved in antiinflammatory events. ß 2015 Diabetes India. Published by Elsevier Ltd. All rights reserved.
1. Introduction Obesity is associated with cardio-metabolic morbidity; however, some obese individuals escape this association, forming a unique obese sub-phenotype. Clinical biomarkers that reflect already manifested co-morbidities, such as dyslipidemia, hyperglycemia, hypertension, and anthropometric or imaging-based assessment of adipose tissue distribution have been used to define
* Corresponding author at: Apartado Postal 23, Maracaibo, 4001-A., Zulia, Venezuela. Tel.: +58 261 7114752; fax: +58 261 7916053. E-mail address: [email protected] (J. Mosquera).
this sub-phenotype [1]. The relationship between co-morbidities and inflammatory events in the obesity has been reported [1]; in addition, the association of leukocytes with chronic inflammation in obesity has also been reported [1,2]. These data suggest that the leukocyte induces inflammation with further development of comorbidities. The initiation of co-morbidities could be related to the beginning and persistent time of the inflammatory events during obesity. Therefore, obese individuals with no co-morbidities can represent an early stage of inflammation. During obesity, chronic inflammation can be mediated by innate and adaptive immune responses [3]. In this regard, the expression of cytokines such as CRP and IL-2 during inflammation could be important in both types of immune response during obesity. CRP is an acute-phase reactant
http://dx.doi.org/10.1016/j.dsx.2015.09.007 1871-4021/ß 2015 Diabetes India. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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that rapidly increases in response to tissue injury and to a variety of inflammatory stimuli [4,5]. This cytokine is involved in several proinflammatory events, including chemotaxis, modulation of nitric oxide and oxygen species, induction of cytokines and proliferation of immune and nonimmune cells [6–8]. IL-2 is a potent proinflammatory cytokine, produced mainly by activated CD4+ and CD8+ T cells, with important effects on the biology of regulatory T cells (Treg cells), and therefore, on the homeostasis of immune response [9–15]. The presence of modulatory compounds on the immune response in obesity has been little studied. Insulin and adiponectin, molecules with anti-inflammatory effect [16–19], could have an important role during obesity inflammatory events. Therefore, the aim of this study was to determine the circulating levels of CRP, IL-2, adiponectin and insulin and evaluate the associations between them and with the leukocyte count in obese individuals with normal clinical and biochemical parameters and with or without IR. 2. Subjects A total of 44 obese individuals chosen through a voluntary call to participate in this study were analyzed in an initial screening. Only 19 obese non-diabetic adult subjects (20–55 years old) without the known risk factors for obesity (no co-morbidities) were selected. Increased serum insulin concentration was found in 10 of these individuals; therefore, obese individuals were divided in two groups: one with normal levels of insulin (n = 10) and the other with increased levels (individuals with IR; n = 9). All obese individuals had a body mass index (BMI) greater than 30 kg/ m2. Nine lean control individuals, whose BMI was less than 25 kg/ m2, were also selected in this study. All subjects fulfilled the inclusion criteria: absence of arterial hypertension, diabetes or other metabolic abnormalities, not evidence of hepatitis or HIV infection (determined by the absence of antibodies against the viruses), absence of any other current infectious processes, not having taken any of the medications known to influence glucose or lipid metabolism or the inflammatory pathway, and in the case of women, not under hormonal contraceptive drugs. Information regarding age, sex, qualifying criteria, current medications, height, weight and waist circumference was collected. Body mass index was calculated from the formula: weight (kg)/height (m2), considering obesity when it was >30 kg/m2. Absence of hypertension was confirmed by means of a sphyngomanometer. Ultrasound was used for the estimation of visceral fat and hepatic steatosis as described by Ryder et al. [20]. Acquisition of blood samples and all scientific studies were approved by the Ethic Committee of the Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’,
University of Zulia in Maracaibo, Venezuela, according with the principles of the Declaration of Helsinki as revised in 2008. Written consent was obtained from all subjects. 3. Materials and methods 3.1. Biochemical, immunological and hematological measurements A fasting blood sample was withdrawn from each subject for the determination of studied parameters. Glucose, total cholesterol and triglycerides were measured enzymatically; high density lipoprotein (HDL)-cholesterol was measured after precipitation of the apo B-containing lipoproteins (Human GmbH, Germany). Plasma insulin was determined by chemiluminescence immunoassay (IMMULITE 1000, Siemens Diagnostics, USA) and total glycated hemoglobin by the method of Bioscience Medical SL, Spain. HOMA-IR was calculated as: fasting insulin (IU/L) fasting glucose (mmol/L)/22.5 according to Mathews et al. [21], and the cut off point to consider IR was 2.6 [22,23]. A highly sensitive CRP enzyme immunoassay was used to quantify circulating levels of CPR (Hemagen Diagnostics, Inc., Columbia, USA) and the results were expressed as mg/L. IL-2 was measured using a commercial kit (Mersham Life Science, England) and the results expressed as pg/ mL. Serum content of adiponectin was also determined by a commercial ELISA (SPI-BIO Bertin Pharma Branch France) and the results expressed as mg/mL. The number of leukocytes and leukocyte subtypes were computed with an autoanalyzer (Beckman Coulter Counter, Coulters Corporation, FL, USA). The neutrophil: lymphocyte ratio was defined as the loge neutrophil count/ loge lymphocyte count. 3.2. Statistical analysis The data were normally distributed and were expressed as the mean standard deviation. Differences among groups were analyzed by ANOVA and Bonferroni’s test. Pearson’s correlations were estimated for various blood cell counts or for insulin or adiponectin content with CRP and IL-2 content using PRISM statistical software (GraphPad Software). Differences were considered statistically significant at p < 0.05. 4. Results None of the lean and obese individuals in this study had hypertension, hyperglycemia or elevated glycated hemoglobin, and their mean values for total and LDL cholesterol were similar among the three groups. As expected, the BMI, HOMA-IR, waist
Table 1 Clinical and laboratory parameters in obese individuals with or without IR and in healthy lean control individuals. Parameter
Control (A)
Ob IR (B)
Ob no IR(C)
A vs. B P value
A vs. C P value
B vs. C P value
N Age (years) Body mass index (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Homa-IR Waist circumference (cm) Insulin (mU/mL) Glycemia (mg/dL) Glycated hemoglobin (%) Triglycerides (mg/dL) Cholesterol (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Visceral fat (cm)
9 29.7 3.3 22.4 0.5 112.5 2.5 75 1.9 0.83 0.2 77 2.0 4.1 1.0 80.3 3.8 6.11 0.14 80.3 3.8 163.9 7.9 51.8 2.5 99.8 6.2 2.36 0.3
9 33.3 4.2 37.8 0.8 122.2 2.8 85.1 1.7 5.83 0.7 114.94.2 25.3 2.8 92.8 3.4 6.37 0.36 126.614.7 186 14.0 41 2.4 119.511.0 6.2 0.6
10 38.7 3.4 34.6 1.2 114.0 2.7 76 1.6 1.48 0.2 106 2.4 6.93 0.9 86.4 1.6 6.44 0.14 127.9 13.2 187.1 14.4 42 3.3 117.4 14.5 5.4 0.4
NS <0.01 <0.05 <0.01 <0.01 <0.01 <0.01 <0.05 NS <0.05 NS <0.05 NS <0.01
NS <0.01 NS NS NS <0.01 NS NS NS <0.05 NS <0.05 NS <0.01
NS <0.05 NS <0.01 <0.01 NS <0.01 NS NS NS NS NS NS NS
Ob IR: obese with insulin resistance; Ob no IR: obese without insulin resistance; values are expressed as mean standard deviation.
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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circumference and visceral fat of obese with IR and obese without IR were elevated in relation to the control group; however, obese with IR showed significantly increased values of BMI, HOMA-IR and insulin compared to obese without IR. Triglyceride values were higher and HDL cholesterol lower in both obese groups; however, the mean values were in the normal range for this population (Table 1). Increased serum CRP content in obese individuals with and without IR was observed (Fig. 1A). The values of IL-2 in all obese individuals were decreased (Fig. 1B). Serum analysis showed decreased values of adiponectin content in obese individuals (lean: 10.33 1.59; obese with IR: 7.49 0.88*; obese without IR: 8.20 0.66* mg/mL. *p < 0.001 vs. lean). Positive correlations between total leukocyte, neuthrophil, lymphocyte and monocyte counts with CRP concentration in obese individuals with IR were observed; however, these correlations in obese individuals without IR were absent (Fig. 2). No correlations between the different leukocyte type counts and the IL-2 values were observed in obese individuals (Fig. 2). Values of serum insulin in all obese groups were positively correlated with the CRP values and negatively correlated with IL-2 concentration in obese individuals with IR (Fig. 3). Decreased values of circulating adiponectin in obese groups were negatively correlated with CRP,
Fig. 1. Serum concentrations of CRP and IL-2 in obese and lean individuals. (A) Increased CRP concentration was found in obese individuals. (B) Decreased IL-2 values were found in obese individuals.
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with stronger correlation in obese individuals with IR (Fig. 4). There were no statistical significances when the neutrophil: lymphocyte ratio was correlated with CRP or IL-2 content (Fig. 2). 5. Discussion Obesity is a major risk factor for cardio-metabolic morbidity. Co-morbidities such as hyperglycemia, hypertension, dyslipidemia and alterations in anthropometric or imaging-based for adipose tissue distribution parameters in obesity have been widely studied. In addition, pro-inflammatory events in human obesity have been shown to correlate with adipose tissue inflammation and with obesity-associated health risks [1]. In this study two groups of obese subjects (with and without IR) were analyzed. Alterations such as dyslipidemia, hyperglycemia and hypertension were absent suggesting lack of cardio-metabolic morbidity and probably an initial stage of inflammatory induced morbidity events. Inflammation in obesity results from leukocyte infiltration of adipose tissue, inducing the production of cytokines and other products capable of inducing inflammation [1,2]. Therefore, associations between several risk factors and obesity parameters have been determined. The association of leukocytes with biochemical and clinical parameters during the metabolic syndrome and diabetes has been previously documented. Metaanalysis and correlation studies in individuals with IR or with type 2 diabetes suggest a role for circulating leukocytes on the pathophysiological changes associated with those alterations [24–26]. In this study, several leukocyte subtype counts from obese individuals with increased concentration of serum insulin, showed positive correlations with serum CRP values, suggesting a role of these cells on CRP levels and a role for inflammation in obesity with no co-morbidities. Previously the association of leukocyte count with obesity parameters has also been reported. In this regard, correlations of leukocyte count with anthropometric parameters of obesity, serum HDL concentration and increased levels of insulin have been reported, suggesting a role of circulating leukocytes on the induction of risk factors, when co-morbidities were not present [27]. Similar findings in individuals with increased risk of diabetes have been documented, showing increased leukocyte subtype count related to insulin sensitivity rather than subclinical inflammation [28]. Since our findings were present only in obese individuals with IR, a common pathogenic mechanism in obesity and diabetes could be suggested. Accordingly, stronger correlation between insulin content and CRP was found in this study, suggesting a possible additional role of insulin in CRP levels and inflammation. However, leukocyte count from obese individual without IR was also associated with CRP content, suggesting other factors involved in CRP production in obesity. Increased CRP concentration in obese individuals could be related to decreased values of adiponectin. Adiponectin is an antiinflammatory marker [16], and CRP increases in response to an inflammatory process [4,5]. The association of CRP with obesity parameters in individuals with alterations of the arterial thickness of intima-media have been reported [29–32], suggesting a possible inflammatory effect of CRP in obesity. Since, adiponectin concentration showed significant inverse correlations with CRP in obese individuals in this study; the decreased presence of this molecule in blood could reduce its anti-inflammatory effect with further increment of CRP values, suggesting lacking of adiponectin protective role during the inflammatory events in obesity. Decreased levels of IL-2 were not associated with leukocyte counts; however, a negative correlation between insulin and IL-2 content was observed, suggesting an anti-inflammatory role of this hormone decreasing IL-2, a powerful inflammatory cytokine. Accordingly, obese individuals with IR did not present increased inflammatory markers compared with increased values in obese
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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Fig. 2. Correlations of leukocyte type counts with CRP and IL-2 concentrations. Superior panel: positive correlation graphics. CRP was positively correlated with total leukocyte (A), neutrophil (B), lymphocyte (C) and monocyte (D) counts in obese individuals with IR. Inferior panel: Leukocytes from obese individuals did not correlate with IL-2 values. Framed values represent positive correlations of leukocyte types with CRP.
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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Fig. 3. Correlations of insulin values with CRP and IL-2 concentrations. Superior panel: positive correlation graphics. Insulin values were positively correlated with CRP (A) and negatively correlated with IL-2 (B) in obese individuals with IR. In obese individuals without IR, insulin values were also positively correlated with CRP (C). Inferior panel: Insulin values from obese individuals without IR did not correlate with IL-2 values. Framed values represent significant insulin correlations.
individuals without IR [33]. The anti-inflammatory effect of insulin reflected in the suppression of plasma concentration of cytokines, adhesion molecules, matrix metalloproteinase, tissue factor, plasminogen activator inhibitor-1, vascular endothelial growth factor, intranuclear nuclear factor-kB and Egr-1 binding in peripheral blood mononuclear cells has been documented [17–19]. Increased levels of IL-2 seem to be irrelevant during the early stages of obesity inflammation and probably in latter periods, since, no increased values of serum IL-2 in obese individuals with several co-morbidities have been reported [34–36]; however, low levels of IL-2 could contribute to persistent inflammatory stage. In this regard, the key finding showing the important role for IL-2 in Treg cell development and homeostasis could be important. Low or absent presence of IL-2 can induce decreased Treg cells and failure to control autoreactive and inflammatory responses by inducing over-activity of T cell response instead of immunodeficiency [37– 39]. IR during obesity is secondary to an inflammatory response caused by the infiltration of monocytes and other circulating leukocytes in the adipose tissue [40–43], resulting in altered insulin-mediated signaling pathway and in hyperglycemia, hypertension and hyperlipidemia [44–46]. In the present study, individuals with high levels of insulin, and clinical and metabolic parameters in the normal range, did not show the expected consequences related to IR, suggesting a sustained homeostasis that could be due to an early presence of IR status or antiinflammatory effect of insulin. The N/L, a new marker for predicting steatohepatitis and fibrosis in patients with nonalcoholic fatty liver disease is higher in these types of patients [47]. In this study, no significant differences were found when N/L values were correlated with the CRP or IL-2 content, suggesting that N/F is not a useful marker for these compounds.
As described above, obese individuals without co-morbidities expressed increased levels of CRP and decreased levels of IL-2. CRP is an acute-phase reactant, whose plasma levels rapidly increase in response to tissue injury and to a variety of inflammatory stimuli; it is predominantly synthesized by the liver and regulated by proinflammatory cytokines [4,5]. During an acute-phase response, rapid increase in the production of CRP is observed [48]. In addition to being a sensitive marker of inflammation, CRP has direct proinflammatory effects, decreasing nitric oxide and prostacyclin release and increasing the expression levels of monocyte chemoattractant protein-1, IL-8, and plasminogen activator inhibitor-1 in endothelial cells. In monocytes, CRP induces tissue factor secretion, increases reactive oxygen species and pro-inflammatory cytokine release and induces monocyte chemotaxis and adhesion. In vascular smooth muscle cells (VSMC), CRP increases inducible nitric oxide production, NF-kB and mitogen-activated protein kinase activities and up-regulates angiotensin II receptor (AT1) resulting in increased reactive oxygen species and VSMC proliferation [6– 8]. Obesity has been associated with chronic low grade inflammation [49], triggered by white adipose tissue, which is an active factor in the metabolism linked to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-a), interleukins (IL-1 and IL-6) and CRP [50–53]. CRP has been reported increased in morbid obesity and metabolic syndrome [54,55]. The increased presence of CRP in obesity reflects the inflammatory events and seems to influence metabolic disorders [56]. In this regard, it has been reported that CRP has a direct inhibitory effect on insulin signaling [57], suggesting that CPR could have an important role inducing co-morbidities in obesity. Therefore, this cytokine could be an early marker to be detected in obesity without co-morbidities.
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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Fig. 4. Correlations of adiponectin values with CRP and IL-2 concentrations. Superior panel: negative correlation graphics. In obese individuals, adiponectin was negatively correlated with CRP (A and B). Inferior panel: Adiponectin from obese individuals did not correlate with IL-2 values. Framed values represent significant adiponectin correlations.
IL-2 was originally characterized as a potent T cell growth factor, promoting the expansion of antigen activated CD4+ and CD8+ T cells. This cytokine is produced mainly by activated CD4+ and CD8+ T cells and consumed primarily by cells expressing the high-affinity form of the IL-2 receptor. IL-2 has pro-inflammatory and anti-apoptotic effects and up-regulated CD25 expression [9–13]. The influence of IL-2 on the homeostasis of immune response is very important, acting on regulatory T cells (Treg cells), which control the activity of effector cells [14,15]. In this study, low levels of circulating IL-2 in obese individuals were found. Similar finding was observed in in vitro studies, where lipopolysaccharide treated human primary adipocytes showed, increased production of several cytokines/ chemokines; however, IL-2 was down-regulated [58]. These data suggest an altered functional biology in adipocytes that could be involved in the low circulating levels of IL-2 in obese individuals in this study. In addition, CPR could be modulated by the IL-2 content. Immunoregulatory effects of CRP are different during the acute phase of inflammation and remote periods of immune response. At the early stages, when the production of IL-2 is negligible, CRP can act as a mitogen inducing polyclonal activation of lymphocytes, while later it acts as a factor limiting clonal expansion of committed immunocompetent cells [59]. Therefore, the combination of high levels of CRP and low levels of IL-2, found in this study, could represent early stages of inflammation. In conclusion, this study showed obese individuals without abnormal biochemical and clinical parameters with increased CRP and decreased IL-2 contents, suggesting a role for these cytokines in the early stage of inflammation. Molecules such as adiponectin
and insulin may be involved in anti-inflammatory events, retarding the co-morbidity expression. These obese individuals with different levels of insulin may represent two different initial stages of the chronic inflammation found in obesity. Further studies are required to determine the evolution of obese individuals with high insulin levels, but with normal clinical and biochemical parameters and the presence of inflammatory and anti-inflammatory events over time. Acknowledgements This manuscript was supported by Consejo de Desarrollo Cientı´fico y Humanı´stico de la Universidad del Zulia (CC-0440-10, Maracaibo, Venezuela) and it had no role in the design, analysis or writing of this article. We thank Lissette Connell and Nelson Fernandez for excellent laboratory assistance (Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela), and Nora Palazzi and Volga Mijac for clinical assistance. Conflicts of interest: The authors have none to declare. References [1] Pecht T, Gutman-Tirosh A, Bashan N, Rudich A. Peripheral blood leukocyte subclasses as potential biomarkers of adipose tissue inflammation and obesity subphenotypes in humans. Obes Rev 2014;15:322–37. http://dx.doi.org/ 10.1111/obr.12133. [2] Carvalheira JB, Qiu Y, Chawla A. Blood spotlight on leukocytes and obesity. Blood 2013;122:3263–7. http://dx.doi.org/10.1182/blood-2013-04-459446.
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Original Article
Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content Renata Vargas a, Elena Ryder a, Marı´a Diez-Ewald a, Jesu´s Mosquera a,*, Anyelo Dura´n a, ˜ ez b, Caterina Pen ˜ a c, Erika Ferna´ndez a Nereida Valero a, Adriana Pedrean a
Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela Ca´tedra de Inmunologı´a. Escuela de Bionanalisis, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela c Ca´tedra de Gene´tica, Escuela de Bionanalisis, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela b
A R T I C L E I N F O
A B S T R A C T
Keywords: Obesity Inflammation Cytokines Insulin
Aims: Chronic inflammation in obesity is associated with co-morbidities such as, hyperglycemia, hypertension and hyperlipidemia. Leukocytes play an important role in this inflammation and C-reactive protein (CRP) and Interleukin-2 (IL-2) can be important effectors during the immune response in obesity; however, the initial inflammatory events in obesity remain unclear. The aim of this study was to determine the circulating levels of CRP, IL-2, insulin and adiponectin, their association and the association with leukocyte count in obese individuals without co-morbidities and with or without insulin resistance (IR). Materials and methods: Nineteen obese non-diabetic and 9 lean subjects were studied for serum levels of CRP, IL-2, insulin, adiponectin, lipids, glycated hemoglobin, glycemia, for homeostasis model assessment of insulin resistance (HOMA-IR), arterial pressure and anthropometric parameters, and for leukocyte counts. Neutrophil/lymphocyte ratio (N/L) was calculated using the loge of leukocyte counts. Associations were determined by Pearson’s correlation. Results: None of the studied groups presented co-morbidities and two groups of obese individuals with normal or high levels of insulin (IR) were found. Increased CRP concentration and decreased IL-2 and adiponectin concentrations in obese were observed. Positive correlation between leukocyte type counts with CRP in obese with IR was found; however, no correlations with IL-2 in obese were observed. Insulin in obese were positively correlated with CRP and negatively correlated with IL-2 in IR obese individuals. Adiponectin in obese was negatively correlated with CRP. Conclusion: CRP and IL-2 may represent two important effectors in the early inflammatory events in obese individuals without co-morbidities. Adiponectin and insulin may be involved in antiinflammatory events. ß 2015 Diabetes India. Published by Elsevier Ltd. All rights reserved.
1. Introduction Obesity is associated with cardio-metabolic morbidity; however, some obese individuals escape this association, forming a unique obese sub-phenotype. Clinical biomarkers that reflect already manifested co-morbidities, such as dyslipidemia, hyperglycemia, hypertension, and anthropometric or imaging-based assessment of adipose tissue distribution have been used to define
* Corresponding author at: Apartado Postal 23, Maracaibo, 4001-A., Zulia, Venezuela. Tel.: +58 261 7114752; fax: +58 261 7916053. E-mail address: [email protected] (J. Mosquera).
this sub-phenotype [1]. The relationship between co-morbidities and inflammatory events in the obesity has been reported [1]; in addition, the association of leukocytes with chronic inflammation in obesity has also been reported [1,2]. These data suggest that the leukocyte induces inflammation with further development of comorbidities. The initiation of co-morbidities could be related to the beginning and persistent time of the inflammatory events during obesity. Therefore, obese individuals with no co-morbidities can represent an early stage of inflammation. During obesity, chronic inflammation can be mediated by innate and adaptive immune responses [3]. In this regard, the expression of cytokines such as CRP and IL-2 during inflammation could be important in both types of immune response during obesity. CRP is an acute-phase reactant
http://dx.doi.org/10.1016/j.dsx.2015.09.007 1871-4021/ß 2015 Diabetes India. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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that rapidly increases in response to tissue injury and to a variety of inflammatory stimuli [4,5]. This cytokine is involved in several proinflammatory events, including chemotaxis, modulation of nitric oxide and oxygen species, induction of cytokines and proliferation of immune and nonimmune cells [6–8]. IL-2 is a potent proinflammatory cytokine, produced mainly by activated CD4+ and CD8+ T cells, with important effects on the biology of regulatory T cells (Treg cells), and therefore, on the homeostasis of immune response [9–15]. The presence of modulatory compounds on the immune response in obesity has been little studied. Insulin and adiponectin, molecules with anti-inflammatory effect [16–19], could have an important role during obesity inflammatory events. Therefore, the aim of this study was to determine the circulating levels of CRP, IL-2, adiponectin and insulin and evaluate the associations between them and with the leukocyte count in obese individuals with normal clinical and biochemical parameters and with or without IR. 2. Subjects A total of 44 obese individuals chosen through a voluntary call to participate in this study were analyzed in an initial screening. Only 19 obese non-diabetic adult subjects (20–55 years old) without the known risk factors for obesity (no co-morbidities) were selected. Increased serum insulin concentration was found in 10 of these individuals; therefore, obese individuals were divided in two groups: one with normal levels of insulin (n = 10) and the other with increased levels (individuals with IR; n = 9). All obese individuals had a body mass index (BMI) greater than 30 kg/ m2. Nine lean control individuals, whose BMI was less than 25 kg/ m2, were also selected in this study. All subjects fulfilled the inclusion criteria: absence of arterial hypertension, diabetes or other metabolic abnormalities, not evidence of hepatitis or HIV infection (determined by the absence of antibodies against the viruses), absence of any other current infectious processes, not having taken any of the medications known to influence glucose or lipid metabolism or the inflammatory pathway, and in the case of women, not under hormonal contraceptive drugs. Information regarding age, sex, qualifying criteria, current medications, height, weight and waist circumference was collected. Body mass index was calculated from the formula: weight (kg)/height (m2), considering obesity when it was >30 kg/m2. Absence of hypertension was confirmed by means of a sphyngomanometer. Ultrasound was used for the estimation of visceral fat and hepatic steatosis as described by Ryder et al. [20]. Acquisition of blood samples and all scientific studies were approved by the Ethic Committee of the Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’,
University of Zulia in Maracaibo, Venezuela, according with the principles of the Declaration of Helsinki as revised in 2008. Written consent was obtained from all subjects. 3. Materials and methods 3.1. Biochemical, immunological and hematological measurements A fasting blood sample was withdrawn from each subject for the determination of studied parameters. Glucose, total cholesterol and triglycerides were measured enzymatically; high density lipoprotein (HDL)-cholesterol was measured after precipitation of the apo B-containing lipoproteins (Human GmbH, Germany). Plasma insulin was determined by chemiluminescence immunoassay (IMMULITE 1000, Siemens Diagnostics, USA) and total glycated hemoglobin by the method of Bioscience Medical SL, Spain. HOMA-IR was calculated as: fasting insulin (IU/L) fasting glucose (mmol/L)/22.5 according to Mathews et al. [21], and the cut off point to consider IR was 2.6 [22,23]. A highly sensitive CRP enzyme immunoassay was used to quantify circulating levels of CPR (Hemagen Diagnostics, Inc., Columbia, USA) and the results were expressed as mg/L. IL-2 was measured using a commercial kit (Mersham Life Science, England) and the results expressed as pg/ mL. Serum content of adiponectin was also determined by a commercial ELISA (SPI-BIO Bertin Pharma Branch France) and the results expressed as mg/mL. The number of leukocytes and leukocyte subtypes were computed with an autoanalyzer (Beckman Coulter Counter, Coulters Corporation, FL, USA). The neutrophil: lymphocyte ratio was defined as the loge neutrophil count/ loge lymphocyte count. 3.2. Statistical analysis The data were normally distributed and were expressed as the mean standard deviation. Differences among groups were analyzed by ANOVA and Bonferroni’s test. Pearson’s correlations were estimated for various blood cell counts or for insulin or adiponectin content with CRP and IL-2 content using PRISM statistical software (GraphPad Software). Differences were considered statistically significant at p < 0.05. 4. Results None of the lean and obese individuals in this study had hypertension, hyperglycemia or elevated glycated hemoglobin, and their mean values for total and LDL cholesterol were similar among the three groups. As expected, the BMI, HOMA-IR, waist
Table 1 Clinical and laboratory parameters in obese individuals with or without IR and in healthy lean control individuals. Parameter
Control (A)
Ob IR (B)
Ob no IR(C)
A vs. B P value
A vs. C P value
B vs. C P value
N Age (years) Body mass index (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Homa-IR Waist circumference (cm) Insulin (mU/mL) Glycemia (mg/dL) Glycated hemoglobin (%) Triglycerides (mg/dL) Cholesterol (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Visceral fat (cm)
9 29.7 3.3 22.4 0.5 112.5 2.5 75 1.9 0.83 0.2 77 2.0 4.1 1.0 80.3 3.8 6.11 0.14 80.3 3.8 163.9 7.9 51.8 2.5 99.8 6.2 2.36 0.3
9 33.3 4.2 37.8 0.8 122.2 2.8 85.1 1.7 5.83 0.7 114.94.2 25.3 2.8 92.8 3.4 6.37 0.36 126.614.7 186 14.0 41 2.4 119.511.0 6.2 0.6
10 38.7 3.4 34.6 1.2 114.0 2.7 76 1.6 1.48 0.2 106 2.4 6.93 0.9 86.4 1.6 6.44 0.14 127.9 13.2 187.1 14.4 42 3.3 117.4 14.5 5.4 0.4
NS <0.01 <0.05 <0.01 <0.01 <0.01 <0.01 <0.05 NS <0.05 NS <0.05 NS <0.01
NS <0.01 NS NS NS <0.01 NS NS NS <0.05 NS <0.05 NS <0.01
NS <0.05 NS <0.01 <0.01 NS <0.01 NS NS NS NS NS NS NS
Ob IR: obese with insulin resistance; Ob no IR: obese without insulin resistance; values are expressed as mean standard deviation.
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circumference and visceral fat of obese with IR and obese without IR were elevated in relation to the control group; however, obese with IR showed significantly increased values of BMI, HOMA-IR and insulin compared to obese without IR. Triglyceride values were higher and HDL cholesterol lower in both obese groups; however, the mean values were in the normal range for this population (Table 1). Increased serum CRP content in obese individuals with and without IR was observed (Fig. 1A). The values of IL-2 in all obese individuals were decreased (Fig. 1B). Serum analysis showed decreased values of adiponectin content in obese individuals (lean: 10.33 1.59; obese with IR: 7.49 0.88*; obese without IR: 8.20 0.66* mg/mL. *p < 0.001 vs. lean). Positive correlations between total leukocyte, neuthrophil, lymphocyte and monocyte counts with CRP concentration in obese individuals with IR were observed; however, these correlations in obese individuals without IR were absent (Fig. 2). No correlations between the different leukocyte type counts and the IL-2 values were observed in obese individuals (Fig. 2). Values of serum insulin in all obese groups were positively correlated with the CRP values and negatively correlated with IL-2 concentration in obese individuals with IR (Fig. 3). Decreased values of circulating adiponectin in obese groups were negatively correlated with CRP,
Fig. 1. Serum concentrations of CRP and IL-2 in obese and lean individuals. (A) Increased CRP concentration was found in obese individuals. (B) Decreased IL-2 values were found in obese individuals.
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with stronger correlation in obese individuals with IR (Fig. 4). There were no statistical significances when the neutrophil: lymphocyte ratio was correlated with CRP or IL-2 content (Fig. 2). 5. Discussion Obesity is a major risk factor for cardio-metabolic morbidity. Co-morbidities such as hyperglycemia, hypertension, dyslipidemia and alterations in anthropometric or imaging-based for adipose tissue distribution parameters in obesity have been widely studied. In addition, pro-inflammatory events in human obesity have been shown to correlate with adipose tissue inflammation and with obesity-associated health risks [1]. In this study two groups of obese subjects (with and without IR) were analyzed. Alterations such as dyslipidemia, hyperglycemia and hypertension were absent suggesting lack of cardio-metabolic morbidity and probably an initial stage of inflammatory induced morbidity events. Inflammation in obesity results from leukocyte infiltration of adipose tissue, inducing the production of cytokines and other products capable of inducing inflammation [1,2]. Therefore, associations between several risk factors and obesity parameters have been determined. The association of leukocytes with biochemical and clinical parameters during the metabolic syndrome and diabetes has been previously documented. Metaanalysis and correlation studies in individuals with IR or with type 2 diabetes suggest a role for circulating leukocytes on the pathophysiological changes associated with those alterations [24–26]. In this study, several leukocyte subtype counts from obese individuals with increased concentration of serum insulin, showed positive correlations with serum CRP values, suggesting a role of these cells on CRP levels and a role for inflammation in obesity with no co-morbidities. Previously the association of leukocyte count with obesity parameters has also been reported. In this regard, correlations of leukocyte count with anthropometric parameters of obesity, serum HDL concentration and increased levels of insulin have been reported, suggesting a role of circulating leukocytes on the induction of risk factors, when co-morbidities were not present [27]. Similar findings in individuals with increased risk of diabetes have been documented, showing increased leukocyte subtype count related to insulin sensitivity rather than subclinical inflammation [28]. Since our findings were present only in obese individuals with IR, a common pathogenic mechanism in obesity and diabetes could be suggested. Accordingly, stronger correlation between insulin content and CRP was found in this study, suggesting a possible additional role of insulin in CRP levels and inflammation. However, leukocyte count from obese individual without IR was also associated with CRP content, suggesting other factors involved in CRP production in obesity. Increased CRP concentration in obese individuals could be related to decreased values of adiponectin. Adiponectin is an antiinflammatory marker [16], and CRP increases in response to an inflammatory process [4,5]. The association of CRP with obesity parameters in individuals with alterations of the arterial thickness of intima-media have been reported [29–32], suggesting a possible inflammatory effect of CRP in obesity. Since, adiponectin concentration showed significant inverse correlations with CRP in obese individuals in this study; the decreased presence of this molecule in blood could reduce its anti-inflammatory effect with further increment of CRP values, suggesting lacking of adiponectin protective role during the inflammatory events in obesity. Decreased levels of IL-2 were not associated with leukocyte counts; however, a negative correlation between insulin and IL-2 content was observed, suggesting an anti-inflammatory role of this hormone decreasing IL-2, a powerful inflammatory cytokine. Accordingly, obese individuals with IR did not present increased inflammatory markers compared with increased values in obese
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Fig. 2. Correlations of leukocyte type counts with CRP and IL-2 concentrations. Superior panel: positive correlation graphics. CRP was positively correlated with total leukocyte (A), neutrophil (B), lymphocyte (C) and monocyte (D) counts in obese individuals with IR. Inferior panel: Leukocytes from obese individuals did not correlate with IL-2 values. Framed values represent positive correlations of leukocyte types with CRP.
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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Fig. 3. Correlations of insulin values with CRP and IL-2 concentrations. Superior panel: positive correlation graphics. Insulin values were positively correlated with CRP (A) and negatively correlated with IL-2 (B) in obese individuals with IR. In obese individuals without IR, insulin values were also positively correlated with CRP (C). Inferior panel: Insulin values from obese individuals without IR did not correlate with IL-2 values. Framed values represent significant insulin correlations.
individuals without IR [33]. The anti-inflammatory effect of insulin reflected in the suppression of plasma concentration of cytokines, adhesion molecules, matrix metalloproteinase, tissue factor, plasminogen activator inhibitor-1, vascular endothelial growth factor, intranuclear nuclear factor-kB and Egr-1 binding in peripheral blood mononuclear cells has been documented [17–19]. Increased levels of IL-2 seem to be irrelevant during the early stages of obesity inflammation and probably in latter periods, since, no increased values of serum IL-2 in obese individuals with several co-morbidities have been reported [34–36]; however, low levels of IL-2 could contribute to persistent inflammatory stage. In this regard, the key finding showing the important role for IL-2 in Treg cell development and homeostasis could be important. Low or absent presence of IL-2 can induce decreased Treg cells and failure to control autoreactive and inflammatory responses by inducing over-activity of T cell response instead of immunodeficiency [37– 39]. IR during obesity is secondary to an inflammatory response caused by the infiltration of monocytes and other circulating leukocytes in the adipose tissue [40–43], resulting in altered insulin-mediated signaling pathway and in hyperglycemia, hypertension and hyperlipidemia [44–46]. In the present study, individuals with high levels of insulin, and clinical and metabolic parameters in the normal range, did not show the expected consequences related to IR, suggesting a sustained homeostasis that could be due to an early presence of IR status or antiinflammatory effect of insulin. The N/L, a new marker for predicting steatohepatitis and fibrosis in patients with nonalcoholic fatty liver disease is higher in these types of patients [47]. In this study, no significant differences were found when N/L values were correlated with the CRP or IL-2 content, suggesting that N/F is not a useful marker for these compounds.
As described above, obese individuals without co-morbidities expressed increased levels of CRP and decreased levels of IL-2. CRP is an acute-phase reactant, whose plasma levels rapidly increase in response to tissue injury and to a variety of inflammatory stimuli; it is predominantly synthesized by the liver and regulated by proinflammatory cytokines [4,5]. During an acute-phase response, rapid increase in the production of CRP is observed [48]. In addition to being a sensitive marker of inflammation, CRP has direct proinflammatory effects, decreasing nitric oxide and prostacyclin release and increasing the expression levels of monocyte chemoattractant protein-1, IL-8, and plasminogen activator inhibitor-1 in endothelial cells. In monocytes, CRP induces tissue factor secretion, increases reactive oxygen species and pro-inflammatory cytokine release and induces monocyte chemotaxis and adhesion. In vascular smooth muscle cells (VSMC), CRP increases inducible nitric oxide production, NF-kB and mitogen-activated protein kinase activities and up-regulates angiotensin II receptor (AT1) resulting in increased reactive oxygen species and VSMC proliferation [6– 8]. Obesity has been associated with chronic low grade inflammation [49], triggered by white adipose tissue, which is an active factor in the metabolism linked to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-a), interleukins (IL-1 and IL-6) and CRP [50–53]. CRP has been reported increased in morbid obesity and metabolic syndrome [54,55]. The increased presence of CRP in obesity reflects the inflammatory events and seems to influence metabolic disorders [56]. In this regard, it has been reported that CRP has a direct inhibitory effect on insulin signaling [57], suggesting that CPR could have an important role inducing co-morbidities in obesity. Therefore, this cytokine could be an early marker to be detected in obesity without co-morbidities.
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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Fig. 4. Correlations of adiponectin values with CRP and IL-2 concentrations. Superior panel: negative correlation graphics. In obese individuals, adiponectin was negatively correlated with CRP (A and B). Inferior panel: Adiponectin from obese individuals did not correlate with IL-2 values. Framed values represent significant adiponectin correlations.
IL-2 was originally characterized as a potent T cell growth factor, promoting the expansion of antigen activated CD4+ and CD8+ T cells. This cytokine is produced mainly by activated CD4+ and CD8+ T cells and consumed primarily by cells expressing the high-affinity form of the IL-2 receptor. IL-2 has pro-inflammatory and anti-apoptotic effects and up-regulated CD25 expression [9–13]. The influence of IL-2 on the homeostasis of immune response is very important, acting on regulatory T cells (Treg cells), which control the activity of effector cells [14,15]. In this study, low levels of circulating IL-2 in obese individuals were found. Similar finding was observed in in vitro studies, where lipopolysaccharide treated human primary adipocytes showed, increased production of several cytokines/ chemokines; however, IL-2 was down-regulated [58]. These data suggest an altered functional biology in adipocytes that could be involved in the low circulating levels of IL-2 in obese individuals in this study. In addition, CPR could be modulated by the IL-2 content. Immunoregulatory effects of CRP are different during the acute phase of inflammation and remote periods of immune response. At the early stages, when the production of IL-2 is negligible, CRP can act as a mitogen inducing polyclonal activation of lymphocytes, while later it acts as a factor limiting clonal expansion of committed immunocompetent cells [59]. Therefore, the combination of high levels of CRP and low levels of IL-2, found in this study, could represent early stages of inflammation. In conclusion, this study showed obese individuals without abnormal biochemical and clinical parameters with increased CRP and decreased IL-2 contents, suggesting a role for these cytokines in the early stage of inflammation. Molecules such as adiponectin
and insulin may be involved in anti-inflammatory events, retarding the co-morbidity expression. These obese individuals with different levels of insulin may represent two different initial stages of the chronic inflammation found in obesity. Further studies are required to determine the evolution of obese individuals with high insulin levels, but with normal clinical and biochemical parameters and the presence of inflammatory and anti-inflammatory events over time. Acknowledgements This manuscript was supported by Consejo de Desarrollo Cientı´fico y Humanı´stico de la Universidad del Zulia (CC-0440-10, Maracaibo, Venezuela) and it had no role in the design, analysis or writing of this article. We thank Lissette Connell and Nelson Fernandez for excellent laboratory assistance (Instituto de Investigaciones Clı´nicas ‘‘Dr. Ame´rico Negrette’’, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela), and Nora Palazzi and Volga Mijac for clinical assistance. Conflicts of interest: The authors have none to declare. References [1] Pecht T, Gutman-Tirosh A, Bashan N, Rudich A. Peripheral blood leukocyte subclasses as potential biomarkers of adipose tissue inflammation and obesity subphenotypes in humans. Obes Rev 2014;15:322–37. http://dx.doi.org/ 10.1111/obr.12133. [2] Carvalheira JB, Qiu Y, Chawla A. Blood spotlight on leukocytes and obesity. Blood 2013;122:3263–7. http://dx.doi.org/10.1182/blood-2013-04-459446.
Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007
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Please cite this article in press as: Vargas R, et al. Increased C-reactive protein and decreased Interleukin-2 content in serum from obese individuals with or without insulin resistance: Associations with leukocyte count and insulin and adiponectin content. Diab Met Syndr: Clin Res Rev (2015), http://dx.doi.org/10.1016/j.dsx.2015.09.007