Influence of weight reduction on blood levels of C-reactive protein, tumor necrosis factor-α, interleukin-6, and oxylipins in obese subjects

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Abstract

Introduction

Obesity is associated with inflammation and weight reduction has been shown to influence the inflammatory process. Besides classic inflammatory markers, oxidized polyunsaturated fatty acid (PUFA) metabolites (oxylipins) are potent mediators of inflammation. Little is known about endogenous levels of oxylipins, e.g. hydroxy, epoxy and dihydroxy FA in obese subjects with persistent low-grade inflammation. We aimed to evaluate levels of inflammatory markers and blood oxylipins in obese subjects before and after weight reduction.

Subjects and methods

In the present study, 42 obese (BMI 32.7±0.22 kg/m2) men and women were classified in groups according to high-sensitivity C-reactive protein (hsCRP) levels (no inflammation<1 mg/L; low-grade inflammation≥3 mg/L). Subjects underwent an intervention for eight weeks, which consisted of two phases: (1) week 1 and 2: total replacement of three meals by a formula diet and (2) six week partial formula diet (replacement of 1–2 meals). Blood samples were taken prior and post intervention for analysis of plasma protein levels of hsCRP, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Plasma Levels of free (unesterified) hydroxy, epoxy, and dihydroxy FAs as well as several prostanoids were analyzed in plasma by means of LC-MS-based targeted metabolomics.

Results

At baseline subjects with low-grade inflammation (hsCRP 8.95±1.39 mg/L) showed significant higher levels of IL-6 (22.7±1.15 ng/L) and TNF-α (17.4±0.75 ng/L) compared to subjects with no inflammation (hsCRP: 0.69±0.05 mg/L; IL-6: 15.9±1.18 ng/L; TNF-α: 14.6±0.80 ng/L). In both group’s body weight was significantly reduced (p<0.001) after intervention (no inflammation group: −7.19±0.86 kg, −7.3±0.89%, p<0.001; low-grade inflammation group: −6.78±0.87 kg, −6.7±0.81%, p<0.001). Moreover, we observed significant decreases in levels of hsCRP (4.66±0.64 mg/L; p=0.006), IL-6 (6.81±1.15 ng/L; p<0.001) and TNF-α (6.09±0.47 ng/L; p<0.001) in subjects with low-grade inflammation. Of 60 quantified oxylipins, 11 linoleic acid (LA)-, 1 dihomo-γ-linolenic acid (DGLA)-, 7 alpha linolenic acid (ALA)-, 15 arachidonic acid (AA)-, 8 eicosapentaenoic acid (EPA)- and 18 docosahexaenoic acid (DHA)-metabolites could be detected in plasma. For most oxylipins no differences were found between the low and high hsCRP groups before and after weight reduction. Interestingly, in subjects with low- grade inflammation several AA-derived oxylipins (5-, 8-, 12-hydroxyeicosatetraenoic acids (HETE)) were significantly higher compared to subjects with no inflammation before weight reduction and significantly reduced after weight reduction.

Conclusion

Even moderate weight loss in obese subjects correlates to a significant improvement in the inflammatory state, by reducing hsCRP, IL-6, TNF-α and few oxylipins. The biological consequences of these changes remain to be further investigated.

Introduction

The rapidly growing rate of obesity is a severe health problem [1]. The basic cause of this problem is mostly due to the imbalance between energy intake and expenditure which induces an excessive accumulation of body fat [2]. Specifically, the abdominal obesity plays a crucial role in the development of obesity-related complications. Increased visceral adipose tissue is the central feature of the Metabolic Syndrome (MetS), while other components include hyperglycemia, atherogenic dyslipidaemia and hypertension [3], [4]. In addition, metabolic disturbances in obese subjects trigger a persistent low-grade inflammatory response, accompanied by an induction of several markers of inflammation [5], [6]. The adipose tissue, as an active endocrine organ, activates pro-inflammatory serine kinase cascade signaling pathways which promote the secretion of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [6], [7], [8]. IL-6 and TNF-α in turn are both stimulators of C-Reactive Protein (CRP) release from hepatocytes [8], [9]. Therefore, higher levels of high-sensitivity (hs)CRP, IL-6 and TNF-α indicate an inflammatory state in obese subjects [8], [10], [11], [12], which plays an important role in atherosclerotic processes [13]. Values of CRP≥3 mg/L are thought to reflect elevated low-grade inflammation and considered as predictor for future cardiovascular and metabolic diseases [14], [15]. Thus, lowering inflammation is important for improving obesity-related risk factors [16].

Oxidized arachidonic acid (AA 20:4n-6)-derived metabolites, i.e. eicosanoids, are well known to act as potent mediators of inflammation [17], [18], [19]. However, the family of oxidized polyunsaturated fatty acids (PUFA), oxylipins, is broad and only limited information is available about the biological role of several hydroxy, epoxy and dihydroxy FAs derived from AA and other PUFAs such as linoleic acid (LA 18:2n-6), dihomo-γ-linolenic acid (DGLA 20:3n-6), α-linolenic acid (ALA 18:3n-3), eicosapentaenoic acid (EPA 20:5n-3) or docosahexaenoic acid (DHA 22:6n-3). PUFAs can be enzymatically oxidized to oxylipins by conversion of cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP) enzymes in the AA cascade [20]. A body of evidence indicates that 5-LOX products of AA (e.g., 5-hydroxyeicosatetraenoic acid (5-HETE) or 5-oxo-eicosatetraenoic acid (5-oxo-ETE)) exert pro-inflammatory effects by increasing the production of pro-inflammatory cytokines and inducing chemotaxis to attract inflammatory cells in the blood vessels [21], [22]. Accordingly, 5-LOX products are considered to be detrimental factors in several pathological conditions, including cardiovascular diseases [22], [23]. 12-LOX may play a direct role in obesity-induced-inflammation and the pathology of insulin resistance [19], [24], [17]. This enzyme is primarily involved in the production of pro-inflammatory AA-derived oxylipins (e.g., 12-HETE), and metabolizing LA to 13-hydroxyoctadecadienoic acid (13-HODE) [20]. As discussed previously, the majority of 12-LOX products detected in plasma probably results from platelet activation during sample preparation [25], [26]. Little is known about the endogenous levels of hydroxy, epoxy and dihydroxy FA in obese subjects with low-grade inflammation. Moreover, dietary adjustment resulting in weight reduction may influence the oxylipin pattern.

The aim of this study was to determine the reduction of inflammation markers in stage I obesity subjects (body mass index (BMI) between 30.0 and 34.9 kg/m²) with low-grade inflammation (hsCRP≥3 mg/L) compared to subjects with no inflammation (hsCRP<1 mg/L) and their response to formula-based low-fat low-calorie diet-induced weight reduction. In addition, we aimed to evaluate plasma oxylipin levels in obese subjects with no and low-grade inflammation as well as the effect of diet-induced weight loss on oxylipin patterns.

Section snippets

Subjects and study design

The subjects were selected from a collective of 160 men and women taking part in a weight reduction study. The study protocol, subject selection and anthropometric measurements methodology is described in [27].

The criteria for selection of subjects for this study were: (1) BMI between 30 and 34.9 kg/m2 and (2) hsCRP levels<1 mg/L or≥3 mg/L. 22 subjects (13 men and 9 women) with hsCRP levels<1 mg/L (no inflammation) as well as 20 subjects (7 men and 13 women) with hsCRP levels≥3 mg/L (low-grade

Results

Included subjects (22 male; 20 female) had an average age of 50 years (range: 33–64 years) and were allocated to obesity stage I (30.0–34.9 kg/m2). The formula diet as meal replacements was well tolerated. Four subjects (10%) dropped out of the study because of acute illness, no time or no reply. The serum concentrations of hsCRP, IL-6 and TNF-α as well as free oxylipins in plasma were quantified in blood samples of 42 subjects before and of 38 subjects after eight weeks of weight reduction.

Discussion

Low-grade systemic inflammation is characteristic in various chronic diseases such as MetS, cardiovascular disease (CVD), type 2 diabetes, hypertension, and non-alcoholic fatty liver disease, which are all associated with obesity [34]. Inflammation plays a key role in the development of atherosclerotic plaques [35]. A particular focus has been spent on elevated inflammatory markers, specifically hsCRP, but also IL-6 and TNF-α, which are associated with an increased cardiovascular risk [14], [36]

Conclusions

This study showed that a low-fat low-caloric diet significantly lowers weight and elevated hsCRP, IL-6 and TNF-α levels in obese subjects with low-grade inflammation, which suggests a beneficial clinical effect for CVD. We observed no systematic differences in plasma oxylipin concentrations between obese subjects with and without low-grade inflammation. This indicates that the formation and degradation of this class of lipid metabolites are under tight endogenous control. However, levels of the

Acknowledgments

The provision of the test products by Certmedica International GmbH (Aschaffenburg, Germany) is kindly acknowledged. The authors are solely responsible for the design and conduct of the study, collection, management, analysis, and interpretation of the data, as well as preparation of the manuscript. We would like to thank the participants who contributed their time to this project. This study was supported by a Marie Curie Career Integration Grant to NHS (CIG 293536), a grant of the fellowship

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