Obesity-related dysregulation of the Tryptophan–Kynurenine metabolism: Role of age and parameters of the metabolic syndrome

Authors


  • Funding agencies: This work was funded by the “Zukunftsfond Steiermark” Project “STYJOBS”. Furthermore, this work was partially supported by the Austrian Science Fund (FWF) grant No. P20455.

  • Disclosure: The authors declared no conflict of interest.

Abstract

Objective

Obesity-related immune mediated systemic inflammation was associated with the development of the metabolic syndrome by induction of the tryptophan (TRP)–kynurenine (KYN) pathway. The study aimed to assess whether this holds true across the lifespan from juvenility to adulthood.

Design and Methods

Five hundred twenty-seven participants aged between 10 and 65 years were analyzed. Standard anthropometric measures, carotid ultrasound, and laboratory analysis including interleukin-6, ultra-sensitive C-reactive protein, lipids, glucose metabolism, neopterin, TRP, KYN levels, and the KYN/TRP ratio were performed.

Results

Overweight/obese (ow/ob) adults had significantly increased KYN serum levels and a significantly increased KYN/TRP ratio. In sharp contrast, ow/ob juvenile males aged ≤18 years showed decreased, females similar KYN and KYN/TRP ratio in comparison to their control counterparts. Also, adult ow/ob subjects with metabolic syndrome showed markedly increased KYN/TRP ratios contrary to decreased KYN/TRP ratios in ow/ob juveniles. Abdominal fat content, characterized by age normalized waist circumference, and not body mass index, had the strongest effect for an increase of the KYN/TRP ratio in adults.

Conclusions

TRP metabolism and obesity-related immune mediated inflammation differs markedly between juveniles and adults. While childhood obesity seems to be dominated by a Th2-driven activation, an accelerated production of Th1-type cytokines may pave the way for later atherosclerotic endpoints.

Introduction

Obesity is a common, deadly, and costly disease in developed countries which impacts all age groups, race, and gender. Obesity can be classified as an inflammatory disease because it is associated with immune activation and a chronic, low-grade systemic inflammation (cLGI) [1-3]. The basic impetus for obesity is over nutrition and a lack of physical exercise. Over nutrition leads to an excess intake of tryptophan (TRP)—an essential amino acid, a precursor for serotonin (5-HT) and melatonin, and a key player in the caloric intake regulation [4, 5]. Yet, the circulating levels of TRP have been shown to be low in morbidly obese subjects [6].

TRP can be metabolized through the kynurenine (KYN) and methoxyindole pathways. The first, taking 95% of TRP, is mediated by the rate-limiting enzymes TRP 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). TDO is induced by TRP and stress hormones, while IDO is induced by pro-inflammatory, Th-1 type cytokines such as Interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) [7]. IDO induction causes TDO activity suppression and vice versa [8]. KYN, the product of IDO/TDO activity, acts as positive feedback for IDO activity, and, most importantly, inhibits TRP transport via the blood–brain barrier [7, 9]. The ratio of KYN (the product) to TRP (the substrate) is an established and good estimator of IDO activity [6].

An upregulation of IDO activity, essentially caused by chronic immune-mediated inflammation, could be a key component in the initiation and propagation of obesity and the associated metabolic syndrome (MetS) [4]. The induction of pro-inflammatory pathways in obesity usually results in a state of Th-1 weighted, low-grade inflammation, and a suppressed Th-2 immune response [2, 4, 10, 11]. Neopterin, produced by macrophages upon stimulation with the Th1-type cytokine, IFN-γ, is a sensitive and reliable indicator of Th1-type immune response [12].

The second TRP metabolism pathway leads to the formation of serotonin (5-HT) and melatonin, known satiety and satiation regulators. Upregulated Th1-type inflammation and IDO activity have been suggested to cause the decreased TRP availability for the methoxyindole pathway, and thus moderate several critical symptoms indirectly and directly linked to obesity [4]. Serotonin regulates carbohydrate and fat intake [13], relieves stress which is another caloric intake trigger [14], and inhibits neuropeptide Y (NYP)—one of the most potent orexigenic peptides in the hypothalamus [15]. In connections with sleep, melatonin also plays a critical role in caloric intake regulation. Sleep duration has been inversely linked to leptin levels and food consumption. Sleep deprivation upregulates orexin activity, which then activates NYP and induces hunger [16, 17].

In the foregoing, we examined chronic inflammatory activation [2], pre-atherosclerosis [2, 10, 18], and serum neopterin levels [19] in obese juveniles, and discovered that serum neopterin levels were not increased in this group [19]. This indicated that early onset low grade inflammation, as found in obese juveniles is different, and probably a more Th2-driven process in contrast to obese adults, where a Th1-weighted inflammation predominates. So far, there are no data available on TRP metabolism and its potential association with cLGI in obese juveniles and adults.

Thus, this study was designed to examine the hypothesis whether biomarkers of TRP metabolism and inflammation (i.e., neopterin, TRP, KYN, ultrasensitive C-reactive protein (US-CRP), and IL-6) differ in overweight/obese (ow/ob) youth as compared to ow/ob adults.

Methods

Study participants are from the prospective, observational study STYJOBS/EDECTA (STYrian Juvenile OBesity Study/Early DEteCTion of Atherosclerosis; ClinicalTrials.gov Identifier NCT00482924), which investigates metabolic/cardiovascular parameters in obese individuals who were free of chronic health conditions, except MetS. We included individuals aged between 10 and 65 years old (Table 1). The inclusion criterion for overweight/obese youth (≤18 years old) was body mass index (BMI) ≥85th to 94th (obese ≥95th) percentile, and for overweight/obese adults BMI > 25 < 29.9 kg/m2 (obese ≥30 kg/m2). Juvenile controls had a BMI between 5th and 84.9th percentile according to Cole et al. [20], adult controls a BMI between 18.5 and 24.9, respectively. The analysis of normal weighted controls was included in the design of this study to provide a reference for the metabolically healthy ow/ob. Participants were excluded if they received medications or hormone replacement therapy. Written consent was obtained from all adult participants, and parental consent was obtained from youth. Procedures: Standard anthropometric data (height, weight, waist-, hip-circumference, waist to hip-, waist to height-ratio) were obtained from each subject as described elsewhere [21]. Waist-circumference was measured midway between the lower costal margin and the iliac crest; hip-circumference at the maximum circumference over the buttocks [21], and resting blood pressure in a sitting position in the right arm at the end of the physical examination.

Table 1. Gender dependent characteristics of study participants (n = 571, normal-, versus overweight, obese)
 ≤18 years>18 years
 FemaleMale FemaleMale 
 ControlsOverweight/obeseControlsOverweight/obesePf/PmControlsOverweight/obeseControlsOverweight/obesePf/Pm
  1. KYN/TRP = Kynurenine (µmol/l)/Tryptophan (mmol/l) ratio.

Numbers141021380 129955682 
Tryptophan, µmol/l62.3 ± 9.364.4 ± 11.762.2 ± 10.664.5 ± 9.60.446/0.14258.1 ± 11.455.0 ± 7.964.5 ± 12.762.6 ± 9.00.024/0.331
Kynurenine, µmol/l2.2 ± 0.52.1 ± 0.62.9 ± 0.92.3 ± 0.70.837/0.0072.3 ± 0.72.6 ± 0.82.6 ± 0.73.0 ± 0.8<0.001/0.008
KYN/TRP35.6 ± 11.333.9 ± 11.646.5 ± 12.536.1 ± 11.90.627/0.00539.7 ± 10.748.5 ± 13.741.4 ± 11.047.6 ± 11.4<0.001/0.002
US-CRP, mg/l1.5 ± 2.23.3 ± 4.40.9 ± 1.22.8 ± 2.80.018/0.0171.7 ± 1.86.0 ± 7.11.7 ± 3.52.9 ± 2.8<0.001/0.033
Interleukin 6, pg/ml2.2 ± 1.03.9 ± 3.03.5 ± 3.53.7 ± 3.00.002/0.8821.9 ± 1.03.3 ± 2.32.5 ± 1.82.8 ± 2.5<0.001/0.396
Neopterin, nmol/l4.5 ± 2.64.6 ± 1.16.5 ± 2.05.0 ± 1.70.984/0.0305.8 ± 2.16.1 ± 1.76.8 ± 3.56.9 ± 5.90.345/0.943

Determination of metabolic syndrome

For youth, MetS was determined according to the updated criteria of Alberti et al. [22] and for adults according to Zimmet et al. [23]; i.e. waist circumference ≥90th percentile (for youth); male ≥94 cm, female ≥80 cm (for adults); and two of the four criteria fulfilled: fasting glucose ≥100 mg/dl (5.6 mmol/l); triglycerides ≥150 mg/dl (1.7 mmol/l); high density lipoprotein (HDL)-cholesterol <40 mg/dl (1.03 mmol/l), males; <50 mg/dl (1.29 mmol/l), females; blood pressure ≥130 systolic or ≥85 mmHg diastolic.

Laboratory analysis

Fasting blood samples were collected from 08:00 to 10:30 am. IL-6 was analyzed by an electrochemiluminescence immunoassay (Roche Diagnostics, Germany). US-CRP was analyzed with a Tina-quant® C-reactive protein latex ultrasensitive assay (Roche Diagnostics, Germany). Cholesterol, HDL-cholesterol, and triglycerides were measured by enzymatic photometric methods (Roche Diagnostics, Germany), LDL cholesterol calculated by the Friedewald formula. Oxidized low dense lipoprotein (oxLDL) was measured by ELISA (Mercodia oxidized LDL Competitive ELISA, SE-754 50, Uppsala, Sweden). Intra- and inter-assay variation coefficients for all ELISAs in our study were below 10 percent. Plasma insulin was measured by ELISA (Mercodia, Uppsala, Sweden), plasma glucose by the glucose hexokinase method. Homeostatic model assessment-insulin resistance (HOMA-IR) was calculated using the formula by Mathews et al. (1985) [24]. A cut off of greater than 2.5 was used for insulin resistance identification. Alkaline phosphatase (AP), aspartate transaminase (AST/GOT), alanine transaminase (ALT/GPT), gamma-GT (gGT), cholinesterase (CHE), creatinine, and uric acid (UA) were analyzed on a Cobas 8000. Neopterin concentrations were measured by a competitive enzyme-linked immunosorbent assay according to the manufacturer's instructions (BRAHMS Diagnostics, Berlin, Germany). Free TRP and KYN serum concentrations, as well as concentrations of phenylalanine (PHE) and tyrosine (TYR), were determined by high-performance liquid chromatography, as described elsewhere [25, 26]. The ratios of KYN/TRP and PHE/TYR were calculated as indexes of IDO and PHE activities, respectively.

Statistics

All statistical analyses were carried out using PASW Statistics 20.0 for Windows. Normal distribution of all clinical and biochemical measures was controlled by the Kolmogorov–Smirnov test. In cases of skewed distribution of variables logarithmic transformation was applied for significance calculation. Differences between groups were analyzed by an unpaired t-test. When more than two groups were compared, an analysis of variance (ANOVA) with a three-stage factor [normal weighted controls, ow/ob, gender] was performed. Differences were calculated by Bonferroni post hoc test.

Ethics

STYJOBS/EDECTA was approved by the ethical committee of the Medical University of Graz, and conducted in compliance of human studies with the Helsinki Declaration of 1975, as revised in 1996.

Results

Ow/ob adults (age >18 years and <65 years) had significantly increased KYN serum levels and a significantly increased KYN/TRP ratio in comparison to controls (Table 1, Figure 1). In sharp contrast, ow/ob juvenile males (age ≤ 18 years) showed decreased KYN and KYN/TRP values in comparison to their control counterparts (Table 1, Figure 1). In addition, juveniles fulfilling the criteria of the MetS had unaltered KYN and KYN/TRP, whereas adults with MetS had markedly increased KYN and KYN/TRP with the highest level of significance (P = 0.003, Table 2).

Figure 1.

Gender-dependent KYN/TRP ratio in overweight/obese probands compared to normal weight controls.

Table 2. Characteristics of overweight, obese study participants (metabolic syndrome)
 ≤18 years>18 years
 Overweight/obesePOverweight/ObeseP
  1. KYN/TRP = Kynurenine (µmol/L)/Tryptophan (mmol/L) ratio.

Metabolic syndromeNoYes NoYes 
Numbers13151 10671 
Age, y12.3 ± 3.013.0 ± 3.0 38.2 ± 12.639.4 ± 9.4 
BMI, kg/m²28.0 ± 4.833.8 ± 8.3<0.00130.6 ± 5.135.3 ± 7.0<0.001
Tryptophan, µmol/l63.7 ± 11.866.3 ± 9.20.16757.7 ± 9.859.7 ± 8.30.154
Kynurenine, µmol/l2.2 ± 0.72.2 ± 0.60.9062.6 ± 0.83.0 ± 0.70.003
KYN/TRP35.5 ± 12.233.8 ± 11.50.4246.4 ± 13.150.6 ± 11.40.03
US-CRP, mg/l2.7 ± 3.04.2 ± 5.30.0203.7 ± 4.85.8 ± 6.80.02
Interleukin 6, pg/ml3.4 ± 2.25.1 ± 4.70.0082. 8 ± 2.53.5 ± 2.20.09
Neopterin, nmol/l4.8 ± 1.74.7 ± 1.00.8716.5 ± 5.36.4 ± 1.90.933

With the exception of a decrease in adult ow/ob females, TRP serum levels were not significantly different from normal weighted controls in the ow/ob groups or between ow/ob subjects with or without MetS (Tables 1 and 2).

Levels of US-CRP were significantly increased in ow/ob juveniles and adults, but IL-6 levels were only increased in ow/ob juvenile and adult females. Subjects with MetS had increased US-CRP levels, in juveniles and in adults (Table 2). Neopterin was significantly decreased in ow/ob juvenile males (Table 1).

The scatter plot in Figure 2 shows that waist circumference, a surrogate measure for abdominal fat content, normalized by age was significantly, positively correlated with KYN/TRP in adult normal weighted controls. Compared to ow/ob subjects, the correlation plot between KYN/TRP and normalized waist circumference showed a markedly stronger positive slope with increased age and abdominal fat content in normal weighted subjects.

Figure 2.

Age > 18 years: controls versus overweight/obese. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3 shows that adult ow/ob subjects who fulfilled the criteria for MetS had, over the age of 18 years, a stronger correlated and larger increase of KYN/TRP as compared to ow/ob subjects without MetS.

Figure 3.

Age 0-80 years: overweight/obese, MetS no versus MetS yes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Discussion

Overweight and obesity are associated with a systemic cLGI [2, 10]. Besides dyslipidemia and insulin resistance, cLGI is centrally involved in obesity-associated cardiovascular disease (CVD), which leads to myocardial infarction (MI) and/or stroke—the most common causes of death in the western world [10, 27]. CVD is usually caused by atherosclerosis (AS), a focal inflammatory process of the vascular wall around lipid deposits called AS-plaque [27-29]. Besides monocytes and macrophages, activated T-cells of the Th-1 subtype are involved as important effectors in both cLGI and in AS-plaques [30].

AS-plaques may remain stable for many years or develop to unstable, “vulnerable” lesions. These lesions potentially cause fatal clinical end points like MI or stroke, which is seen more frequently in subjects with overweight/obesity usually suffering from the MetS [31]. The Th-1/Th-2 cell balance of the immune response is considered to play an important role in the process of “vulnerabilization” of AS-plaques [32, 33]. Increased neopterin levels in patients with AS and CVD highlights the relevance of the monocyte and T-cell interplay in the pathogenesis of coronary artery disease [34]. In a former study, we analyzed serum neopterin in obese juveniles with pre-atherosclerotic symptoms to investigate the immunological component of chronic inflammation. The pre-atherosclerotic symptoms were identified by an increased intima media thickness (IMT) of the common carotid arteries (CCA). Interestingly, we found that neopterin levels were not increased, as expected, but were decreased in the obese juveniles with increased IMT [19].

In accordance with our former observations, in this study neopterin was found decreased in males aged ≤ 18 years. Further, the KYN to TRP ratio (KYN/TRP) was also significantly decreased in ow/ob juvenile males. In sharp contrast to this, the KYN/TRP ratio was greatly increased in ow/ob adults of both sexes.

The increased KYN/TRP indicates an increase in IDO activity, which is usually stimulated by IFN-γ from Th-1 cell–monocyte interactions. Since the enzyme TDO physiologically catabolizing TRP in the liver is only active when TRP concentrations exceed basal requirements, circulating plasma TRP concentrations are extremely stable [35]. Therefore, the increased KYN/TRP must be caused by upregulation of IDO activity as the solely available natural and rate limiting TRP catabolizing enzyme and is a possible indirect indicator of immune-mediated inflammation, which also plays an important role in atherosclerotic vascular lesions of ow/ob adults [5].

In previous studies we could show that cLGI and pre-atherosclerotic vascular abnormalities were present in ow/ob subjects as early as in childhood [2, 10]. In the current study that encompasses a markedly higher number of investigated subjects that span a large age range, US-CRP was increased in both ow/ob juveniles and ow/ob adults. This underlines the presence of cLGI in early and advanced phases of obesity. As dysfunctions of TRP metabolism, including increased KYN/TRP, were already reported in obese adults in association with cLGI [36, 37], it was surprising that a significantly increased KYN/TRP ratio could only be observed in ow/ob adults but not in ow/ob juveniles.

Low or even significantly decreased neopterin and KYN/TRP concentrations coincide with low or subnormal activity of Th1-type immunity which may be due to a Th-2-type prevalence. Thus, the cross-talk between the different immune compartments may suppress cytokine production by Th-1 cells in ow/ob juveniles. Consequently, our results suggest an age related switch toward more a Th-1-weighted characteristic of obesity which is associated with cLGI that can start at the early onset of adulthood. Possibly, the Th-1 skewing of cLGI sets the course toward clinical endpoints like MI and stroke, which are prevalent in elder subjects.

Importantly, we observed that the degree of abdominal obesity correlates better with the KYN/TRP ratio than the classification of overweight/obesity from the BMI calculation. From this, the more abdominal obesity means a greater emphasis on Th-1 inflammation. This is especially relevant for “normal weight controls” with BMI <25 kg/m2 but a large waist circumference, thus indicating an elevated intra-abdominal fat accumulation and Th-1 inflammation identified by increased KYN levels. Further, in the ow/ob group, adult subjects with fulfilled criteria of the MetS showed a significantly stronger increase in serum KYN levels and KYN/TRP ratio compared to ow/ob without MetS. Notably, this was not seen in ow/ob juveniles with MetS.

Limitations of our data are given by the fact that we did not perform a direct analysis of Th1/Th2 activity (e.g., Luminex determination of cytokine profiles) in our study. However, KYN/TRP provides a reliable indirect evidence of increased IDO activity due to IFN-γ production of Th1 cells. Further, the process of growing may influence TRP metabolism in juveniles. Indeed, KYN levels were found higher in juvenile males of this study which had a stronger growing identified by a significantly higher AP compared to their juvenile female counterparts (not shown). However, despite this fact, no increased TRYP breakdown was seen in overweight/obese juveniles. Quite in contrary, KYN/TRP was even decreased in overweight/obese juvenile males (Table 1). This underlines the presence of a potent compensatory mechanism especially active in overweight/obese juvenile males which prevents a TRP breakdown. Later, in adulthood, the opposite will become true. It is still unclear whether such a mechanism relates to immune mediated inflammation and/or to differences in TRP breakdown in hepatic cells. Thus, it will be interesting to investigate this effect more in depth in a larger collective of overweight/obese juveniles compared to adults in future studies.

Taken together, our results clearly support indirect evidence coming from the TRP metabolism that the obesity-related immune mediated inflammation differs markedly between juveniles and adults. In more early phases of life, a more Th2-driven activation may predominate, later on, a Th-1 weighted inflammation with accelerated production of Th1-type cytokines like IFN-γ may pave the way for dangerous atherosclerotic endpoints.

Now these facts have potential clinical relevance. First, a conservative management of obesity by a consequent lifestyle change with sustained physical activity in childhood and/or adolescence might prevent a critical switch to a more aggressive “quality” of immune mediated inflammation well before irreversible clinical harm occurs. This might hold true especially in cases of “metabolically unhealthy” juvenile obese subjects with fulfilled criteria of MetS because they show a yet undisturbed TRP metabolism.

Second, the analytically simple determination of neopterin and KYN/TRP concentrations may provide diagnostic evidence for the presence of a Th-1 driven, more “aggressive inflammation” irrespective of age—especially in obese patients who fulfill the criteria for MetS. A targeted, anti-inflammatory regimen may be necessary in these patients.

Such therapeutic approaches have been recently suggested such as: [1] The inhibition of IDO activity with minocycline [7] and 1-methyl-dl-TRP [7], or [2] the administration of methoxyindoles (melatonin, N-acetylserotonin) which modulate the KYN-TRP pathway due to their inhibitory effect on production of cortisol [7, 38]. Further, methoxyindoles (melatonin, in particular) might attenuate excitatory, glutamate-mediated responses triggered by KYN pathway metabolites [7], and melatonin agonists (e.g., Ramelteon), age-associated hypertension and body weight gain [39].

Of course, all these pharmacologic therapies remain to be evaluated for potential damaging side effects. However, if they withstand these critical examinations, they may be challenging tools for a preclinical management of obesity-related life threatening conditions. In fact they address aspects of the MetS, including critical immune-mediated inflammation and uncontrolled body weight gain caused by a less control of hedonic inputs as well.

Ancillary