ESTADO DEL ARTE INTERNACIONAL

Acute phase reactants; interleukin-6 (IL-6) and C-reactive protein (CRP) are increased during acute and chronic inflammation and are the most studied and widely used markers of inflammation.

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2.3.1 C-reactive protein (CRP)

C-reactive protein (CRP) can be measured in blood and has been used as an indicator of acute inflammation for decades. Macrophages and adipose cells secrete factors (interleukin-6) which cause synthesis of CRP in the liver. In physiological states, CRP binds to phosphocholine expressed on the surface of dead or dying cells and bacteria in order to activate the complement system. This facilitates phagocytosis by macrophages and ultimately clears the body of necrotic and apoptotic cells (812).

More recently, inflammatory markers have received attention for their ability to predict CVD risk (287). Among these, CRP is one of the more powerful with a recent meta-analysis showing that for every 1-standard deviation (SD) increase in CRP, vascular risk (adjusted for age and sex) increases by more than 60% (813). CRP is stable in plasma or whole blood at 4 and 21 degrees C for at least three days and for several years at -80C. Moreover, it is stable after five freeze- thaw cycles and is therefore a stable marker of inflammation (814).

A review of the literature indicates that CRP levels in blood are influenced by a number of environmental and lifestyle factors including age, gender, cholesterol level, body mass index, blood pressure, insulin resistance, smoking and sleep deprivation(815). There is also genetic variation in CRP levels (816). Several single-nucleotide polymorphisms (SNPs) in the CRP gene have been shown to directly influence steady state CRP levels in blood and are inherited independent of the above risk factors (816;817) with effects across the lifespan. Earlier studies showed conflicting result with regard to the relationship between serum CRP and SNPs with cardiovascular risk (816;817) but more recent larger studies have been consistent in showing no association between serum CRP as

determined by Mendelian randomization and CVD (especially hypertension) (818- 820), casting some doubt on the causality of the association.

In animal studies it has been reported that chronic elevation of CRP is associated with a greater risk of hypertension. Vongpatanasin et al reported that Ang II leads to exaggerated blood pressure elevation in CRP transgenic mice, and this response was reversed by a nitric oxide (NO) donor, indicating a role for NO deficiency in the process (821). Schwartz et al also showed in mice that CRP

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downregulates endothelial NO synthase (eNOS) and attenuates re

endothelialization (822). Other in vitro and animal studies also show inhibition of (eNOS) and impaired endothelial vasoreactivity following CRP administration (823). In rats, delivery of adeno- associated virus overexpression of CRP (AAV- hCRP) increased BP and impaired vasoreactivity (824;825), as well as increasing oxidative stress, expression of angiotensin 1 receptors and endothelin-1. The same genetic manipulation also decreased expression of eNOS and impaired endothelium dependent vaso-relaxation (824;825). These experimental observations suggest that CRP may have physiologically-relevant biological activity at least in animals.

However, there are many discrepancies between human and animal studies in relation to CRP. For example, statins have been shown to decrease BP without lowering CRP in mice (825). However in humans, statins lower CRP (826) but do not affect BP (827). Nevertheless, some epidemiological studies support a relationship between high levels of CRP and hypertension (827).

So even if animal studies show a causal association between CRP and the

development of HTN (824;825), the evidence of a causal association in humans is not strong (818-820;828). It remains uncertain whether CRP might increase BP directly or via some other mechanism (e.g. obesity, insulin resistance) or whether both are affected by some other feature of the metabolic syndrome (829). CRP is unlikely to be on the causal pathway in relation to development of hypertension, but rather a risk marker for chronic low grade inflammation.

2.3.2 Interleukin-6 (IL-6)

IL-6 is a cytokine released from a number of cells ranging from adipocytes, skeletal muscle cells, monocytes, lymphocytes etc. IL-6 acts both as a pro- inflammatory and anti-inflammatory cytokine. The main anti-inflammatory effects are the release of IL-1ra, IL-10 and sTNF-R (830). The pro-inflammatory effects of IL-6 are through expansion and activation of T cells, differentiation of B cells, and the induction of acute-phase reactants by hepatocytes (831). Serum samples of IL-6 can be stored at -20oC for several years and are not significantly altered by repeated freeze-thaw cycles (832); it is therefore a stable marker of inflammation.

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In disease states, IL-6 is released in response to tumour necrosis alpha (TNF-α) and has pro-inflammatory actions. It is raised to chronically high levels which have pro-inflammatory and damaging effects (833). However, it can also be released physiologically during exercise (in a TNF-- independent manner ) which can have protective and anti-inflammatory effects (830). Skeletal muscle- derived IL-6 is beneficial in modulating glucose and fatty acid metabolism during exercise. It stimulates adipose tissue lipolysis, fat utilization and has positive effects on pancreatic β-cells function, contributing to improved glycaemia following exercise (833;834).

Healthy adipose tissue is populated with 5–10% macrophages but this macrophage infiltration increases up to 60% in obesity (507). Activated

macrophages release TNF-α and IL-6 resulting in insulin resistance (835). Obesity leads to activation of inflammatory pathways in all insulin target tissues,

including fat, liver and muscle, signifying a role for inflammation in driving the pathogenesis of systemic insulin resistance (831). Proposed mechanisms leading to inflammation in obesity include oxidative stress, lipotoxicity, glucotoxicity, endoplasmic reticulum stress, hypoxia, amyloid and lipid deposition (831).

There is therefore considerable evidence that IL6 secretion promotes insulin resistance and that its concentration is elevated in obesity and type 2 diabetes mellitus (836). Moreover, in a study in a non-diabetic Caucasian population, Succurro et al found that increased IL6 levels were related to an increased risk of developing insulin resistance (837). However, there are also studies which question this finding. When muscle cells are treated with IL-6 in vitro there was increased glucose uptake and translocation of glucose transporter GLUT4 - an insulin-sensitising action (830). Carey et al also found that IL-6 was not elevated in lean subjects with insulin resistance and suggested that fat mass was the proximal cause for raised IL6 in T2DM (836). In keeping with this suggestion, an epidemiological study suggested that IL-6 lost its association with insulin

resistance after adjustment for BMI and waist-to-hip ratio; however, the majority of participants were men so the results cannot be generalized (838). Contradicting these results, Andreozzi et al found a negative correlation between IL-6 and clamp-derived insulin stimulated glucose disposal (M). The correlation remained significant even after adjustment for age, sex, BMI and

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free fatty acids (839). In another study, IL-6 also correlated negatively with the Insulin sensitivity index (840). Given these uncertainties, further investigation is required in well–characterised human cohorts.

2.4 Measurement of diabetes