Katrien H.J. Gaens

Advanced glycation endproducts and its receptor for advanced glycation endproducts in obesity.

Purpose of review To highlight the potential importance of advanced glycation endproducts (AGEs) and advanced-lipoxidation endproducts (ALEs) in obesity and obesity-related complications, and the contribution of the receptor for advanced glycation endproducts (RAGE) and the glyoxylase defense system therein. Recent findings Formation of AGEs/ALEs and its precursors, including methylglyoxal (MGO), are increased in conditions characterized by hyperglycemia, hyperlipidemia and enhanced oxidative stress. This metabolic profile is generally considered typical for obesity. Increased plasma and/or tissue levels of MGO and of specific AGEs/ALEs, such as N&egr;-(carboxymethyl)lysine (CML), in obesity have recently been described. In addition to increased formation, the suppressed defense system in obesity against AGEs/ALEs formation, that is, the glyoxylase system, will further contribute to AGEs/ALEs formation in obesity. AGEs/ALEs are not inert. In-vitro studies showed that AGEs induced the production of inflammatory mediators in adipocytes and macrophages via RAGE activation, which may subsequently contribute to the development of obesity-related complications. Summary The recognition of an enhanced AGEs/ALEs formation in adipose tissue and the biological consequences thereof may lead to a further understanding of underlying mechanisms in dysregulated production of adipokines in obesity.

Association of Polymorphism in the Receptor for Advanced Glycation End Products (RAGE) Gene with Circulating RAGE Levels

OBJECTIVE The receptor for advanced glycation end products (RAGE)-ligand interaction has been linked to vascular complications. The family of soluble forms of RAGE (sRAGE) consists of splice variants and proteolytically cleaved and shed forms of RAGE. sRAGE may be a reflection of cell-bound RAGE. Because genetic variation in the RAGE gene may be associated with individual differences in sRAGE concentration and outcome, we investigated whether RAGE single-nucleotide polymorphisms (SNPs) were associated with circulating levels of sRAGE. METHODS Nine SNPs, covering the common RAGE gene variation, were genotyped in a Dutch cohort of subjects with normal glucose metabolism (n = 301), impaired glucose metabolism (n = 127), and type 2 diabetes mellitus (n = 146). We used linear regression analyses adjusted for age, sex, and glucose metabolism status to compare sRAGE levels across genotypes. RESULTS SNP rs2060700 (Gly82Ser) showed an association with sRAGE levels. Specifically, after adjustments for age, sex, and glucose metabolism, subjects with CT genotype had -527 pg/ml (95% confidence interval -724 to -330, P < 0.001) lower sRAGE levels compared with the CC genotype (age, sex, and glucose metabolism adjusted mean +/- SE values of 836 +/- 99 and 1369 +/- 26 pg/ml, respectively, P < 0.001). These results were confirmed in a subsample of a second cohort study of subjects with CT (n = 37) and CC genotype (n = 37). Immunoblotting using antibodies against amino acids 39-55 and 100-116 of RAGE also showed a similar decrease of sRAGE levels in the CT genotypes. No other SNPs showed an association with sRAGE levels. In addition, no associations between SNPs and the advanced glycation end products N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine were found. CONCLUSION The CC genotype of SNP rs2070600 (Gly82Ser) was strongly associated with higher sRAGE levels in a Dutch population. The mechanism by which Gly82Ser polymorphism alters the sRAGE levels remains to be elucidated.

Nε-(Carboxymethyl)lysine-Receptor for Advanced Glycation End Product Axis Is a Key Modulator of Obesity-Induced Dysregulation of Adipokine Expression and Insulin Resistance

Objective—Dysregulation of inflammatory adipokines by the adipose tissue plays an important role in obesity-associated insulin resistance. Pathways leading to this dysregulation remain largely unknown. We hypothesized that the receptor for advanced glycation end products (RAGE) and the ligand N&egr;-(carboxymethyl)lysine (CML) are increased in adipose tissue and, moreover, that activation of the CML–RAGE axis plays an important role in obesity-associated inflammation and insulin resistance. Approach and Results—In this study, we observed a strong CML accumulation and increased expression of RAGE in adipose tissue in obesity. We confirmed in cultured human preadipocytes that adipogenesis is associated with increased levels of CML and RAGE. Moreover, CML induced a dysregulation of inflammatory adipokines in adipocytes via a RAGE-dependent pathway. To test the role of RAGE in obesity-associated inflammation further, we constructed an obese mouse model that is deficient for RAGE (ie, RAGE–/–/LeptrDb–/– mice). RAGE–/–/LeptrDb–/– mice displayed an improved inflammatory profile and glucose homeostasis when compared with RAGE+/+/LeptrDb–/– mice. In addition, CML was trapped in adipose tissue in RAGE+/+/LeptrDb–/– mice but not in RAGE–/–/LeptrDb–/–. RAGE-mediated trapping in adipose tissue provides a mechanism underlying CML accumulation in adipose tissue and explaining decreased CML plasma levels in obese subjects. Decreased CML plasma levels in obese individuals were strongly associated with insulin resistance. Conclusions—RAGE-mediated CML accumulation in adipose tissue and the activation of the CML–RAGE axis are important mechanisms involved in the dysregulation of adipokines in obesity, thereby contributing to the development of obesity-associated insulin resistance.

Endogenous formation of Nε-(carboxymethyl)lysine is increased in fatty livers and induces inflammatory markers in an in vitro model of hepatic steatosis

BACKGROUND & AIMS Increased lipid peroxidation and inflammation are major factors in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A lipoxidation product that could play a role in the induction of hepatic inflammation is N(ε)-(carboxymethyl)lysine (CML). The aim of the present study was to investigate the relationship between steatosis and CML and to study the role of CML in hepatic inflammation. METHODS We included 74 obese individuals, which were categorized into 3 groups according to the grade of hepatic steatosis. CML accumulation in liver biopsies was assessed by immunohistochemistry and plasma CML levels were measured by mass spectrometry. Plasma CML levels were also determined in the hepatic artery, portal, and hepatic vein of 22 individuals, and CML fluxes across the liver were calculated. Hepatocyte cell lines were used to study CML formation during intracellular lipid accumulation and the effect of CML on pro-inflammatory cytokine expression. Gene expression levels of the inflammatory markers were determined in liver biopsies of the obese individuals. RESULTS CML accumulation was significantly associated with the grade of hepatic steatosis, the grade of hepatic inflammation, and gene expression levels of inflammatory markers PAI-1, IL-8, and CRP. Analysis of CML fluxes showed no release/uptake of CML by the liver. Lipid accumulation in hepatocytes, induced by incubation with fatty acids, was associated with increased CML formation and expression of the receptor for advanced glycation endproducts (RAGE), PAI-1, IL-8, IL-6, and CRP. Pyridoxamine and aminoguanidine inhibited the endogenous CML formation and the increased RAGE, PAI-1, IL-8, IL-6, and CRP expression. Incubation of hepatocytes with CML-albumin increased the expression of RAGE, PAI-1, and IL-6, which was inhibited by an antibody against RAGE. CONCLUSIONS Accumulation of CML and a CML-upregulated RAGE-dependent inflammatory response in steatotic livers may play an important role in hepatic steatosis and in the pathogenesis of NAFLD.

The association between the-374T/A polymorphism of the receptor for advanced glycation endproducts gene and blood pressure and arterial stiffness is modified by glucose metabolism status: the Hoorn and CoDAM studies

Objectives Receptor for advanced glycation endproducts (RAGE)–ligand interaction may lead to vascular complications. Genetic variation in RAGE has been shown to alter expression, activity of RAGE or both. We, therefore, investigated whether RAGE single-nucleotide polymorphisms (SNPs) and haplotypes were associated with vascular disease. Methods Nine tag SNPs that cover the common RAGE gene variation were genotyped in 1291 individuals from two Dutch population-based cohort studies, aged 64.5 ± 8.6 years, with normal glucose metabolism (44%), impaired glucose metabolism (23%) or type 2 diabetes mellitus (33%). We used multiple regression analyses to compare prevalent cardiovascular disease and markers of atherosclerosis, blood pressure and arterial stiffness across genotypes, and examine effect modification by glucose metabolism status. Results In unstratified analyses, no consistent associations between RAGE SNPs and prevalent cardiovascular disease and markers of atherosclerosis were found. However, the AA genotype of SNP rs1800624 (−374T/A) was consistently associated with lower SBP [−5.0 mmHg (95% confidence interval −10.4 to 0.3)] and DBP [−4.2 (−7.2 to −1.3)], pulse pressure [−0.8 (−5.0 to 3.4)] as well as with less arterial stiffness [−0.56 SD (−1.04 to −0.09)] in individuals with normal glucose metabolism, but with higher SBP [6.2 (0.9–11.5)], DBP [2.1 (−0.7 to 5.0)] and pulse pressure [4.1 (−0.2 to 8.4)] in individuals with impaired glucose metabolism or type 2 diabetes mellitus (P for interaction ≤0.05 in all analyses). Similar results were found for a haplotype that includes the −374A allele. Conclusion In individuals with normal glucose metabolism, the −374A allele of the RAGE gene is protectively associated with blood pressure and arterial stiffness, whereas in individuals with impaired glucose metabolism or type 2 diabetes mellitus, it is adversely associated with these variables.

Receptor for Advanced Glycation End Product Polymorphisms and Type 2 Diabetes : The CODAM Study

Genetic variation in the receptor for advanced glycation end products (RAGE) gene may alter the expression and function of RAGE and affect disease development and outcome. We investigated whether single nucleotide polymorphisms (SNPs) in RAGE were associated with diabetes and parameters of glucose homeostasis. In total, nine SNPs of RAGE were analyzed in individuals with and without type 2 diabetes in CODAM: a cohort study of diabetes and atherosclerosis, Maastricht. A significant difference in genotype frequency of SNP rs3134945 was observed between the nondiabetic control subjects, subjects with impaired glucose metabolism, and diabetic patients. The C allele of this polymorphism was significantly associated with higher fasting glucose concentrations, 2‐h postload glucose concentrations, insulin levels, and homeostasis model assessment of insulin resistance. These results indicate that SNP rs3134945 or a locus in linkage disequilibrium with this polymorphism may be involved in the development of insulin resistance and diabetes. Because the functionality of this polymorphism is not known, the mechanism whereby this polymorphism contributes to the development of insulin resistance and diabetes has to be further elucidated.

Circulating classical monocytes are associated with CD11c+ macrophages in human visceral adipose tissue

Immune cell accumulation in adipose tissue (AT) is associated with the development of AT inflammation, resulting in metabolic dysfunction. Circulating immune cell patterns may reflect immune cell accumulation in expanding AT. However, data linking human leukocytes in blood and AT is lacking. We investigated whether blood immune cell populations are associated with their counterparts in subcutaneous (scAT) or visceral AT (vAT). Flow cytometry was performed on blood, scAT and vAT from 16 lean and 29 obese men. Circulating natural killer (NK)-cells, classical monocytes and nonclassical monocytes were higher in obese individuals. vAT, but not scAT, of obese individuals contained more inflammatory CD11c+ “M1” macrophages and NK cells compared to lean individuals. Blood classical monocytes were associated with CD11c+ macrophages in vAT but not scAT. This association was unrelated to expression of the adhesion molecules CD11b and CD11c or of the chemokine receptor CX3CR1 on these monocytes. Other AT immune cells were not associated with their respective counterparts in blood. Finally, CD11c+ macrophages and CD4+ T-cells in vAT were associated with their counterparts in scAT. In conclusion, blood classical monocytes reflect CD11c+ macrophages in vAT.

Protein-Bound Plasma Nε-(Carboxymethyl)lysine Is Inversely Associated With Central Obesity and Inflammation and Significantly Explain a Part of the Central Obesity–Related Increase in Inflammation The Hoorn and CODAM Studies

Objective—Adipose tissue inflammation contributes to the development of complications, such as insulin resistance and type 2 diabetes mellitus. We previously reported that plasma levels of N&egr;-(carboxymethyl)lysine (CML) were decreased in obese subjects resulting from CML accumulation in adipose tissue and that this CML accumulation plays an important role in adipose tissue inflammation. The objective of this study is to investigate associations between obesity (body mass index, waist circumference, and trunk fat mass), plasma CML (as an inversely correlated marker of CML accumulation in adipose tissue), and low-grade inflammation (LGI) in a large sample of individuals whose weight status ranged from normal to morbid obesity. Approach and Results—We studied 1270 individuals of the Cohort on Diabetes and Atherosclerosis Maastricht Study and Hoorn Study, in whom protein-bound CML levels were measured by UPLC-Tandem MS (ultra performance liquid chromatography-tandem mass spectrometry), and 6 inflammatory markers were measured with multiarrays. These inflammatory markers were compiled into an LGI score. Multiple linear regression, adjusted for covariates, showed that (1) waist circumference was inversely associated with protein-bound CML plasma levels (standardized regression coefficient [&bgr;]=−0.357 [95% confidence interval: −0.414; −0.301]); (2) protein-bound CML was inversely associated with LGI score (&bgr;=−0.073 [−0.130;-0.015]); and (3) the association between waist circumference and LGI (&bgr;=0.262 [0.203;0.321]) was attenuated after adjustment for protein-bound CML plasma levels and other potential mediators (to &bgr;=0.202 [0.138;0.266]), with CML explaining the greatest portion of the attenuation (≈12%). Further analysis with dual-energy X-ray absorptiometry measures of body composition confirmed a strong inverse association of fat mass preferentially accumulated in the trunk with protein-bound CML plasma levels, significantly explaining ≈21% of the trunk fat–LGI association. Conclusions—Obesity, in particular central obesity, is characterized by greater levels of LGI but by lower levels of circulating CML; the latter significantly explaining a portion of the positive association between central obesity and inflammation.

Delayed Intervention With Pyridoxamine Improves Metabolic Function and Prevents Adipose Tissue Inflammation and Insulin Resistance in High-Fat Diet-Induced Obese Mice

Obesity is associated with an increased risk for the development of type 2 diabetes and vascular complications. Advanced glycation end products are increased in adipose tissue and have been associated with insulin resistance, vascular dysfunction, and inflammation of adipose tissue. Here, we report that delayed intervention with pyridoxamine (PM), a vitamin B6 analog that has been identified as an antiglycating agent, protected against high-fat diet (HFD)–induced body weight gain, hyperglycemia, and hypercholesterolemia, compared with mice that were not treated. In both HFD-induced and db/db obese mice, impaired glucose metabolism and insulin resistance were prevented by PM supplementation. PM inhibited the expansion of adipose tissue and adipocyte hypertrophy in mice. In addition, adipogenesis of murine 3T3-L1 and human Simpson-Golabi-Behmel Syndrome preadipocytes was dose- and time-dependently reduced by PM, as demonstrated by Oil Red O staining and reduced expression of adipogenic differentiation genes. No ectopic fat deposition was found in the liver of HFD mice. The high expression of proinflammatory genes in visceral adipose tissue of the HFD group was significantly attenuated by PM. Treatment with PM partially prevented HFD-induced mild vascular dysfunction. Altogether, these findings highlight the potential of PM to serve as an intervention strategy in obesity.

Higher Plasma Concentrations of the Methylglyoxal Metabolite D-lactate are Independently Associated with Insulin Resistance: The CODAM Study

Objective: Methylglyoxal (MGO), a highly reactive dicarbonyl compound generated by the spontaneous degradation of glycolytic intermediates, is a major precursor for advanced glycation endproducts and can potentially disrupt cellular functions. MGO can be detoxified by the glyoxalase system into D-lactate. Although experimental studies have shown that increased levels of MGO are associated with insulin resistance, epidemiological evidence of such an association in human studies is lacking. The aim of this study was to investigate the association between plasma D-lactate, as a reflection of plasma MGO concentrations, and insulin resistance. Methods: Cross-sectional, complete case analyses were performed in the Cohort study on Diabetes and Atherosclerosis Maastricht (CODAM). 513 participants were included in the study population: 59.4 ± 6.9 years of age, 63% men, and, by design, 23% impaired glucose metabolism (IGM) and 23% type 2 diabetes mellitus (T2DM). Plasma D-lactate was measured by UPLC-MS/MS. The main outcome measure was HOMA2-IR, as a measure of insulin resistance. The association between plasma D-lactate and HOMA2-IR was studied by multiple linear regression analysis. Results: The prevalence of T2DM increased significantly over the tertiles of plasma D-lactate concentrations. Moreover, plasma D-lactate was positively associated with HOMA2-IR, when adjusted for age and sex (β=0.429; 95% CI: 0.350-0.507; p<0.001), as well as in the fully adjusted model, additionally adjusted for glucose metabolism status, smoking status, prior cardiovascular disease, use of medication (glucose-, lipid-, and blood pressure lowering), estimated glomerular filtration rate, waist circumference, glycated hemoglobin (HbA1C) and plasma L-lactate levels (β=0.145; 95% CI: 0.051-0.239; p=0.003). Additional data indicate that other sources of D-lactate can be excluded. Conclusion: We found a positive association between plasma D-lactate and HOMA2-IR, independently of putative confounders. These results suggest that MGO plays a role in insulin resistance, although direct measurement of MGO is necessary to confirm this.