Pyridoxamine reduces methylglyoxal and markers of glycation and endothelial dysfunction, but does not improve insulin sensitivity or vascular function in abdominally obese individuals: A randomized double-blind placebo-controlled trial.

AIM: To investigate the effects of pyridoxamine (PM), a B6 vitamer and dicarbonyl scavenger, on glycation and a large panel of metabolic and vascular measurements in a randomized double-blind placebo-controlled trial in abdominally obese individuals. MATERIALS AND METHODS: Individuals (54% female; mean age 50 years; mean body mass index 32 kg/m2 ) were randomized to an 8-week intervention with either placebo (n = 36), 25 mg PM (n = 36) or 200 mg PM (n = 36). We assessed insulin sensitivity, ß-cell function, insulin-mediated microvascular recruitment, skin microvascular function, flow-mediated dilation, and plasma inflammation and endothelial function markers. PM metabolites, dicarbonyls and advanced glycation endproducts (AGEs) were measured using ultra-performance liquid chromatography tandem mass spectrometry. Treatment effects were evaluated by one-way ANCOVA. RESULTS: In the high PM dose group, we found a reduction of plasma methylglyoxal (MGO) and protein-bound Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), as compared to placebo. We found a reduction of the endothelial dysfunction marker soluble vascular cell adhesion molecule-1 (sVCAM-1) in the low and high PM dose group and of soluble intercellular adhesion molecule-1 (sICAM-1) in the high PM dose, as compared to placebo. We found no treatment effects on insulin sensitivity, vascular function or other functional outcome measurements. CONCLUSIONS: This study shows that PM is metabolically active and reduces MGO, AGEs, sVCAM-1 and sICAM-1, but does not affect insulin sensitivity and vascular function in abdominally obese individuals. The reduction in adhesion markers is promising because these are important in the pathogenesis of endothelial damage and atherosclerosis.

A 4-Week Diet Low or High in Advanced Glycation Endproducts Has Limited Impact on Gut Microbial Composition in Abdominally Obese Individuals: The deAGEing Trial.

Dietary advanced glycation endproducts (AGEs), abundantly present in Westernized diets, are linked to negative health outcomes, but their impact on the gut microbiota has not yet been well investigated in humans. We investigated the effects of a 4-week isocaloric and macronutrient-matched diet low or high in AGEs on the gut microbial composition of 70 abdominally obese individuals in a double-blind parallel-design randomized controlled trial (NCT03866343). Additionally, we investigated the cross-sectional associations between the habitual intake of dietary dicarbonyls, reactive precursors to AGEs, and the gut microbial composition, as assessed by 16S rRNA amplicon-based sequencing. Despite a marked percentage difference in AGE intake, we observed no differences in microbial richness and the general community structure. Only the Anaerostipes spp. had a relative abundance >0.5% and showed differential abundance (0.5 versus 1.11%; p = 0.028, after low- or high-AGE diet, respectively). While the habitual intake of dicarbonyls was not associated with microbial richness or a general community structure, the intake of 3-deoxyglucosone was especially associated with an abundance of several genera. Thus, a 4-week diet low or high in AGEs has a limited impact on the gut microbial composition of abdominally obese humans, paralleling its previously observed limited biological consequences. The effects of dietary dicarbonyls on the gut microbiota composition deserve further investigation.

A 4-week high-AGE diet does not impair glucose metabolism and vascular function in obese individuals.

BACKGROUNDAccumulation of advanced glycation endproducts (AGEs) may contribute to the pathophysiology of type 2 diabetes and its vascular complications. AGEs are widely present in food, but whether restricting AGE intake improves risk factors for type 2 diabetes and vascular dysfunction is controversial.METHODSAbdominally obese but otherwise healthy individuals were randomly assigned to a specifically designed 4-week diet low or high in AGEs in a double-blind, parallel design. Insulin sensitivity, secretion, and clearance were assessed by a combined hyperinsulinemic-euglycemic and hyperglycemic clamp. Micro- and macrovascular function, inflammation, and lipid profiles were assessed by state-of-the-art in vivo measurements and biomarkers. Specific urinary and plasma AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were assessed by mass spectrometry.RESULTSIn 73 individuals (22 males, mean ± SD age and BMI 52 ± 14 years, 30.6 ± 4.0 kg/m2), intake of CML, CEL, and MG-H1 differed 2.7-, 5.3-, and 3.7-fold between the low- and high-AGE diets, leading to corresponding changes of these AGEs in urine and plasma. Despite this, there was no difference in insulin sensitivity, secretion, or clearance; micro- and macrovascular function; overall inflammation; or lipid profile between the low and high dietary AGE groups (for all treatment effects, P > 0.05).CONCLUSIONThis comprehensive RCT demonstrates very limited biological consequences of a 4-week diet low or high in AGEs in abdominally obese individuals.TRIAL REGISTRATIONClinicaltrials.gov, NCT03866343; trialregister.nl, NTR7594.FUNDINGDiabetesfonds and ZonMw.

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(ε)-(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(-/-)/Leptr(Db-/-) mice). RAGE(-/-)/Leptr(Db-/-) mice displayed an improved inflammatory profile and glucose homeostasis when compared with RAGE(+/+)/Leptr(Db-/-) mice. In addition, CML was trapped in adipose tissue in RAGE(+/+)/Leptr(Db-/-) mice but not in RAGE(-/-)/Leptr(Db-/-). 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.

Glyoxalase-1 overexpression reduces endothelial dysfunction and attenuates early renal impairment in a rat model of diabetes.

AIMS/HYPOTHESIS: In diabetes, advanced glycation end-products (AGEs) and the AGE precursor methylglyoxal (MGO) are associated with endothelial dysfunction and the development of microvascular complications. In this study we used a rat model of diabetes, in which rats transgenically overexpressed the MGO-detoxifying enzyme glyoxalase-I (GLO-I), to determine the impact of intracellular glycation on vascular function and the development of early renal changes in diabetes. METHODS: Wild-type and Glo1-overexpressing rats were rendered diabetic for a period of 24 weeks by intravenous injection of streptozotocin. Mesenteric arteries were isolated to study ex vivo vascular reactivity with a wire myograph and kidneys were processed for histological examination. Glycation was determined by mass spectrometry and immunohistochemistry. Markers for inflammation, endothelium dysfunction and renal dysfunction were measured with ELISA-based techniques. RESULTS: Diabetes-induced formation of AGEs in mesenteric arteries and endothelial dysfunction were reduced by Glo1 overexpression. Despite the absence of advanced nephrotic lesions, early markers of renal dysfunction (i.e. increased glomerular volume, decreased podocyte number and diabetes-induced elevation of urinary markers albumin, osteopontin, kidney-inflammation-molecule-1 and nephrin) were attenuated by Glo1 overexpression. In line with this, downregulation of Glo1 in cultured endothelial cells resulted in increased expression of inflammation and endothelium dysfunction markers. In fully differentiated cultured podocytes incubation with MGO resulted in apoptosis. CONCLUSIONS/INTERPRETATION: This study shows that effective regulation of the GLO-I enzyme is important in the prevention of vascular intracellular glycation, endothelial dysfunction and early renal impairment in experimental diabetes. Modulating the GLO-I pathway therefore may provide a novel approach to prevent vascular complications in diabetes.

Mild oxidative damage in the diabetic rat heart is attenuated by glyoxalase-1 overexpression.

Diabetes significantly increases the risk of heart failure. The increase in advanced glycation endproducts (AGEs) and oxidative stress have been associated with diabetic cardiomyopathy. We recently demonstrated that there is a direct link between AGEs and oxidative stress. Therefore, the aim of the current study was to investigate if a reduction of AGEs by overexpression of the glycation precursor detoxifying enzyme glyoxalase-I (GLO-I) can prevent diabetes-induced oxidative damage, inflammation and fibrosis in the heart. Diabetes was induced in wild-type and GLO-I transgenic rats by streptozotocin. After 24-weeks of diabetes, cardiac function was monitored with ultrasound under isoflurane anesthesia. Blood was drawn and heart tissue was collected for further analysis. Analysis with UPLC-MSMS showed that the AGE Nε-(1-carboxymethyl)lysine and its precursor 3-deoxyglucosone were significantly elevated in the diabetic hearts. Markers of oxidative damage, inflammation, and fibrosis were mildly up-regulated in the heart of the diabetic rats and were attenuated by GLO-I overexpression. In this model of diabetes, these processes were not accompanied by significant changes in systolic heart function, i.e., stroke volume, fractional shortening and ejection fraction. This study shows that 24-weeks of diabetes in rats induce early signs of mild cardiac alterations as indicated by an increase of oxidative stress, inflammation and fibrosis which are mediated, at least partially, by glycation.

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.

Overexpression of glyoxalase-I reduces hyperglycemia-induced levels of advanced glycation end products and oxidative stress in diabetic rats.

The reactive advanced glycation end product (AGE) precursor methylglyoxal (MGO) and MGO-derived AGEs are associated with diabetic vascular complications and also with an increase in oxidative stress. Glyoxalase-I (GLO-I) transgenic rats were used to explore whether overexpression of this MGO detoxifying enzyme reduces levels of AGEs and oxidative stress in a rat model of diabetes. Rats were made diabetic with streptozotocin, and after 12 weeks, plasma and multiple tissues were isolated for analysis of AGEs, carbonyl stress, and oxidative stress. GLO-I activity was significantly elevated in multiple tissues of all transgenic rats compared with wild-type (WT) littermates. Streptozotocin treatment resulted in a 5-fold increase in blood glucose concentrations irrespective of GLO-I overexpression. Levels of MGO, glyoxal, 3-deoxyglucosone, AGEs, and oxidative stress markers nitrotyrosine, malondialdehyde, and F2-isoprostane were elevated in the diabetic WT rats. In diabetic GLO-I rats, glyoxal and MGO composite scores were significantly decreased by 81%, and plasma AGEs and oxidative stress markers scores were significantly decreased by ∼50%. Hyperglycemia induced a decrease in protein levels of the mitochondrial oxidative phosphorylation complex in the gastrocnemius muscle, which was accompanied by an increase in the lipid peroxidation product 4-hydroxy-2-nonenal, and this was counteracted by GLO-I overexpression. This study shows for the first time in an in vivo model of diabetes that GLO-I overexpression reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress. The reduction of oxidative stress by GLO-I overexpression directly demonstrates the link between glycation and oxidative stress.

Smoothelin-B deficiency results in reduced arterial contractility, hypertension, and cardiac hypertrophy in mice.

BACKGROUND: Smoothelins are actin-binding proteins that are abundantly expressed in healthy visceral (smoothelin-A) and vascular (smoothelin-B) smooth muscle. Their expression is strongly associated with the contractile phenotype of smooth muscle cells. Analysis of mice lacking both smoothelins (Smtn-A/B(-/-) mice) previously revealed a critical role for smoothelin-A in intestinal smooth muscle contraction. Here, we report on the generation and cardiovascular phenotype of mice lacking only smoothelin-B (Smtn-B(-/-)). METHODS AND RESULTS: Myograph studies revealed that the contractile capacity of the saphenous and femoral arteries was strongly reduced in Smtn-B(-/-) mice, regardless of the contractile agonist used to trigger contraction. Arteries from Smtn-A/B(-/-) compound mutant mice exhibited a similar contractile deficit. Smtn-B(-/-) arteries had a normal architecture and expressed normal levels of other smooth muscle cell-specific genes, including smooth muscle myosin heavy chain, alpha-smooth muscle actin, and smooth muscle-calponin. Decreased contractility of Smtn-B(-/-) arteries was paradoxically accompanied by increased mean arterial pressure (20 mm Hg) and concomitant cardiac hypertrophy despite normal parasympathetic and sympathetic tone in Smtn-B(-/-) mice. Magnetic resonance imaging experiments revealed that cardiac function was not changed, whereas distension of the proximal aorta during the cardiac cycle was increased in Smtn-B(-/-) mice. However, isobaric pulse wave velocity and pulse pressure measurements indicated normal aortic distensibility. CONCLUSIONS: Collectively, our results identify smoothelins as key determinants of arterial smooth muscle contractility and cardiovascular performance. Studies on mutations in the Smtn gene or alterations in smoothelin levels in connection to hypertension in humans are warranted.

Smoothelin in vascular smooth muscle cells.

Smoothelin-A and -B have only been found in fully differentiated contractile smooth muscle cells. They are increasingly used to monitor the smooth muscle cell differentiation process to a contractile or synthetic phenotype. Vascular-specific smoothelin-B is the first smooth muscle cell marker that disappears when vascular tissues are compromised, for example, in atherosclerosis or restenosis. Recently obtained data show that smoothelin deficiency results in a considerable loss of contractile potential and hence in impaired smooth muscle function and suggest that smoothelins are part of the contractile apparatus.

Contribution of serum response factor and myocardin to transcriptional regulation of smoothelins.

OBJECTIVE: Smoothelin-A and -B isoforms are highly restricted to contractile smooth muscle cells (SMCs). Serum response factor (SRF) and myocardin are essential for contractile SMC differentiation. We evaluated the contribution of SRF/myocardin to transcriptional regulation of smoothelins. METHODS: Rat vascular SMCs were transfected with smoothelin-A and smoothelin-B promoter reporter constructs and promoter activity was analyzed. The effects of mutations in the smoothelin-A promoter CArG-boxes and co-transfections with a myocardin expression plasmid were assessed. Electrophoretic mobility shift assays and chromatin immunoprecipitations were performed to investigate SRF-binding to the smoothelin-A CArG-boxes. RESULTS: Smoothelin promoter activity was detected in vascular SMCs. Comparative sequence analysis revealed two conserved CArG elements in the smoothelin-A promoter that bind SRF as shown by chromatin immunoprecipitation. The proximal CArG-near bound SRF stronger than CArG-far in gel shift assays. Mutagenesis studies also indicated that CArG-near is more important than CArG-far in regulating smoothelin-A promoter activity. Myocardin augmented smoothelin-A promoter activity 2.5-fold in a CArG-near-dependent manner. In contrast, myocardin had little effect on the smoothelin-B promoter. CONCLUSION: Smoothelin-A expression is controlled by an intragenic promoter whose activity is, in part, dependent on two CArG boxes that bind SRF. Our data show a role for SRF/myocardin in regulating smoothelin-A whereas the higher smoothelin-B expression appears to be SRF/myocardin-independent.