Thomas Gawlowski

Effects of n–3 fatty acids on macro- and microvascular function in subjects with type 2 diabetes mellitus

BACKGROUND Recent evidence supports the protective effects of n-3 (omega-3) fatty acids (n-3 FAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on vascular function. OBJECTIVE We investigated the effects of EPA and DHA on postprandial vascular function in subjects with type 2 diabetes mellitus. DESIGN In a double-blind, placebo-controlled, randomized, crossover manner, 34 subjects with type 2 diabetes mellitus received daily either 2 g purified EPA/DHA (termed n-3 FAs) or olive oil (placebo) for 6 wk. At the end of this period, we measured macrovascular (brachial ultrasound of flow-mediated dilatation; FMD) and microvascular [laser-Doppler measurements of reactive hyperemia (RH) of the hand] function at fasting and 2, 4, and 6 h after a high-fat meal (600 kcal, 21 g protein, 41 g carbohydrates, 40 g fat). RESULTS Fasting vascular function remained unchanged after n-3 FAs and placebo. Postprandial FMD decreased from fasting after placebo, with a maximum decrease (38%) at 4 h-an effect that was significantly reduced (P = 0.03 for time x treatment interaction) by n-3 FA supplementation (maximum decrease in FMD was at 4 h: 13%). RH remained unchanged after placebo, whereas it improved significantly (P = 0.04 for time x treatment interaction) after n-3 FA supplementation (maximum increase was at 2 h: 27%). CONCLUSIONS In subjects with type 2 diabetes mellitus, 6 wk of supplementation with n-3 FAs reduced the postprandial decrease in macrovascular function relative to placebo. Moreover, n-3 FA supplementation improved postprandial microvascular function. These observations suggest a protective vascular effect of n-3 FAs.

Dietary Advanced Glycation Endproducts and Oxidative Stress

Advanced glycation endproducts (AGEs) and oxidative stress (OS) contribute to the development and progression of diabetic complications. We have reported that dietary AGEs and OS induce acute endothelial dysfunction in vivo, but little is known about their effects on adipokines. Twenty inpatients with type 2 diabetes mellitus (mean age: 55.9; range: 32–71 years), received a standard diabetes diet for 6 days. On days 4 and 6, the acute effects of a high‐AGE (HAGE) or a low‐AGE (LAGE) meal (15.100 vs. 2.750 kU AGE) were studied in a randomized, cross‐over, investigator‐blinded design. Measurements were performed after an overnight fast, at baseline (B) and at 2, 4, and 6 h after the HAGE or LAGE meals. Both meals had the same ingredients and differed only by the cooking method. Two h following HAGE, a significant decrease from baseline occurred in adiponectin (−10%*‡ vs. +0%) and leptin (−22%*‡ vs. −13%*), and a significant increase occurred in vascular cell adhesion molecule 1 (+19%*‡ vs. −5%) and thiobarbituric acid reactive substances (+23%*‡ vs. +6%). These changes did not occur, or occurred to a lesser extent, following LAGE. At 4 h following HAGE, an increase in methylglyoxal (+20%‡ vs. −5%) and E‐selectin (+54%*‡ vs. −3%) occurred. Urinary AGEs increased only after HAGE (+51%*‡ vs. −2%; values presented as HAGE vs. LAGE; *P < 0.05 vs. baseline, ‡P < 0.05 vs. LAGE). The postprandial excursions in glucose, insulin, and triglycerides were similar between both meals. A meal rich in AGEs induces acute endothelial and adipocyte dysfunction. These effects were prevented by changing the cooking method.

Advanced glycation end products strongly activate platelets.

BackgroundDiabetes mellitus is characterized by hyperglycemia that plays an important role in the pathogenesis of diabetic complications including cardiovascular diseases. Moreover, hyperglycemia induces increased generation of advanced glycation end products (AGEs). The activation of platelets is associated with the development of cardiovascular diseases.Aim of the studyThe question whether AGEs acutely induce platelet activation as a response to exogenous stimulus is addressed.Materials and methodsThe effect of AGEs derived from food and human serum being purified by lysozyme affinity chromatography was examined by incubating in vitro freshly isolated blood platelets from fasted subjects at various concentrations and different time points. Platelet activation, determined as expression of surface markers CD62 and CD63, and the presence of the receptor for AGEs (RAGE) in platelet membranes was measured by flow cytometric analysis using specific antibodies.ResultsIncubation with food-derived as well as serum-derived AGEs stimulated significantly the expression of CD62 up to 7.1-fold and CD63 up to 2.2-fold at the platelet surface membrane as a function of concentration and time. Incubation with thrombin or AGEs significantly increased RAGE expression twofold at the platelet surface membrane.ConclusionsThe increase in surface activation marker and RAGE expression in platelets, resulting from concentrations of AGEs that occur in vivo after a meal or a drink as a source of exogenous AGEs, points to signaling mechanisms for food AGEs that could favor the precipitation of acute postprandial ischemic events.

Heat shock protein 27 modification is increased in the human diabetic failing heart.

Chronic conditions like diabetes mellitus (DM) leading to altered metabolism might cause cardiac dysfunction. Hyperglycemia plays an important role in the pathogenesis of diabetic complications including accumulation of methylglyoxal (MG), a highly reactive alpha-dicarbonyl metabolite of glucose degradation pathways and increased generation of advanced glycation endproducts (AGEs). The aim of this investigation was to study the extent of the MG-modification argpyrimidine in human diabetic heart and in rat cardiomyoblasts grown under hyperglycemic conditions. Left ventricular myocardial samples from explanted hearts of patients with cardiomyopathy with (n=8) or without DM (n=8) as well as nonfailing donor organs (n=6), and rat cardiac myoblasts H9c2 treated with glucose were screened for the MG-modification argpyrimidine. The small heat shock protein 27 (Hsp27) revealed to be the major argpyrimidine containing protein in cardiac tissue. Additionally, the modification of arginine leading to argpyrimidine and the phosphorylation of Hsp27 are increased in the myocardium of patients with DM. In H9c2 cells hyperglycemia leads to a decrease of the Hsp27-expression and an increase in argpyrimidine content and phosphorylation of Hsp27, which was accompanied by the induction of oxidative stress and apoptosis. This study shows an association between diabetes and increased argpyrimidine-modification of myocardial Hsp27, a protein which is involved in apoptosis, oxidative stress, and cytoskeleton stabilization.

Leptin decreases postprandially in people with type 2 diabetes, an effect reduced by the cooking method.

Leptin modulates satiety and increases in obesity and type 2 diabetes mellitus in parallel with leptin resistance. Postprandial leptin regulation has been previously postulated to depend on meal composition, but data are controversial. The hypothesis of our study was that in people with type 2 diabetes mellitus, a postprandial leptin regulation exists that can be regulated not only by meal composition but also by the cooking method. In 20 inpatients with type 2 diabetes (mean age: 55.9 years), the acute effects of 2 meals, a high-heat-processed meal HHPM or a low-heat-processed meal LHPM, on leptin levels were studied on 2 different days in a randomized, crossover design. Both test meals had similar ingredients and differed only in the cooking method used. Parameters were measured after an overnight fast and at 2, 4, and 6 h postprandially. The HHPM induced a marked decrease in leptin levels, from 8 717+/-2 079 pg/ml at baseline to 6 788+/-1 598 pg/ml at 2 h postprandially (-1 929 pg/ml, -22%*), an effect significantly reduced by the LHPM, where values were 8 563+/-1 900 pg/ml at baseline and 7 425+/-1 591 pg/ml at 2 h postprandially (-1 138 pg/ml, -13%* (double dagger)) (*p<0.05 vs. baseline, (double dagger)p<0.05 vs. HHPM). Parameters of oxidative stress and blood AGEs increased only following the HHPM, while postprandial glucose, triglycerides, and insulin excursions were similar between meals. Postprandial leptin decreases following a HHPM meal in people with T2DM, an effect reduced by the cooking method.

Diabetic Cardiomyopathy – to Take a Long Story Serious

Being independent of coronary artery disease and hypertension, diabetic cardiomyopathy is a distinct primary disease process, which precedes the development of congestive heart failure. Epidemiologic as well as clinical studies confirmed the close link between diabetes mellitus and heart failure. Altered cardiac structure and function are common diagnoses in patients with type 2 diabetes mellitus. Hyperglycemia leading to the formation of advanced glycation end products and hyperlipidemia resulting in lipotoxicity are of structural and functional impact on cardiac muscle and cardiomyocytes. New and more sensitive methods of diagnosis identify early diastolic dysfunction as a precursor of the development of congestive heart failure. This review focuses on the mechanistic approach to understand the molecular basis of diabetic cardiomyopathy in patients with type 2 diabetes mellitus.ZusammenfassungAls von koronarer Herzkrankheit und Bluthochdruck unabhängige Krankheitsentität wird die diabetische Kardiomyopathie als Vorstufe der terminalen Herzinsuffizienz verstanden. Sowohl epidemiologische als auch klinische Studien bestätigen den engen Zusammenhang zwischen Diabetes mellitus und terminaler Herzinsuffizienz. Veränderte kardiale Struktur und Funktion sind häufige Diagnosen bei Patienten mit Diabetes mellitus. Hyperglykämie, die mit der Bildung von „advanced glycation end products“ einhergeht, und die in Lipotoxizität mündende Hyperlipidämie beeinflussen Funktion und Struktur des Herzmuskels und der Kardiomyozyten. Die frühe diastolische Dysfunktion wird durch neue und sensitivere Diagnosemethoden als Vorläufer der terminalen Herzinsuffizienz identifiziert. Dieser Übersichtsartikel stellt die wesentlichen Gesichtspunkte der molekularen Zusammenhänge, die zur Entstehung einer diabetischen Kardiomyopathie bei Patienten mit Diabetes mellitus beitragen, zusammen.

Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates.

Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.

Impact of GLO1 Knock Down on GLUT4 Trafficking and Glucose Uptake in L6 Myoblasts

Methylglyoxal (MG), a highly reactive α-dicarbonyl metabolite of glucose degradation pathways, protein and fatty acid metabolism, plays an important role in the pathogenesis of diabetic complications. Hyperglycemia triggers enhanced production of MG and increased generation of advanced glycation endproducts (AGEs). In non-enzymatic reactions, MG reacts with arginine residues of proteins to form the AGEs argpyrimidine and hydroimidazolone. Glyoxalase 1 (GLO1), in combination with glyoxalase 2 and the co-factor glutathione constitute the glyoxalase system, which is responsible for the detoxification of MG. A GLO1 specific knock down results in accumulation of MG in targeted cells. The aim of this study was to investigate the effect of intracellularly accumulated MG on insulin signaling and on the translocation of the glucose transporter 4 (GLUT4). Therefore, L6 cells stably expressing a myc-tagged GLUT4 were examined. For the intracellular accumulation of MG, GLO1, the first enzyme of the glyoxalase pathway, was down regulated by siRNA knock down and cells were cultivated under hyperglycemic conditions (25 mM glucose) for 48 h. Here we show that GLO1 knock down augmented GLUT4 level on the cell surface of L6 myoblasts at least in part through reduction of GLUT4 internalization, resulting in increased glucose uptake. However, intracellular accumulation of MG had no effect on GLUT4 concentration or modification. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. Tiron, which is also a well-known antioxidant, had no impact on MG-induced GLUT4 translocation.

Methylglyoxal Impairs GLUT4 Trafficking and Leads to Increased Glucose Uptake in L6 Myoblasts

Methylglyoxal (MG) is a highly reactive dicarbonyl compound derived mainly from glucose degradation pathways, but also from protein and fatty acid metabolism. MG modifies structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic complications. Hyperglycemia-associated accumulation of MG might be associated with generation of oxidative stress and subsequently insulin resistance. Therefore, the effects of MG on insulin signaling and on translocation of glucose transporter 4 (GLUT4) were investigated in the rat skeletal muscle cell line L6-GLUT4myc stably expressing myc-tagged GLUT4. Twenty four-hour MG treatment resulted in elevated GLUT4 presentation on the surface of L6 myoblasts and in an increased uptake of glucose even without insulin stimulation. Exogenously added MG neither effected IRS-1 expression nor IRS-1 phosphorylation. A decreased expression of Akt1 but not Akt2 and concomitantly increased apoptosis were detected following MG treatment. To exclude that oxidative stress caused by MG treatment leads to increased GLUT4 translocation, effects of pretreatment with 2 antioxidants were investigated. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. In contrast, tiron, a well-known antioxidant that does not exert MG-scavenger function, had no impact on MG-induced GLUT4 translocation supporting the hypothesis of a direct effect of MG on GLUT4 trafficking. In conclusion, prolonged treatment with MG augments GLUT4 level on the surface of L6 myoblasts, at least in part through a higher translocation of GLUT4 from the intracellular compartment as well as a reduction of GLUT4 internalization, resulting in increased glucose uptake.