Erwin Schleicher

Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging.

N(epsilon)-(Carboxymethyl)lysine (CML), a major product of oxidative modification of glycated proteins, has been suggested to represent a general marker of oxidative stress and long-term damage to proteins in aging, atherosclerosis, and diabetes. To investigate the occurrence and distribution of CML in humans an antiserum specifically recognizing protein-bound CML was generated. The oxidative formation of CML from glycated proteins was reduced by lipoic acid, aminoguanidine, superoxide dismutase, catalase, and particularly vitamin E and desferrioxamine. Immunolocalization of CML in skin, lung, heart, kidney, intestine, intervertebral discs, and particularly in arteries provided evidence for an age-dependent increase in CML accumulation in distinct locations, and acceleration of this process in diabetes. Intense staining of the arterial wall and particularly the elastic membrane was found. High levels of CML modification were observed within atherosclerotic plaques and in foam cells. The preferential location of CML immunoreactivity in lesions may indicate the contribution of glycoxidation to the processes occurring in diabetes and aging. Additionally, we found increased CML content in serum proteins in diabetic patients. The strong dependence of CML formation on oxidative conditions together with the increased occurrence of CML in diabetic serum and tissue proteins suggest a role for CML as endogenous biomarker for oxidative damage.

Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response

While the initiation of the adaptive and innate immune response is well understood, less is known about cellular mechanisms propagating inflammation. The receptor for advanced glycation end products (RAGE), a transmembrane receptor of the immunoglobulin superfamily, leads to perpetuated cell activation. Using novel animal models with defective or tissue-specific RAGE expression, we show that in these animal models RAGE does not play a role in the adaptive immune response. However, deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. Such protection is reversed by reconstitution of RAGE in endothelial and hematopoietic cells. These results indicate that the innate immune response is controlled by pattern-recognition receptors not only at the initiating steps but also at the phase of perpetuation.

High glucose-induced transforming growth factor beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells.

Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.

1997 volvo award winner in basic science studies: Immunohistologic markers for age-related changes of human lumbar intervertebral discs

Study Design. The authors performed a correlative macroscopic, histologic, and immunohistochemical investigation on human lumbar intervertebral discs using complete motion segment slices, including all age groups and stages of degeneration. Objectives. To identify markers for age‐related changes of human lumbar intervertebral discs. In particular, to investigate changes in the distribution pattern of collagen Types I, II, III, IV, V, VI, IX, and X. In addition, to study posttranslational protein modification by the immunolocalization of N‐(carboxylmethyl)lysine (CML), which is regarded as a biomarker for oxidative stress. Summary of Background Data. Data on a correlation of age‐related changes in disc morphology and disc matrix composition is sparse. So far, no comprehensive analysis considered a correlation of macroscopic, histologic, and biochemical age‐related alterations using complete sections of intervertebral discs (i.e., including nucleus pulposus, anulus fibrosus, endplates, and vertebral bodies). In addition, there is need for specific markers for these disc changes to allow for a better correlation with disc function. Methods. After photodocumentation of the macroscopic appearance, 229 sagittal lumbar motion segments obtained from 47 individuals (fetal to 86 years) during routine autopsy were processed for histologic and immunohistochemical analysis. All slices were investigated for histologic alterations of disc degeneration. A randomly selected subset of these specimens (n = 45) was used for a correlative analysis of interstitial collagens and molecular modifications of matrix proteins. Results. The presence of CML‐modification of extracellular matrix proteins, mainly collagen, was observed first in the nucleus pulposus of a 13‐year‐old individual and increased significantly with age. In elderly people, both the nucleus pulposus and the anulus fibrosus showed extensive CML deposition. This CML deposition was accentuated in areas of macroscopic and histologic disc degeneration. After the occurrence of CML in the nucleus pulposus, we found a change in the collagen type pattern. An initial increase in nuclear collagen Types II, III, and VI staining was followed by a loss of collagen Type II, the occurrence of collagen Type I, and the persistence of high collagen Type III and VI levels, which were finally decreased again. The nuclear chondrocytes revealed significant changes in their immediate pericellular matrix, indicating phenotypic changes. Thus, exclusively in the nucleus pulposus of adolescents and young adults a significant proportion of cells positively stained for the basement membrane collagen Type IV. Collagen Type X was expressed by nuclear chondrocytes at a higher age and was associated with advanced degenerative disc alterations. Conclusions. The authors present the first study in which age‐related changes are correlated on a macroscopic, histologic, and molecular level using complete sections of lumbar motion segments. They reconfirm the notion that disc degeneration starts as early as in the second decade of life. Therefore, only early prevention of disc damage may inhibit disc degeneration and its sequelae. Phenotypic alterations of nuclear chondrocytes as monitored by collagen Type IV in young adults with minor lesions and collagen Type X in advanced lesions indicate distinct cellular reactions, possibly as a reaction to enhanced oxidative stress. The degree of this oxidative stress is reflected by the CML‐staining pattern which, in turn, indicates that the disc undergoes an accumulative stress, possibly leading to altered properties of the collagen fibrils and, thereby, tissue destruction. The deposition of CML proved to be the best marker for ongoing age‐related changes in the intervertebral disc.

Loss of pain perception in diabetes is dependent on a receptor of the immunoglobulin superfamily

Molecular events that result in loss of pain perception are poorly understood in diabetic neuropathy. Our results show that the receptor for advanced glycation end products (RAGE), a receptor associated with sustained NF-kappaB activation in the diabetic microenvironment, has a central role in sensory neuronal dysfunction. In sural nerve biopsies, ligands of RAGE, the receptor itself, activated NF-kappaBp65, and IL-6 colocalized in the microvasculature of patients with diabetic neuropathy. Activation of NF-kappaB and NF-kappaB-dependent gene expression was upregulated in peripheral nerves of diabetic mice, induced by advanced glycation end products, and prevented by RAGE blockade. NF-kappaB activation was blunted in RAGE-null (RAGE(-/-)) mice compared with robust enhancement in strain-matched controls, even 6 months after diabetes induction. Loss of pain perception, indicative of long-standing diabetic neuropathy, was reversed in WT mice treated with soluble RAGE. Most importantly, loss of pain perception was largely prevented in RAGE(-/-) mice, although they were not protected from diabetes-induced loss of PGP9.5-positive plantar nerve fibers. These data demonstrate, for the first time to our knowledge, that the RAGE-NF-kappaB axis operates in diabetic neuropathy, by mediating functional sensory deficits, and that its inhibition may provide new therapeutic approaches.

Stimulation of Suicidal Erythrocyte Death by Methylglyoxal

Diabetes increases the percentage of circulating erythrocytes exposing phosphatidylserine (PS) at the cell surface. PS-exposing erythrocytes are recognized, bound, engulfed and degraded by macrophages. Thus, PS exposure, a feature of suicidal erythrocyte death or eryptosis, accelerates clearance of affected erythrocytes from circulating blood. Moreover, PS-exposing erythrocytes bind to the vascular wall thus interfering with microcirculation. The present study explored mechanisms involved in the triggering of PS exposure by methylgloxal, an extra- and intracellular metabolite which is enhanced in diabetes. PS exposure, cell size and cytosolic Ca2+-activity after methylglyoxal treatment were measured by FACS analysis of annexin V binding, forward scatter and Fluo-3-fluorescence, respectively, and it was shown that the treatment significantly enhanced the percentage of PS-exposing erythrocytes at concentrations (0.3 µM) encountered in diabetic patients. Surprisingly, methylglyoxal did not significantly increase cytosolic Ca2+ concentration, and at concentrations up to 3 µM, did not decrease the forward scatter. Instead, exposure to methylglyoxal inhibited glycolysis thus decreasing ATP and GSH concentrations. In conclusion, methylglyoxal impairs energy production and anti-oxidative defense, effects contributing to the enhanced PS exposure of circulating erythrocytes and eventually resulting in anemia and deranged microcirculation.

Differential accumulation of advanced glycation end products in the course of diabetic retinopathy

Aims/hypothesis. Glycated proteins, formed by reaction of glucose and protein, react further yielding numerous, mostly undefined advanced glycation end products (AGE). The recently characterized imidazolone-type AGE (AG-1) is non-oxidatively formed involving 3-deoxyglucosone whereas some AGEs, particularly Nɛ-(carboxymethyl)lysine (CML), are formed only in the presence of oxygen. Methods. To study the possible contribution of oxidative and non-oxidative AGE formation in the development of diabetic retinopathy antibodies directed against CML-type and imidazolone-type AGEs were characterized by dot blot analysis and used to localize these well-characterized epitops in the retinas from diabetic rats (early course) and from human Type I (insulin-dependent) diabetes mellitus with laser-treated proliferative diabetic retinopathy (late course). Results. In non-diabetic rats CML was moderately positive in neuroglial and vascular structures of non-diabetic rat retinas and increased strongly in diabetic retinas. Anti-imidiazolone antibody staining was strongly positive only in diabetic capillaries. Advanced human diabetic retinopathy showed strong CML-immunolabelling of the entire retina whereas control samples showed moderate staining of neuroglial structures only with the polyclonal CML-antibody. Anti-imidiazolone antibody staining was faint in the inner retina of control sections but were strong throughout the entire diabetic retina. Immunolabelling for the AGE-receptor was congruent with a marker of Müller cells. Conclusion/interpretation. Our data indicate that the oxidatively formed CML is present in non-diabetic retinas as a regular constituent but increases in diabetes both in neuroglial and vascular components. Imidazolone-type AGE are restricted to microvessels and spread during later stages over the entire retina, co-localizing with the expression of AGE-receptor. [Diabetologia (1999) 42: 728–736]

Regulation of erythrocyte survival by AMP-activated protein kinase

AMP‐activated protein kinase (AMPK), an energy‐sensing enzyme, counteracts energy depletion by stimulation of energy production and limitation of energy utilization. On energy depletion, erythrocytes undergo suicidal death or eryptosis, triggered by an increase in cytosolic Ca2+ activity ([Ca2+]i) and characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. The present study explored whether AMPK participates in the regulation of eryptosis. Western blotting and confocal microscopy disclosed AMPK expression in erythrocytes. [Ca2+]i (Fluo3 fluorescence), cell volume (forward scatter), and PS exposure (annexin V binding) were determined by fluorescence‐activated cell sorting (FACS) analysis. Glucose removal increased [Ca2+]i, decreased cell volume, and increased PS exposure. The AMPK‐inhibitor compound C (20 µM) did not significantly modify eryptosis under glucose‐replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca2+]i by Ca2+ ionophore ionomycin triggered eryptosis, an effect blunted by the AMPK activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR;1 mM). As compared with erythrocytes from wild‐type littermates (ampk+/+), erythrocytes from AMPKctl‐deficient mice (ampk−/−) were significantly more susceptible to the eryptotic effect of energy depletion. The ampk−/− mice were anemic despite excessive reticulocytosis, and they suffered from severe splenomegaly, again pointing to enhanced erythrocyte turnover. The observations disclose a critical role of AMPK in the survival of circulating erythrocytes.—Föller, M., Sopjani, M., Koka, S., Gu, S., Mahmud, H., Wang, K., Floride, E., Schleicher, E., Schulz, E., Münzel, T., Lang, F. Regulation of erythrocyte survival by AMP‐activated protein kinase. FASEB J. 23, 1072–1080 (2009)

The chemopreventive polyphenol Curcumin prevents hematogenous breast cancer metastases in immunodeficient mice.

Dissemination of metastatic cells probably occurs long before diagnosis of the primary tumor. Metastasis during early phases of carcinogenesis in high risk patients is therefore a potential prevention target. The plant polyphenol Curcumin has been proposed for dietary prevention of cancer. We therefore examined its effects on the human breast cancer cell line MDA-MB-231 in vitroand in a mouse metastasis model. Curcumin strongly induces apoptosis in MDA-MB-231 cells in correlation with reduced activation of the survival pathway NFĸB, as a consequence of diminished ΙĸB and p65 phosphorylation. Curcumin also reduces the expression of major matrix metalloproteinases (MMPs) due to reduced NFĸ B activity and transcriptional downregulation of AP-1. NFĸ B/p65 silencing is sufficient to downregulate c-jun and MMP expression. Reduced NFĸ B/AP-1 activity and MMP expression lead to diminished invasion through a reconstituted basement membrane and to a significantly lower number of lung metastases in immunodeficient mice after intercardiac injection of 231 cells (p=0.0035). 68% of Curcumin treated but only 17% of untreated animals showed no or very few lung metastases, most likely as a consequence of down-regulation of NFĸ B/AP-1 dependent MMP expression and direct apoptotic effects on circulating tumor cells but not on established metastases. Dietary chemoprevention of metastases appears therefore feasible.

Practical approach for the identification and isomer elucidation of biomarkers detected in a metabonomic study for the discovery of individuals at risk for diabetes by integrating the chromatographic and mass spectrometric information

Sensitive and high-resolution chromatographic-driven metabonomomics studies experienced major growth with the aid of new analytical technologies and bioinformatics software packages. Hence, data collections by LC-MS and data analyses by multivariate statistical methods are by far the most straightforward steps, and the detection of biomarker candidates can easily be achieved. However, the unequivocal identification of the detected metabolite candidates, including isomer elucidation, is still a crux of current metabonomics studies. Here we present a comprehensive analytical strategy for the elucidation of the molecular structure of metabolite biomarkers detected in a metabonomics study, exemplified analyzing spot urine of a cohort of healthy, insulin sensitive subjects and clinically well characterized prediabetic, insulin resistant individuals. An integrated approach of LC-MS fingerprinting, multivariate statistic analysis, LC-MSn experiments, micro preparation, FTICR-MS, GC retention index, database search, and generation of an isotope labeled standard was applied. Overall, we could demonstrate the efficiency of our analytical approach by the unambiguous elucidation of the molecular structure of an isomeric biomarker candidate detected in a complex human biofluid. The proposed strategy is a powerful new analytical tool, which will allow the definite identification of physiologically important molecules in metabonomics studies from basic biochemistry to clinical biomarker discovery.