Glucotoxicity, Protein Glycation and Adipose Tissue Dysfunction

Introduction: Glycation is a non-enzymatic reaction between a sugar and a free-amino group contained in molecules such as proteins, amino acids, DNA, RNA, and lipids. In the initial phase, the carbonyl group of the reducing carbohydrate condenses with the free-amino group on the biomolecule to form a reversible glycosylamine, which is then converted to a more stable Amadori product. Once formed, these products can with time undergo to dehydration, cyclization, oxidation, and rearrangements forming a polymorphic group of compounds collectively referred as Advanced Glycation End-products (AGEs). The accumulation of AGEs in vivo is considered the most important link between diabetes and oxidative disease. The rate of glycation could account for the long-term diabetic complications. Nevertheless, the role of AGEs is yet to be determined and it is, still nowadays, an important and challenging field of study. Little is known about their presence and their consequences on adipose tissue; in particular, the role of AGEs on adipogenesis and fat cell functionality remains fully unexplored. Methods: In this project, 3T3-L1 cell line and stromal vascular fraction cells isolated from human adipose tissue were used. The purpose was to set up an in vitro model for investigating the effects of a chronic exposure to a glycation-inducing environment on the adipogenetic process. Morphological analyses, E.L.I.S.A. assay for AGEs, expression analysis of lipid and glucose metabolism genes, glucose uptake, and immunofluorescence for RAGE, GLP1r, GLUT4 and AGEs, were performed on mature adipocytes. Moreover, the gene expression profile obtained in vitro, was compared to that of subcutaneous and visceral adipose tissues obtained from 64 obese subjects with type 2 diabetes or with normal glucose tolerance underwent to bariatric surgery. In collaboration with the Mass Spectrometry Service (CNR – IENI Padova), the use of mass spectrometry to characterize post-translation modifications induced by glycation was evaluated. Results and conclusions: Following an exposition to a glycation-inducing environment, an increase of intracellular AGEs both by immunofluorescence and E.L.I.S.A. was observed in our in vitro model. Preliminary analysis on cell cultures suggested that a glycation-inducing environment importantly impacts on gene expression profile. In glycating conditions, an increased expression of Glut4 and, on the contrary, an insulin-induced glucose uptake reduction was observed. These results allow us to hypothesize an influence of AGEs on GLUT4 translocation to the plasma-membrane and/or on its functionality. Finally, although mass spectrometry represents a high potential technique was shown that it has several disadvantages and technical challenges. In particular, several post-translational modifications can affect simultaneously the same protein, thus the total mass increase will reflect the sum of all modifications. Furthermore, spectra analysis of a whole cellular or tissue lysates resulted excessively complex to underline small differences. Multiple steps in which proteins of a specific molecular weight range would be isolated, concentrated and separately assayed are therefore required in order to simplify the analysis by mass spectrometry.

Endothelial dysfunction and inflammation in diabetic nephropathy

In this thesis we investigated various mechanisms by which the immune system plays a role in the development of diabetic nephropathy. In chapter 2 we describe that APOC1-tg mice develop glomerulosclerosis at 15 months of age, with increased number of glomerular macrophages. Our results suggest that apoCI may exacerbate the development of DN by increasing the inflammatory response in activated glomerular macrophages. In chapter 3 we demonstrate that sFLT-1 significantly improves kidney function, resolves diabetes-related kidney damage, and reduces endothelial cell activation and inflammation, suggesting that sFLT-1 can reduce the severity of DN by reducing glomerular inflammation and supporting cellular repair mechanisms.In chapter 4 we show that reducing endoglin levels diminished VEGF-A-induced endothelial activation by increasing pAkt and reducing pATF-2. These data suggest that targeting endoglin may have therapeutic value in patients who are at risk for developing DN.In chapter 5 we demonstrate that renal complement activation is associated with more severe classes of DN, reduced kidney function, and the presence of histological lesions.In chapter 6 we describe that transfecting APOC1-tg mice with sFlt-1 does not accelerate development of glomerulosclerosis, but lowered the number of glomerular macrophages. These data suggest that sFLT-1 treatment has an anti-inflammatory effect.Dutch Kidney Foundation; Chipsoft; Stichting Preventie Diabetes; Department of pathology, LUMCLUMC / Geneeskunde Repositoriu

HIGH RESOLUTION MASS SPECTROMETRIC STRATEGIES IN DRUG DISCOVERY FOR THE INVESTIGATION OF COVALENT AND NON-COVALENT INTERACTIONS

High resolution mass spectrometric strategies in drug discovery for the investigation of covalent and non covalent interactions Dott. Danilo De Maddis PhD tutor: Prof. Giancarlo Aldini The research work here described was focused on the set-up and application of analytical methods for studying compounds effective as inhibitors of the advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs) as well as as antagonists of the receptors of AGEs (RAGE). AGEs and ALEs represent a quite complex and heterogeneous class of compounds that are formed by different mechanisms, by heterogeneous precursors and can be formed either exogenously or endogenously. AGEs represent a class of covalently modified proteins generated by oxidative and non-oxidative pathways, involving sugars or their degradation products. The term ALEs includes a variety of covalent adducts which are generated by the non-enzymatic reaction of reactive carbonyl species (RCS), produced by lipid peroxidation and lipid metabolism, with the nucleophilic residues of macromolecules, especially proteins. AGEs and ALEs share some common properties, for example, both consist of non-enzymatic, covalently modified proteins and oxidative stress is often (although not always) involved in the mechanism of their formation. Moreover some AGEs and ALEs have the same structure, since they arise from common precursors, as in the case of carboxymethyllysine (CML) which is generated by glyoxal, which in turn is formed by both lipid and sugar oxidative degradation pathways [1]. Besides being considered as reliable biomarkers of oxidative damage, as well as predictors and prognostic factors, more recently, AGEs and ALEs have also been recognized as important pathogenetic factors of some oxidative based diseases, as supported by the following facts: 1) a strict correlation between the amount of AGEs/ALEs in tissues and fluids and disease states has been found, in both animal and human subjects; 2) a substantial amount of literature is now available reporting the molecular and cellular pathogenic mechanisms for the AGEs/ALEs involvement in the onset and progression of different oxidative-based diseases including diabetes [2], chronic renal failure [3], cardiovascular diseases [4] and neurological disorders [5]. The AGEs/ALEs damaging effect is mediated by different mechanisms, including the dysfunction of the proteins undergoing the oxidative modification, protein polymerization, signal transduction, immunoresponse and RAGE activation. Some of the biological effects are due to the loss of function of the target proteins undergoing the covalent modification, such as in the case of extracellular matrix proteins that lose their elastic and mechanical functions when modified as AGEs/ALEs and in particular, when cross-links are involved [6]. Other examples of a direct damaging effect of AGEs/ALEs can be ascribed to the covalent modification of enzymes and receptors that lose their activity due to the covalent modification involving the catalytic or binding site, or following a conformational change of the protein structure. Moreover AGEs and ALEs can be immunogenic. Hence AGEs/ALEs are now considered as promising drug targets and a substantial effort is dedicated to delve the molecular strategies aimed at preventing, reducing or removing these protein oxidation products. The different molecular approaches thus far reported can be grouped by considering at which level of the damaging AGEs/ALEs cascade they are effective and in particular if they act by inhibiting the AGEs/ALEs formation, accelerating their catabolism or blocking their biological effects. The first level of action, the inhibition of AGEs/ALEs formation, also consists of different approaches, which target the different inducers (ROS, metal ions) and intermediate products (mainly reactive carbonyl species, RCS) involved in the AGEs/ALEs formation. Antioxidants, radical scavengers, metal-ion chelators and reactive carbonyl compounds quenchers (RCS sequestering agents) represent the most promising approaches so far reported for inhibiting AGEs/ALEs formation. In some cases, as found both for natural or synthetic compounds, the inhibition of AGEs/ALEs formation does not proceed through a single specific mechanism but implicates multiple mechanisms, involving at least two of the following ones: antioxidant, radical-scavenging, metal ion chelation and RCS trapping. The second level of intervention consists of accelerating the catabolism of already formed AGEs/ALEs and this can be achieved by potentiating the endogenous proteolytic system or by using xenobiotics able to catalytically degrade AGEs/ALEs. The third level of intervention, which is also the most innovative, consists of blocking the biological response of AGEs/ALEs by inhibiting the activation of the RAGE receptors through different classes of antagonists. It should be noted that such an approach permits the blocking of the damaging effect induced not only by endogenously formed AGEs/ALEs but also by exogenously derived AGEs/ALEs. In view of a drug discovery program aim to search bioactive compounds, the first step of the research program was to set up analytical methods based on high resolution MS strategies able to test the efficacy and selectivity of compounds effective as sequestering agents of RCS and also acting as RAGE antagonists. The methods so far reported in the literature to test RCS sequestering compounds consist to measure the consumption of the aldehydes when incubated with the test compounds by a direct spectrophotometric analysis and depending on the aldehyde, the derivatization reaction should also be considered. The main limitations of such approach is that it cannot be applied to mixtures or extracts, it cannot be applied to a high throughput screening and in several cases the experimental conditions such as the acid condition of the derivatization process can dissociate the adducts between the aldehydes and the sequestering agents. Therefore, based on the limits above reported, the initial aim of this research project was focused to set-up and then apply suitable analytical methods able to screen novel RCS sequestering agents. An HPLC method was firstly set-up in order to measure the consumption of RCS (reactivity) and of endogenous carbonyls such as pyridoxal (selectivity) when incubated in presence of the tested compounds. The method was optimized in order to use a mobile phase buffered at pH 7.4 in order to avoid the acid catalyzed degradation of the formed adducts. Reactivity towards RCS was evaluated by testing the ability of the tested compound to quench 4-hydroxy-nonenal (HNE) chosen as a model of alfa,beta-unsaturated aldehydes, glyoxal (GO) and methylglyoxal (MGO) as di-carbonyl derivatives. Selectivity was tested by measuring the consumption of pyridoxal as an endogenous aldehyde. The method was firstly validated by testing the reactivity and selectivity of known RCS scavenger compounds such as edaravone (EDA), hydralazine (HY), aminoguanidine (AG), and pyridoxamine (PYR). Even though they are very effective as RCS detoxifying agents, their usage is limited due to their lack of selectivity because they react with physiological aldehydes such as pyridoxal and by a promiscuous activity: ED is a neuroprotective compound, HY an antihypertensive drug and AG a NOS inhibitor. The HPLC method was then applied to study the reactivity and selectivity of carnosine (CAR) and derivatives such as anserine, n-acetyl carnosine and of carnosine analogues designed to be stable to carnosinase, a specific metal-ion dependent homodimeric dipeptidase (carnosinase, CN1 EC.3.4.13.20),. The screening permitted to identify carnosinol, a carnosine peptidomimetic characterized by the replacement of the carboxyl group with a hydroxyl group, as a selective and reactive RCS sequestering agent, characterized by a suitable PK profile since resistant to serum carnosinase and recognized by HPEPT1 (patent application: WO2011080139). Beside evaluating the reactivity and the selectivity, the approach was then implemented by an ESI-MS approach aimed to fully characterize the covalent adducts between RCS and the sequestering agents. The investigation of the mechanisms of carbonyl quenching have so ensued a broad understanding of the biochemical mechanisms in vitro as well as in vivo that occur between cytotoxic RCS and the "sweepers" detoxifying agents. The method was then applied to test the RCS sequestering activity of proteinogenic histidine-containing dipeptides with the C-terminus capped by a methyl ester or by a primary amido group so as to study dipeptides which should be still recognized by peptide transporters and resistant to proteolysis. Moreover, the study considered diastereoisomeric pairs of dipeptides produced by alternating the absolute configuration of the histidine residue thus revealing the effect of configuration on the quenching activity (7). The second step of the work was to set-up a rapid and accurate method for testing the ability of RCS sequestering agents to inhibit protein carbonylation. To set-up the method, HNE, which is one of the most abundant and reactive lipid-derived RCS, was used as RCS, and ubiquitin [8] as a model of protein substrate. Going into details, ubiquitin was selected as a protein target since it is commercially available at high purity and at a reasonable cost and because its molecular weight is suitable for intact protein analysis using a high resolution mass spectrometer such as the Orbitrap. Despite lacking cysteine residues, ubiquitin exposes a set of reactive lysines and a highly reactive histidine, which make this protein a suitable benchmark to test protein carbonylation and its inhibition. The method was firstly validated by using known RCS sequestering agents and the results well correlate with those obtained by HPLC analysis. The method was found suitable to test mixtures or extracts and to be used for HTS applications. AGEs and most probably ALEs are ligands and activators of the receptor RAGE whose activation is involved in a NF-kB pathway, leading to NADPH oxidase activation, oxidative stress and a condition of inflammatory and pro-fibrotic response. Since the damaging AGEs-RAGE axis has been associated to the onset and/or progression of many chronic and degenerative oxidative based disease and in many metabolic disorders, it has been recently hypothesized that the AGE-RAGE axis represents a promising drug target. One approach to counteract the AGEs-RAGE axis is represented by the design of RAGE antagonists. It should be considered that a drug design approach aimed to screen RAGE antagonists firstly requires a reliable analytical method able to test the binding affinity of small molecules and the set-up of such a method represents the aim of the present research program that is based on the following steps: \uf0d8 RAGE expression as recombinant protein in E. coli strain ORIGAMI B (DE3) with recombinant plasmid pET-15b VC1; \uf0d8 Application of the automated loop injection High Resolution Mass Spectrometry (MS) method, based on the High Resolution Mass Spectrometry method developed to study the Ubiquitin-RCS interaction, to inquire in vitro non-covalent binding relationship between low molecular weight AGEs and recombinant RAGE. The receptor for advanced glycation end products (RAGE) is a type I transmembrane glycoprotein of the mmunoglobulin superfamily of cell surface receptors. RAGE extracellular portion is involved in ligand binding and contains one \u201cV\u201d \u2013type followed by two \u201cC\u201d-type immunoglobulin-like domains (V-C1-C2 structure). V-C1 domains form an integrated unit whereas C2 domain is attached to V-C1 by a flexible linker but is a fully independent unit. As a general strategy, we decided to express the V-C1 portion of the extracellular domain of human RAGE (sRAGE) as protein target since it is water soluble and involved in the molecular recognition and engagement of the AGEs ligands. Therefore, V, V-C1 and s-RAGE expression was focused on the recombinant protein expression carried out by the protein studies obtained through the E. coli expression system. Despite a greater facility to handling the microorganism, E. coli in contrast to eukaryotic expression system, is not capable to perform post-translational modifications, including glycosylation, which are instead present in the human RAGE. Regardless for the lack of this peculiarity, numerous studies reported in literature, indicate that, post-translational modifications have not significantly effect on the RAGE properties regarding for instance the bind activity toward certain ligands and so that, the bacterial E. coli expression system represent one of the most used host for the heterologous proteins expression. A great advantage by using E.coli is its peculiarity of secreting the protein of interest in the medium that it can be easily purified in one-step. This advantage have avoided the use of tags, such as the Poly-His, that could affect the property of sRAGE during high throughput screening of libraries of compounds and making unnecessary the removal step by specific protease treatments. The high resolution mass spectrometric (ESI-MS) approach in top-down and bottom-up approach was then used in order to verify the identity of the protein. The multi-charged ESI-MS spectrum of the recombinant protein corresponding deconvoluted MS spectrum showing the MW of 24580 Da which is consistent with the theoretical one. GSHMAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPV-PLEEVQLVVE. The primary sequence was then identified by a bottom-up approach consisting to enzymatically digest the protein, separate the peptides by reversed phase capillary column. Eluted peptides were then sequenced by MS/MS analyses. The primary structure of the protein corresponding to the predicted sequence on the bases of the designed nucleotide sequence of sRAGE. After having characterized the protein, a MS approach to study the non covalent binding of RAGE with ligands was set-up. The ligand-binding properties of RAGE was studied by a native MS method that is suitable to study the non covalent interactions between ligands and recombinant V-C1. The advantages of native MS over other methods is that it does not require labeled target or ligands, it is characterized by high sensitivity, low sample consumption, fully automatation and that ligands can be screened as mixture. The experiments consist in maintaining the concentration of VC1 constant and increasing the ligand concentration. The ligands used are well known low molecular weight sRAGE ligands such as carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) derived peptide [9]. Unmodified peptides were also used as controls. Validation of the method was obtained by comparing the Kd values obtained by native MS analysis in respect to the Kd values determined from fluorescence titration experiments. The native MS method was found accurate and suitable to test libraries in order to understand the structure requirements for RAGE recognition as well as for searching antagonists. References [1] Baynes JW . Chemical modification of proteins by lipids in diabetes. Clin Chem Lab Med 2003 ; 41 : 1159 \u2013 1165. [2] Yamagishi S , Maeda S , Matsui T , Ueda S , Fukami K , Okuda S . Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes. Biochim Biophys Acta 2012 ; 1820 : 663 \u2013 671. [3] Iacobini C , Menini S , Ricci C , Scipioni A , Sansoni V , Mazzitelli G , et al . Advanced lipoxidation end-products mediate lipid-induced glomerular injury: role of receptormediated mechanisms . J Pathol 2009 ; 218 : 360 \u2013 369. [4] Del Turco S , Basta G . An update on advanced glycation endproducts and atherosclerosis. Biofactors 2012 ; 38 : 266 \u2013 274. [5] Li JL , Liu DN , Sun L , Lu Y , Zhang Z . Advanced glycation end products and neurodegenerative diseases: Mechanisms and perspective. J Neurol Sci 2012 ; 317 : 1 \u2013 5. [6] Ciulla MM , Paliotti R , Carini M , Aldini G . Fibrosis, enzymatic and non-enzymatic cross-links in hypertensive heart disease. Cardiovasc Hematol Disord Drug Targets 2011. [7] Vistoli G, De Maddis D, Straniero V, Pedretti A, Pallavicini M, Valoti E, Carini M, Testa B, Aldini G. Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents. Eur J Med Chem. 2013 Aug;66:153-60 [8] Liang X, Chen Y, Zhuang J, Zhang M, Xiong W, Guo H, Jiang F, Hu P, Guo D, and Shi W, Advanced oxidation protein products as prognostic biomarkers for recovery from acute kidney injury after coronary artery bypass grafting. Biomarkers 17 (2012) 507-12. [9] Xue J, Rai V, Singer D, Chabierski S, Xie J, Reverdatto S, Burz DS, Schmidt AM, Hoffmann R, Shekhtman A. Advanced glycation end product recognition by the receptor for AGEs. Cell Press. 2011 May 11;19(5):722-32

Background Examples of Literature Searches on Topics of Interest

A zip file of various literature searches & some resources related to our work related to exposure after the Chernobyl accident and as we began looking at helping in Semey Kazakhstan----a collection of literature reviews on various topics we were interested in... eg. establishing a registry of those exposed for longterm follow-up, what we knew about certain areas like genetics and some resources like A Guide to Environmental Resources on the Internet by Carol Briggs-Erickson and Toni Murphy which could be found on the Internet and was written to be used by researchers, environmentalists, teachers and any person who is interested in knowing and doing something about the health of our planet. See more at https://archives.library.tmc.edu/dm-ms211-012-0060