In type 2 diabetes mellitus glycated albumin alters macrophage gene expression impairing ABCA1-mediated cholesterol efflux.

Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2.

A preliminary fastview of mitochondrial protein profile from healthy and type 2 diabetic subjects.

Type 2 diabetes results from the development of insulin resistance and a concomitant impairment of insulin secretion. Mitochondrial dysfunctions are thought to be the major contributor to the development of various pathologies, including type 1 and type 2 diabetes mellitus. Mitochondrial oxidative stress has been reported in models of both type 1 and type 2 diabetes mellitus and may play a central role in mitochondrial dysfunction. In the present study, we investigated the occurrence of protein alterations, due to the presence of type 2 diabetes, in mitochondria isolated from human peripheral blood mononuclear cells (PBMCs] by matrix-assisted laser desorp- tion/ionization mass spectrometry (MALDI-MS]. PBMCs may be suitable for this investigation because they have insulin receptors that quickly respond to changes in insulin concentration, and in the presence of insulin rapidly increase their rates of glucose utiliza- tion. In the presence of insulin-resistance conditions, such as type 2 diabetes mellitus, this mechanism is altered and the glycation of cytoplasmic as well as mitochondrial proteins may plausibly appear. Therefore, PBMCs may be useful tools to verify modifications or altered expression of mitochondrial proteins. Human mitochondria were obtained from 32 subjects, 16 healthy controls and 16 type 2 diabetic patients. Two different methods for mitochondria isolation and purification were employed and compared. Some proteins have been found to be differently expressed in the two groups of subjects under investigation and can be classified into two sets: i.e. proteins related to ATP synthase [e.g. 6.8kDa mitochondrial proteolipid [MLQ]; ATP-CF6 [m/z 12,597)] and proteins related to cell proliferation and apoptosis [e.g. TIMM9 [m/z 10,378); Bcl-2-like protein 2 (m/z20,742)].

A preliminary investigation on placenta protein profile reveals only modest changes in well controlled gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is associated with a wide range of tissue-specific changes depending on the quality of glycemic control of the mothers. Here we tested the hypothesis that GDM is associated with alterations in the human term placenta proteome. For this aim, two different approacheswere employed. The placenta homogenates from 20 healthy subjects and those from 20 GDM pregnant women were pooled. The two samples thus obtained were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the proteins detected were tentatively identified by comparison of their molecular weight with the Human Protein Reference Database, restricting the search to the species expressed in the placenta tissue. However this approach led to misleading results: in fact, an in deep analysis of the spectra and tandem mass spectrometry (MS/MS) measurements of the digestion products from the protein detected, unequivocally proved that the species observed are maternal and fetal globins. Consequently, the two pools were analyzed by 1D sodium dodecyl sulphate polyacrylamide gel electrophoresis; the different bands obtained were digested by trypsin and the digestion products were analyzed by MALDI-MS; the protein identification was carried out by comparison of the peptide mass fingerprint with databases. Only modest quantitative differences were observed between the placenta protein profiles of healthy and GDM subjects, indicating that GDM, if well controlled, induces only minor changes in the placental proteome. One example of differently expressed proteins in the placenta homogenate pool from GDM and the controls was the SRRM1 protein, a member of the serine-arginine protein kinase family; for GDM samples, the MALDI spectrum of its digestion products showed the presence of molecular species attributable to glycation and glyco-oxidation processes.

Some views on proteomics in diabetes.

Mass spectrometry has been widely used in the field of diabetes. The development of new ionization methods and the effective coupling of mass spectrometry with liquid chromatography have enabled the protein modifications due to glycation processes to be investigated. Matrix assisted laser desorption/ionization mass spectrometry (MALDI/MS) has been used to evaluate the degree of glycation of specific plasma proteins. In contrast, the classic proteomic approach has been used to identify glycation sites and condensed sugar modifications. The same methods have been applied to studies on urinary protein profiles, enabling changes due to the development of long-term, diabetes-induced nephropathy to be identified. Published studies demonstrate that mass spectrometry is an important analytical tool for monitoring diabetes, capable of providing physicians with a new, more complete view of the physiopathological changes occurring as the disease develops.

Tyramine oxidation by copper/TPQ amine oxidase and peroxidase from Euphorbia characias latex.

Tyramine, an important plant intermediate, was found to be a substrate for two proteins, a copper amine oxidase and a peroxidase from Euphorbia characias latex. The oxidation of tyramine took place by two different mechanisms: oxidative deamination to p-hydroxyphenylacetaldehyde by the amine oxidase and formation of di-tyramine by the peroxidase. The di-tyramine was further oxidized at the two amino groups by the amino oxidase, whereas p-hydroxyphenylacetaldehyde was transformed to di-p-hydroxyphenylacetaldehyde by the peroxidase. Data obtained in this study indicate a new interesting scenario in the metabolism of tyramine.

Glycated human serum albumin isolated from poorly controlled diabetic patients impairs cholesterol efflux from macrophages: an investigation by mass spectrometry.

Advanced glycation end-products impair ABCA-1-mediated cholesterol efflux by eliciting inflammation, the generation of reactive oxygen species and endoplasmatic reticulum (ER) stress. The glycation level of human serum albumin (HSA) from type 1 and type 2 diabetic patients was determined by matrix assisted laser desorption/ionization (MALDI) mass spectrometry and related to possible impairment of ER function and cellular cholesterol efflux. Comparison of the MALDI spectra from healthy and diabetic subjects allowed us to determine an increased HSA mean mass of 1297 Da for type 1 and 890 Da for type 2. These values reflect a mean condensation of at least 8 glucose units and 5 glucose units, respectively. Mouse peritoneal macrophages were treated with HSA from control, type 1 and type 2 diabetic subjects in order to measure the expression of Grp78, Grp94, protein disulfide isomerase (PDI), calreticulin (CRT) and ABCA-1. (14)C-cholesterol overloaded-J774 macrophages were treated with HSA from control and diabetic subjects and further incubated with apo A-1 to determine the cholesterol efflux. Combined analyses comprising HSA from type 1 and type 2 diabetic patients were performed in cellular functional assays. In macrophages, PDI expression increased 89% and CRT 3.4 times in comparison to HSA from the control subjects. ABCA-1 protein level and apo A-I mediated cholesterol efflux were, respectively, 50% and 60% reduced in macrophages exposed to HSA from type 1 and type 2 diabetic patients when compared to that exposed to HSA from control subjects. We provide evidence that the level of glycation that occurs in albumin in vivo damages the ER function related to the impairment in macrophage reverse cholesterol transport and so contributes to atherosclerosis in diabetes.

Mass spectrometry for diabetic nephropathy monitoring: new effective tools for physicians.

The main aim of diabetic nephropathy monitoring is to identify molecular markers, that is, to find changes occurring at metabolome and proteome levels indicative of the disease's development. The mass spectrometry methods available today have been successfully applied to this field. This paper provides a short description of the basic aspects of the mass spectrometric methods used for diabetic nephropathy monitoring, reporting and discussing the results obtained using different approaches.

Structural and Functional Characterization of a New Double Variant Haemoglobin (HbG-Philadelphia/Duarte α(2)ß(2)).

WE REPORT THE FIRST CASE OF COSEGREGATION OF TWO HAEMOGLOBINS (HBS): HbG-Philadelphia [α68(E17)Asn → Lys] and HbDuarte [ß62(E6)Ala → Pro]. The proband is a young patient heterozygous also for ß°-thalassaemia. We detected exclusively two haemoglobin variants: HbDuarte and HbG-Philadelphia/Duarte. Functional study of the new double variant HbG-Philadelphia/Duarte exhibited an increase in oxygen affinity, with a slight decrease of cooperativity and Bohr effect. This functional behaviour is attributed to ß62Ala → Pro instead of α68Asn → Lys substitution. Indeed, HbG-Philadelphia isolated in our laboratory from blood cells donor carrier for this variant is not affected by any functional modification, whereas purified Hb Duarte showed functional properties very similar to the double variant. NMR and MD simulation studies confirmed that the presence of Pro instead of Ala at the ß62 position produces displacement of the E helix and modifications of the tertiary structure. The substitution α68(E17)Asn → Lys does not cause significant structural and dynamical modifications of the protein. A possible structure-based rational of substitution effects is suggested.

Increase in urinary purines and pyrimidines in patients with methylmalonic aciduria combined with homocystinuria.

BACKGROUND: Methylmalonic aciduria combined with homocystinuria (MMA-HC) is the biochemical trait of a metabolic disorder resulting from impaired conversion of dietary cobalamin (cbl, or vitamin B12) to its two metabolically active forms. Effects on urinary purine and pyrimidine levels have not been described for this condition. METHODS: Urine samples were collected from three patients with methylmalonic aciduria combined with homocystinuria and from 70 healthy subjects. Urinary purine and pyrimidine levels were quantitated by the use of LC/UV-Vis and LC/ESI/MS. RESULTS: Higher urine levels of pyrimidines were detected with both methods in patients compared to controls. CONCLUSION: Methylmalonic aciduria with homocystinuria is due to deficiency of the enzyme, cobalamin reductase. The enzyme defect leads to altered hepatic metabolism, which appears to modify circulating pyrimidine levels.