Neither Incretin or Amino Acid Responses, nor Casein Content, Account for the Equal Insulin Response Following Iso-Lactose Loads of Natural Human and Cow Milk in Healthy Young Adults.

Human milk contains <50% less protein (casein) than cow milk, but is equally effective in insulin secretion despite lower postingestion hyperaminoacidemia. Such potency of human milk might be modulated either by incretins (glucagon-like polypeptide-1,GLP-1); glucose-inhibitory-polypeptide, GIP), and/or by milk casein content. Healthy volunteers of both sexes were fed iso-lactose loads of two low-protein milks, i.e., human [Hum] (n = 8) and casein-deprived cow milk (Cow [↓Cas]) (n = 10), as well as loads of two high-protein milks, i.e., cow (n = 7), and casein-added human-milk (Hum [↑Cas]) (n = 7). Plasma glucose, insulin, C-peptide, incretins and amino acid concentrations were measured for 240'. All milks induced the same transient hyperglycemia. The early [20'−30'] insulin and C-peptide responses were comparable among all milk types apart from the low-protein (Cow [↓Cas]) milk, where they were reduced by <50% (p < 0.05 vs. others). When comparing the two high-protein milks, GLP-1 and GIP [5'−20'] responses with the (Hum [↑Cas]) milk were lower (by ≈2−3 fold, p < 0.007 and p < 0.03 respectively) than those with cow milk, whereas incretin secretion was substantially similar. Plasma amino acid increments largely reflected the milk protein content. Thus, neither casein milk content, nor incretin or amino acid concentrations, can account for the specific potency of human milk on insulin secretion, which remains as yet unresolved.

Effect of Reversal of Whey-Protein to Casein Ratio of Cow Milk, on Insulin, Incretin, and Amino Acid Responses in Humans.

SCOPE: Milk-proteins, besides lactose, stimulate insulin and incretin secretion. Although whey-proteins (WP) are more efficient than casein (Cas) in hormone secretion, the effects of reversal of the (WP/Cas) ratio in whole-milk are poorly known. METHODS AND RESULTS: Healthy volunteers received two different cow-milk drinks, at identical lactose (0.36 g × kg-1 BW) and total-protein (0.18 g × kg1 BW) loads, but at reversed WP/Cas ratio. One is cow-whole milk with a ≈20/80 [WP/Cas] ratio, the other an experimental cow-milk with a ≈70/30 [WP/Cas] ratio ([↑WP↓Cas]-milk). Both milk-types induced the same mild hyperglycemic response. Following [↑WP↓Cas]-milk, the [20'-90'] insulin incremental area (iAUC) (+ ≈44%, p < 0.035), and the [20'-120'] C-peptide iAUC (+ ≈47%, p < 0.015) are greater than those with cow-milk. Similarly, following [↑WP↓Cas]-milk, the GLP-1 [20'-90'] iAUC (+96%, p < 0.025), and the GIP [30'-60'] iAUC (+140%, p < 0.006), were greater than those with cow-milk. Plasma total and branched-chain amino acids are also greater following the [↑WP↓Cas] than cow-milk. CONCLUSIONS: Reversal of the (WP/Cas) ratio in cow-milk enhanced the insulin response, an effect possibly mediated by incretins and/or amino acids(s). These data may be useful in designing specific milk formulas with different effects on insulin and incretin response(s).

Lumican is overexpressed in lung adenocarcinoma pleural effusions.

Adenocarcinoma (AdC) is the most common lung cancer subtype and is often associated with pleural effusion (PE). Its poor prognosis is attributable to diagnostic delay and lack of effective treatments and there is a pressing need in discovering new biomarkers for early diagnosis or targeted therapies. To date, little is known about lung AdC proteome. We investigated protein expression of lung AdC in PE using the isobaric Tags for Relative and Absolute Quantification (iTRAQ) approach to identify possible novel diagnostic/prognostic biomarkers. This provided the identification of 109 of lung AdC-related proteins. We further analyzed lumican, one of the overexpressed proteins, in 88 resected lung AdCs and in 23 malignant PE cell-blocks (13 lung AdCs and 10 non-lung cancers) using immunohistochemistry. In AdC surgical samples, lumican expression was low in cancer cells, whereas it was strong and diffuse in the stroma surrounding the tumor. However, lumican expression was not associated with tumor grade, stage, and vascular/pleural invasion. None of the lung cancer cell-blocks showed lumican immunoreaction, whereas those of all the other tumors were strongly positive. Finally, immunoblotting analysis showed lumican expression in both cell lysate and conditioned medium of a fibroblast culture but not in those of A549 lung cancer cell line. PE is a valid source of information for proteomic analysis without many of the restrictions of plasma. The high lumican levels characterizing AdC PEs are probably due to its release by the fibroblasts surrounding the tumor. Despite the role of lumican in lung AdC is still elusive, it could be of diagnostic value.

Pros and cons of peptide isolectric focusing in shotgun proteomics.

In shotgun proteomics, protein mixtures are proteolytically digested before tandem mass spectrometry (MS/MS) analysis. Biological samples are generally characterized by a very high complexity, therefore a step of peptides fractionation before the MS analysis is essential. This passage reduces the sample complexity and increases its compatibility with the sampling performance of the instrument. Among all the existing approaches for peptide fractionation, isoelectric focusing has several peculiarities that are theoretically known but practically rarely exploited by the proteomics community. The main aim of this review is to draw the readers' attention to these unique qualities, which are not accessible with other common approaches, and that represent important tools to increase confidence in the identification of proteins and some post-translational modifications. The general characteristics of different methods to perform peptide isoelectric focusing with natural and artificial pH gradients, the existing instrumentation, and the informatics tools available for isoelectric point calculation are also critically described. Finally, we give some general conclusions on this strategy, underlying its principal limitations.

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis.

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10(-9) g/L), pigment epithelium growth factor (10(-8) g/L), hepatocyte growth factor activator (10(-7) g/L) and thrombospondin-1 (10(-6) g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.

Polar electrophoresis: shape of two-dimensional maps is as important as size.

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of "isobaric" spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.

High abundance proteins depletion vs low abundance proteins enrichment: comparison of methods to reduce the plasma proteome complexity.

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol.

Proteomic analysis of clonal interstitial aortic valve cells acquiring a pro-calcific profile.

Calcific degeneration represents the most frequent aortic valve disease observed in industrialized countries. Our aim is to study modifications in the cytosolic and membrane protein profile of aortic interstitial valve cells (VIC) acquiring a pro-calcific phenotype. We studied a clonal population of bovine VIC that expresses bone-related proteins (such as alkaline phosphatase [ALP]) and calcifies a collagen matrix in response to endotoxin (LPS) treatment. A proteomic analysis was performed on proteins extracted from cells treated for 12 days with LPS (100 ng/mL) versus control. We identified 34 unique cytosolic and 10 unique membrane-associated proteins showing significant changes after treatment. These proteins are involved in several cellular functions, such as chaperone-mediated protein folding, protein metabolism and transport, cell redox/nitric oxide homeostasis, and cytoskeletal organization. Reduced expression of proteins involved in NOS bioactivity (such as DDAH-1 and -2) suggested a role for the l-arginine/ADMA ratio in controlling VIC phenotypic profile. In accordance with this hypothesis, we observed that exposure of clonal cells to l-arginine prevented LPS-induced ALP expression and collagen calcification. In conclusion, we identified several proteins involved in structural, metabolic, and signaling functions that are significantly altered in aortic VIC acquiring a pro-calcific profile, thus giving new insights into the pathogenesis of aortic valve degeneration.

Altered chaperone and protein turnover regulators expression in cultured skin fibroblasts from type 1 diabetes mellitus with nephropathy.

In type-1 diabetes mellitus (T1DM) with diabetic nephropathy (DN), accumulation of abnormal proteins in the kidney and other tissues may derive from constitutive alterations of intracellular protein recognition, assembly, and turnover. We characterized the proteins involved in these functions in cultured skin fibroblasts from long-term T1DM patients with [DN+] or without [DN-] nephropathy but similar metabolic control, and from matched healthy subjects. 2-D gel electrophoresis and MS-MALDI analysis were employed. The [DN+] T1DM patients, compared with the two other groups, exhibited increased abundance of a high-molecular weight isoform of protein disulphide-isomerase A3 and a decrease of two low-molecular weight isoforms. They also had increased levels of heat shock protein (HSP) 60 kDa isoform #A4, of HSP71 kDa isoform #A30, and of HSP27 kDa isoform #6, whereas the HSP27 kDa isoforms #A90 and #A71 were decreased. Cathepsin beta-2 (#40), the cation-independent mannose 6-phosphate receptor binding protein 1 (CIMPR) (#A27), and annexin 2 (#A9) were also decreased in the [DN+] T1DM patients, whereas the RNA-binding protein regulatory subunity (#38) and the translationally-controlled tumor protein (TCTP) (#A45) were increased. These changes of chaperone-like proteins in fibroblasts may highlight those of the kidney and be patho-physiologically related to the development of nephropathy in T1DM.

Slow versus fast proteins in the stimulation of beta-cell response and the activation of the entero-insular axis in type 2 diabetes.

BACKGROUND: We tested whether ingestion of whey protein can induce greater post-prandial amino acid (AA) levels in the plasma and a higher beta-cell response than casein ingestion in type 2 diabetes mellitus patients. METHODS: The study was designed as a double-blind, randomized, and controlled cross-over clinical trial. Twelve post-absorptive type 2 diabetic subjects who were withdrawn from their usual hypoglycemic therapy were studied. A medium calorie (approximately 6 kcal/kg BW), high protein (approximately 50% of total kcal) mixed meal, containing whey protein, casein, or a free amino acid (FREE AA) mixture matching the casein AA composition, was randomly administered on three different occasions. RESULTS: Following ingestion of whey protein, plasma concentrations of total, branched chain, and essential AA were 25-50% greater than after ingestion of casein (p < 0.0001), and were similar to those observed after the FREE AA meal. With whey protein, C-peptide, insulin, and pro-insulin concentrations were greater by 12-40% (p < 0.02 or less) than with casein, and similar to those with FREE AA. Glucagon-like polypeptide 1 (GLP-1) response tended to be lower with casein than with whey protein. Glucose-dependent insulinotropic polypeptide (GIP) response was greater with either whey protein or casein than with FREE AA. Post-prandial glucose concentrations were similar after whey protein and casein ingestion, but lower after the FREE AA meal. CONCLUSIONS: In type 2 diabetes, the ingestion of a fast-absorbable protein results in a greater post-prandial aminoacidemia and a higher beta-cell secretion than the ingestion of a 'slow' protein. Whether these changes can be maintained chronically in combination with hypoglycemic therapy, possibly also resulting in better glycemic control, remains to be established.

Proteome analysis of cultured fibroblasts from type 1 diabetic patients and normal subjects.

CONTEXT: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes. OBJECTIVE: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls. DESIGN: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and alpha-actinin 4 and a decrease of tubulin-beta2 and splice isoform 2 of tropomyosin beta-chain were detected. CONCLUSIONS: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.

A rapid method to determine plasma homocysteine concentration and enrichment by gas chromatography/mass spectrometry.

Homocysteine is an independent risk factor for cardio- and/or cerebrovascular diseases. Many methods are used to measure plasma homocysteine levels in physiological fluids. Current gas chromatographic/mass spectrometric (GC/MS) methods allow determination not only of plasma homocysteine concentration, but also of its turnover. However, they have some methodological limitations due to the reduction of disulfide bonds between homocysteine and other thiols or proteins often requiring the use of several very toxic compounds or multi-step procedures that are particularly time-consuming, and/or utilize expensive instruments. Herein is described a rapid and precise GS/MS method to determine homocysteine turnover from a relatively low volume of plasma (200 microL). First disulfide bonds were reduced by 2-mercaptoethanol, which allows the maintenance of the reduced status preventing the rebuilding of the disulfide bond. Then the sample was derivatized to form the bis-tert-butyldimethylsilyl derivative. A deuterated internal standard, DL-[3,3,3',3',4,4,4',4'-2H8]-homocystine, was employed to account for losses associated with each analytical step. To evaluate the 'in vivo' homocysteine metabolic turnover, [1-13C]-methionine was infused and the derived [1-13C]-homocysteine quantitated. So a standard curve of [1-13C]-homocysteine was prepared by the decomposition of the [1-13C] methionine. The ions at m/z 325 and 326 were monitored, corresponding to the unlabeled [12C]-homocysteine and to labeled [13C]-homocysteine, respectively. The ion at m/z 325 ([M-114)]+) probably resulted from the loss of one derivatizing group to regenerate a free amino group. The intra-assay coefficient of variation (CV-intra%) was consistently less than 1.06%, the inter-assay (CV-inter%) less than 1.05%. The method described here seems to be simpler, more rapid, and less toxic than those published so far. In particular, its main strength appears to be the degree of precision obtained. We suggest applying this method to the measurement of the 'in vivo' rate of production of homocysteine (by the plasma 13C-homocysteine enrichment) from its precursor (13C-methionine).