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.

Hematopoietic and Nonhematopoietic p66Shc Differentially Regulates Stem Cell Traffic and Vascular Response to Ischemia in Diabetes.

Aims: Peripheral artery disease (PAD) is a severe complication of diabetes, characterized by defective traffic of hematopoietic stem/progenitor cells (HSPCs). We examined the hematopoietic versus nonhematopoietic role of p66Shc in regulating HSPC traffic and blood flow recovery after ischemia in diabetic mice. Results: Using streptozotocin-induced diabetes, chimeric mice with green fluorescent protein (GFP)+ bone marrow (BM), and the hind limb ischemia model, we found that the physiologic mobilization and homing of HSPCs were abolished by diabetes, along with impaired vascular recovery. Hematopoietic deletion of p66Shc, obtained by transplanting p66Shc-/- BM cells into wild-type (Wt) recipients, but not nonhematopoietic deletion, constrained hyperglycemia-induced myelopoiesis, rescued postischemic HSPC mobilization, and improved blood flow recovery in diabetic mice. In Wt diabetic mice transplanted with BM cells from GFP+p66Shc-/- mice, the amount of HSPCs homed to ischemic muscles was greater than in mice transplanted with GFP+p66Shc+/+ cells, with recruited cells displaying higher expression of adhesion molecules and Vegf. In 40 patients with diabetes, p66Shc gene expression in mononuclear cells was correlated with myelopoiesis and elevated in the presence of PAD. In 13 patients with diabetes and PAD, p66Shc expression in HSPC-mobilized peripheral blood cells was inversely correlated with VEGF expression. Innovation: For the first time, we dissect the role of hematopoietic versus nonhematopoietic p66Shc in regulating HSPC traffic and ischemic responses. Conclusion: Hematopoietic deletion of p66Shc was sufficient to rescue HSPC mobilization and homing in diabetes after ischemia and improved blood flow recovery. Inhibiting p66Shc in blood cells may be a novel strategy to counter PAD in diabetes. Antioxid. Redox Signal. 36, 593-607. Clinical Trial No.: NCT02790957.

Urine-Derived Epithelial Cells as a New Model to Study Renal Metabolic Phenotypes of Patients with Glycogen Storage Disease 1a.

Glycogen storage diseases (GSDs) represent a model of pathological accumulation of glycogen disease in the kidney that, in animal models, results in nephropathy due to abnormal autophagy and mitochondrial function. Patients with Glycogen Storage Disease 1a (GSD1a) accumulate glycogen in the kidneys and suffer a disease resembling diabetic nephropathy that can progress to renal failure. In this study, we addressed whether urine-derived epithelial cells (URECs) from patients with GSD1a maintain their biological features, and whether they can be used as a model to study the renal and metabolic phenotypes of this genetic condition. Studies were performed on cells extracted from urine samples of GSD1a and healthy subjects. URECs were characterized after the fourth passage by transmission electron microscopy and immunofluorescence. Reactive oxygen species (ROS), at different glucose concentrations, were measured by fluorescent staining. We cultured URECs from three patients with GSD1a and three healthy controls. At the fourth passage, URECs from GSD1a patients maintained their massive glycogen content. GSD1a and control cells showed the ciliary structures of renal tubular epithelium and the expression of epithelial (E-cadherin) and renal tubular cells (aquaporin 1 and 2) markers. Moreover, URECs from both groups responded to changes in glucose concentrations by modulating ROS levels. GSD1a cells were featured by a specific response to the low glucose stimulus, which is the condition that more resembles the metabolic derangement of patients with GSD1a. Through this study, we demonstrated that URECs might represent a promising experimental model to study the molecular mechanisms leading to renal damage in GSD1a, due to pathological glycogen storage.

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).

Fenofibrate increases circulating haematopoietic stem cells in people with diabetic retinopathy: a randomised, placebo-controlled trial.

AIM/HYPOTHESIS: In two large RCTs, fenofibrate reduced the progression of diabetic retinopathy. We investigated whether fenofibrate increases circulating haematopoietic stem/progenitor cells (HSPCs), which have vascular properties and have been shown to protect from retinopathy. METHODS: We conducted a 12 week parallel-group RCT comparing fenofibrate vs placebo. Patients with diabetic retinopathy and without other conditions that would affect HSPCs were enrolled at a tertiary diabetes outpatient clinic and randomised to receive fenofibrate or placebo based on a computer-generated sequence. Patients and study staff assessing the outcomes were blinded to group assignment. The primary endpoint was the change in the levels of circulating HSPCs, defined by expression of the stem cell markers CD34 and/or CD133. Secondary endpoints were the changes in endothelial progenitor cells, lipids, soluble mediators and gene expression. We used historical data on the association between HSPCs and retinopathy outcomes to estimate the effect of fenofibrate on retinopathy progression. RESULTS: Forty-two participants with diabetic retinopathy were randomised and 41 completed treatment and were analysed (20 in the placebo group and 21 in the fenofibrate group). Mean age was 57.4 years, diabetes duration was 18.2 years and baseline HbA1c was 60 mmol/mol (7.6%). When compared with placebo, fenofibrate significantly increased levels of HSPCs expressing CD34 and/or CD133. CD34+ HSPCs non-significantly declined in the placebo group (mean ± SD -44.2 ± 31.6 cells/106) and significantly increased in the fenofibrate group (53.8 ± 31.1 cells/106). The placebo-subtracted increase in CD34+ HSPCs from baseline was 30% (99.3 ± 43.3 cells/106; p = 0.027) which, projected onto the relationship between HSPC levels and retinopathy outcomes, yielded an OR of retinopathy progression of 0.67 for fenofibrate vs placebo. Endothelial differentiation of CD34+ cells, estimated by the %KDR (kinase insert domain receptor) expression, was significantly reduced by fenofibrate. Fenofibrate decreased serum triacylglycerols, but the change in triacylglycerols was unrelated to the change in HSPCs. No effect was observed for endothelial progenitor cells, cytokines/chemokines (stromal-cell derived factor-1, vascular endothelial growth factor, monocyte chemoattractant protein-1) and gene expression in peripheral blood mononuclear cells. CONCLUSIONS/INTERPRETATION: Fenofibrate increased HSPC levels in participants with diabetic retinopathy and this mechanism may explain why fenofibrate reduced retinopathy progression in previous studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT01927315.

PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes.

BACKGROUND: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. METHODS: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages. RESULTS: We found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway. CONCLUSIONS: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Systemic and vascular inflammation in an in-vitro model of central obesity.

Metabolic disorders due to over-nutrition are a major global health problem, often associated with obesity and related morbidities. Obesity is peculiar to humans, as it is associated with lifestyle and diet, and so difficult to reproduce in animal models. Here we describe a model of human central adiposity based on a 3-tissue system consisting of a series of interconnected fluidic modules. Given the causal link between obesity and systemic inflammation, we focused primarily on pro-inflammatory markers, examining the similarities and differences between the 3-tissue model and evidence from human studies in the literature. When challenged with high levels of adiposity, the in-vitro system manifests cardiovascular stress through expression of E-selectin and von Willebrand factor as well as systemic inflammation (expressing IL-6 and MCP-1) as observed in humans. Interestingly, most of the responses are dependent on the synergic interaction between adiposity and the presence of multiple tissue types. The set-up has the potential to reduce animal experiments in obesity research and may help unravel specific cellular mechanisms which underlie tissue response to nutritional overload.

miR-30c-5p regulates macrophage-mediated inflammation and pro-atherosclerosis pathways.

AIMS: Atherosclerosis is an inflammatory disease wherein cholesterol-loaded macrophages play a major role. MicroRNAs and microparticles propagate inflammatory pathways and are involved in cardiovascular disease. We aimed to screen and validate circulating microRNAs correlated with atherosclerosis development in humans, and to dissect the molecular mechanisms associated with atherogenesis using in vitro and in vivo approaches. METHODS AND RESULTS: A panel of 179 secreted microRNAs was screened in plasma samples of patients with and without atherosclerosis, and validated cross-sectionally and prospectively in patients followed for up to 11 years. miR-30c-5p was inversely correlated with total and LDL cholesterol, carotid intimal media thickness (CIMT), presence and future development of plaques. Using a human macrophage line and in vitro gene silencing strategies, we found that miR-30c-5p was downregulated by oxidized LDL (oxLDL) via the scavenger receptor CD36 and inhibition miR processing by Dicer. In turn, miR-30c-5p downregulation was responsible for the effects of oxLDL on macrophage IL-1ß release, caspase-3 expression, and apoptosis. miR-30c-5p loaded into microparticles was uptaken by macrophages and regulated target genes, like caspase-3, at transcriptional level. To establish the relevance of this pathway on endothelial damage as the earliest step of atherogenesis, we show that systemic miR-30c-5p knockdown induced caspase-3 and impaired endothelial healing after carotid injury in C57Bl/6 J mice. CONCLUSIONS: With an unbiased screening of secreted microRNAs, we identify reduction of miR-30c-5p in microparticles as a promoter of early atherosclerosis, by conveying pro-inflammatory pro-apoptotic signals and impairing endothelial healing. Therefore, stimulation of miR-30c-5p is a candidate direct anti-atherosclerotic therapy.

Acute Effects of Linagliptin on Progenitor Cells, Monocyte Phenotypes, and Soluble Mediators in Type 2 Diabetes.

CONTEXT: Circulating cells, including endothelial progenitor cells (EPCs) and monocyte subtypes, are involved in diabetic complications. Modulation of these cells may mediate additional benefits of glucose-lowering medications. OBJECTIVE: We assessed whether the dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin acutely modifies EPCs and monocyte subsets in patients with type 2 diabetes. DESIGN: This was a randomized, crossover, placebo-controlled trial. SETTING: The study was conducted at a tertiary referral diabetes outpatient clinic. PATIENTS: Forty-six type 2 diabetes patients with (n = 18) or without (n = 28) chronic kidney disease (CKD) participated in the study. INTERVENTION: Intervention included a 4-day treatment with linagliptin 5 mg or placebo during two arms separated by a 2-week washout. MAIN OUTCOME MEASURES: Before and after each treatment, we determined the levels of circulating progenitor cells (CD34, CD133, KDR) and monocyte subtypes (CD14/CD16, chemokine and scavenger receptors) and the concentrations of soluble mediators. RESULTS: Compared with placebo, linagliptin increased CD34(+)CD133(+) progenitor cells (placebo subtracted effect 40.4 ± 18.7/10(6); P = .036), CD34(+)KDR(+) EPCs (placebo subtracted effect 22.1 ± 10.2/10(6); P = .036), and CX3CR1(bright) monocytes (placebo subtracted effect 1.7 ± 0.8%; P = .032). Linagliptin abated DPP-4 activity by greater than 50%, significantly increased active glucagon-like peptide-1 and stromal cell-derived factor-1α, and reduced monocyte chemotactic protein-1, CCL22, and IL-12. Patients with CKD, as compared with those without, had lower baseline CD133(+) and CD34(+)CD133(+) cells and had borderline reduced CD34(+) and CD34(+)KDR(+) cells. The effects of linagliptin on progenitor cells and monocyte subtypes were similar in patients with or without CKD. Fasting plasma glucose, triglycerides and free fatty acids were unaffected. CONCLUSIONS: DPP-4 inhibition with linagliptin acutely increases putative vasculoregenerative and antiinflammatory cells. Direct effects of DPP-4 inhibition may be important to lower vascular risk in diabetes, especially in the presence of CKD.

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.

Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.

CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50µM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.

No peptide left behind: the "out of range" recovery in IPG-IEF fractionation.

IEF is often used in multidimensional shotgun proteomics and the narrow range of 3.5-4.5 is the recommended pH interval for the fractionation of tryptic peptides. Usually, even if IEF is performed in IPG strip with a narrow range pH, the entire sample must be loaded onto the strip, including the "out of IPG range" peptides. We describe a simple protocol to recover at least a part of these missing peptides and show that this recovery significantly influences the overall fractionation result, increasing the number of the identified proteins and the protein coverage.

Glucose and fatty acid metabolism in a 3 tissue in-vitro model challenged with normo- and hyperglycaemia.

Nutrient balance in the human body is maintained through systemic signaling between different cells and tissues. Breaking down this circuitry to its most basic elements and reconstructing the metabolic network in-vitro provides a systematic method to gain a better understanding of how cross-talk between the organs contributes to the whole body metabolic profile and of the specific role of each different cell type. To this end, a 3-way connected culture of hepatocytes, adipose tissue and endothelial cells representing a simplified model of energetic substrate metabolism in the visceral region was developed. The 3-way culture was shown to maintain glucose and fatty acid homeostasis in-vitro. Subsequently it was challenged with insulin and high glucose concentrations to simulate hyperglycaemia. The aim was to study the capacity of the 3-way culture to maintain or restore normal circulating glucose concentrations in response to insulin and to investigate the effects these conditions on other metabolites involved in glucose and lipid metabolism. The results show that the system's metabolic profile changes dramatically in the presence of high concentrations of glucose, and that these changes are modulated by the presence of insulin. Furthermore, we observed an increase in E-selectin levels in hyperglycaemic conditions and increased IL-6 concentrations in insulin-free-hyperglycaemic conditions, indicating, respectively, endothelial injury and proinflammatory stress in the challenged 3-way system.

An in vitro model of glucose and lipid metabolism in a multicompartmental bioreactor.

The energy balance in vivo is maintained through inter-organ cross-talk involving several different tissues. As a first step towards recapitulating the metabolic circuitry, hepatocytes, endothelial cells and adipose tissue were connected in a multicompartmental modular bioreactor to reproduce salient aspects of glucose and lipid metabolism in vitro. We first examined how the two-way cellular interplay between adipose tissue and endothelial cells affects glucose and lipid metabolism. The hepatocyte cell line HepG2 was then added to the system, creating a three-way connected culture, to determine whether circulating metabolite concentrations were normalized, or whether metabolic shifts, which may arise when endothelial cells and adipose tissue are placed in connection, were corrected. The addition of hepatocytes to the system prevented the drop in the concentrations of glucose, L-alanine and lactate, and the rise in that of free fatty acids. There was no significant change in glycerol levels in either of the connected cultures. The results show that connected cultures recapitulate complex physiological systemic processes, such as glucose and lipid metabolism, and that the HepG2 hepatocytes normalize circulating metabolites in this in vitro environment in the presence of other cell types.

Rosiglitazone reduces glucose-induced oxidative stress mediated by NAD(P)H oxidase via AMPK-dependent mechanism.

OBJECTIVE: Hyperglycemia is the main determinant of long-term diabetic complications, mainly through induction of oxidative stress. NAD(P)H oxidase is a major source of glucose-induced oxidative stress. In this study, we tested the hypothesis that rosiglitazone (RSG) is able to quench oxidative stress initiated by high glucose through prevention of NAD(P)H oxidase activation. METHODS AND RESULTS: Intracellular ROS were measured using the fluoroprobe TEMPO-9-AC in HUVECs exposed to control (5 mmol/L) and moderately high (10 mmol/L) glucose concentrations. NAD(P)H oxidase and AMPK activities were determined by Western blot. We found that 10 mmol/L glucose increased significantly ROS production in comparison with 5 mmol/L glucose, and that this effect was completely abolished by RSG. Interestingly, inhibition of AMPK, but not PPARgamma, prevented this effect of RSG. AMPK phosphorylation by RSG was necessary for its ability to hamper NAD(P)H oxidase activation, which was indispensable for glucose-induced oxidative stress. Downstream of AMPK activation, RSG exerts antioxidative effects by inhibiting PKC. CONCLUSIONS: This study demonstrates that RSG activates AMPK which, in turn, prevents hyperactivity of NAD(P)H oxidase induced by high glucose, possibly through PKC inhibition. Therefore, RSG protects endothelial cells against glucose-induced oxidative stress with an AMPK-dependent and a PPARgamma-independent mechanism.

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.

Insulin generates free radicals in human fibroblasts ex vivo by a protein kinase C-dependent mechanism, which is inhibited by pravastatin.

Insulin can generate oxygen free radicals. Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, exert a powerful antioxidant effect. The present study aimed to clarify the mechanisms through which insulin generates free radicals and to assess whether pravastatin modulates such effects. In cultured skin fibroblasts from human volunteers exposed to high insulin concentration, either in the presence or in the absence of pravastatin, insulin induced translocation of the p47(phox) subunit of NAD(P)H oxidase from the cytosol to the membrane and generation of radical oxygen species through a PKC delta-dependent mechanism. The insulin-induced translocation of p47(phox) was PKC delta dependent and attenuated by pravastatin, but independent of the activation of Akt and Rac1. Insulin-induced Akt phosphorylation was increased by pravastatin and ERK1/2 phosphorylation attenuated. The present study demonstrates a novel mechanism by which insulin stimulates the generation of free radicals in human fibroblasts, ex vivo. It involves phosphatidylinositol 3-kinase, PKC delta, and p47(phox) translocation and promotes ERK1/2 phosphorylation. Pravastatin inhibited radical oxygen species production by inhibiting PKC delta. These observations offer a robust explanation for the positive effects of pravastatin treatment in patients with insulin resistance syndrome.

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.

Metformin prevents glucose-induced protein kinase C-beta2 activation in human umbilical vein endothelial cells through an antioxidant mechanism.

Hyperglycemia determines the vascular complications of diabetes through different mechanisms: one of these is excessive activation of the isoform beta2 of protein kinase C (PKC-beta2). Metformin, a widely used antidiabetic agent, is associated with decreased cardiovascular mortality in obese type 2 diabetic patients. Therefore, we assessed the role of metformin in glucose-induced activation of PKC-beta2 and determined the mechanism of its effect in human umbilical venous endothelial cells grown to either normo- (5 mmol/l) or hyperglycemia (10 mmol/l) and moderately and acutely exposed to 25 mmol/l glucose. We studied PKC-beta2 activation by developing adenovirally expressed chimeras encoding fusion protein between green fluorescent protein (GFP) and conventional beta2 isoform (PKC-beta2-GFP). Glucose (25 mmol/l) induced the translocation of PKC-beta2-GFP from the cytosol to the membrane in cells grown to hyperglycemia but not in those grown in normal glucose medium. Metformin (20 micromol/l) prevented hyperglycemia-induced PKC-beta2-GFP translocation. We also assessed oxidative stress under the same conditions with a 4-((9-acridine-carbonyl)amino)-2,2,6,6-tetramethylpiperidin-oxyl,free radical (TEMPO-9-AC) fluorescent probe. We observed significantly increased radical oxygen species production in cells grown in hyperglycemia medium, and this effect was abolished by metformin. We show that in endothelial cells, metformin inhibits hyperglycemia-induced PKC-beta2 translocation because of a direct antioxidant effect. Our data substantiate the findings of previous large intervention studies on the beneficial effect of this drug in type 2 diabetic patients.

Concomitance of diabetic retinopathy and proteinuria accelerates the rate of decline of kidney function in type 2 diabetic patients.

OBJECTIVE: To evaluate the rate of progression of renal disease in proteinuric type 2 diabetic patients with and without retinopathy. RESEARCH DESIGN AND METHODS: Thirty-eight proteinuric type 2 diabetic patients with diabetic retinopathy and 27 without were enrolled in an observational study for the evaluation of rate of glomerular filtration rate (GFR) decline and followed up for a median period of 6 years. GFR was determined at least once per year, and blood pressure, glycated hemoglobin, and proteinuria were determined every 4 months. RESULTS: Although the two groups had comparable GFR, albuminuria, blood pressure, and HbA(1c) at entry of the study, the rate of decline of GFR was higher in type 2 diabetic patients with retinopathy (-6.5 +/- 4.4 ml/year) than in those without (-1.8 +/- 4.8 ml/year; P < 0.0001). Protein and albumin excretion rate increased significantly in patients with retinopathy, while they did not change in those without. Mean blood pressure between the two groups of patients were similar both at entry and during the follow-up, although the proportion of patients treated with at least two antihypertensive drugs was higher in patients with retinopathy. On a multiple regression analysis, only mean blood pressure and proteinuria were significant determinants of progression of renal disease in type 2 diabetic patients with retinopathy. CONCLUSIONS: The rate of progression of renal disease in proteinuric type 2 diabetic patients with retinopathy is faster than that observed in those without retinopathy. The screening for retinopathy identifies patients at high risk for rapid deterioration of kidney function.