The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose

We report the functional characterization of the galF gene of strain VW187 (Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PPi. We show that, although the GalF protein is expressed in vivo, GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system. Furthermore, using a pair of galF-/galU+ and galF/galU+ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo. We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress.

IHG-1 amplifies TGF-beta1 signaling and is increased in renal fibrosis

Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects. In the diabetic nephropathy specimens, in situ hybridization localized IHG-1 to tubular epithelial cells along with TGF-beta1 and activated Smad3, suggesting a possible role in the development of tubulointerstitial fibrosis. Supporting this possibility, IHG-1 mRNA and protein expression also increased with unilateral ureteral obstruction. In the HK-2 proximal tubule cell line, overexpression of IHG-1 increased TGF-beta1-stimulated expression of connective tissue growth factor and fibronectin. IHG-1 was found to amplify TGF-beta1-mediated transcriptional activity by increasing and prolonging phosphorylation of Smad3. Conversely, inhibition of endogenous IHG-1 with small interference RNA suppressed transcriptional responses to TGF-beta1. In summary, IHG-1, which increases in diabetic nephropathy, may enhance the actions of TGF-beta1 and contribute to the development of tubulointerstitial fibrosis.

Gamma Irradiation and Targeted Radionuclides Enhance the Expression of the Noradrenaline Transporter Transgene Controlled by the Radio-Inducible p21(WAF1/CIP1) Promoter

The use of radiation-inducible promoters to drive transgene expression offers the possibility of temporal and spatial regulation of gene activation. This study assessed the potential of one such promoter element, p21(WAF1/CIP1) (WAF1), to drive expression of the noradrenaline transporter (NAT) gene, which conveys sensitivity to radioiodinated meta-iodobenzylguanidine (MIBG). An expression vector containing NAT under the control of the radiation-inducible WAF1 promoter (pWAF/NAT) was produced. The non-NAT expressing cell lines UVW (glioma) and HCT116 (colorectal cancer) were transfected with this construct to assess radiation-controlled WAF1 activation of the NAT gene. Transfection of UVW and HCT cells with pWAF/NAT conferred upon them the ability to accumulate [(131)I]MIBG, which led to increased sensitivity to the radiopharmaceutical. Pretreatment of transfected cells with ? radiation or the radiopharmaceuticals [(123)I]MIBG or [(131)I]MIBG induced dose- and time-dependent increases in subsequent [(131)I]MIBG uptake and led to enhanced efficacy of [(131)I]MIBG-mediated cell kill. Gene therapy using WAF1-driven expression of NAT has the potential to expand the use of this therapeutic modality to tumors that lack a radio-targetable feature.

IHG-1 must be localised to mitochondria to decrease Smad7 expression and amplify TGF-β1-induced fibrotic responses

TGF-ß1 is a prototypic profibrotic cytokine and major driver of fibrosis in the kidney and other organs. Induced in high glucose-1 (IHG-1) is a mitochondrial protein which we have recently reported to be associated with renal disease. IHG-1 amplifies responses to TGF-ß1 and regulates mitochondrial biogenesis by stabilising the transcriptional co-activator peroxisome proliferator-activated receptor gamma coactivator-1-alpha. Here we report that the mitochondrial localization of IHG-1 is pivotal in amplification of TGF-ß1 signaling. We demonstrate that IHG-1 expression is associated with repression of the endogenous TGF-ß1 inhibitor Smad7. Intriguingly, expression of a non-mitochondrial deletion mutant of IHG-1 (?mts-IHG-1) repressed TGF-ß1 fibrotic signaling in renal epithelial cells. In cells expressing ?mts-IHG-1 fibrotic responses including CCN2/connective tissue growth factor, fibronectin and jagged-1 expression were reduced following stimulation with TGF-ß1. ?mts-IHG-1 modulation of TGF-ß1 signaling was associated with increased Smad7 protein expression. ?mts-IHG-1 modulated TGF-ß1 activity by increasing Smad7 protein expression as it failed to inhibit TGF-ß1 transcriptional responses when endogenous Smad7 expression was knocked down. These data indicate that mitochondria modulate TGF-ß1 signal transduction and that IHG-1 is a key player in this modulation.

IHG-1 promotes mitochondrial biogenesis by stabilizing PGC-1α

Increased expression of Induced-by-High-Glucose 1 (IHG-1) associates with tubulointerstitial fibrosis in diabetic nephropathy. IHG-1 amplifies TGF-ß1 signaling, but the functions of this highly-conserved protein are not well understood. IHG-1 contains a putative mitochondrial-localization domain, and here we report that IHG-1 is specifically localized to mitochondria. IHG-1 overexpression increased mitochondrial mass and stabilized peroxisome proliferator-activated receptor ? coactivator-1a (PGC-1a). Conversely, inhibition of IHG-1 expression decreased mitochondrial mass, downregulated mitochondrial proteins, and PGC-1a-regulated transcription factors, including nuclear respiratory factor 1 and mitochondrial transcription factor A (TFAM), and reduced activity of the TFAM promoter. In the unilateral ureteral obstruction model, we observed higher PGC-1a protein expression and IHG-1 levels with fibrosis. In a gene-expression database, we noted that renal biopsies of human diabetic nephropathy demonstrated higher expression of genes encoding key mitochondrial proteins, including cytochrome c and manganese superoxide dismutase, compared with control biopsies. In summary, these data suggest that IHG-1 increases mitochondrial biogenesis by promoting PGC-1a-dependent processes, potentially contributing to the pathogenesis of renal fibrosis.

A Neutron Diffraction and Molecular Dynamics Investigation of Acetate-based Ionic Liquids as Solvents for Glucose

The liquid state structure of the ionic liquid, 1-ethyl-3-methylimidazolium acetate, and the solute/solvent structure of glucose dissolved in the ionic liquid at a 1: 6 molar ratio have been investigated at 323 K by molecular dynamics simulations and neutron diffraction experiments using H/D isotopically substituted materials. Interactions between hydrogen-bond donating cation sites and polar, directional hydrogen-bond accepting acetate anions are examined. Ion-ion radial distribution functions for the neat ionic liquid, calculated from both MD and derived from the empirical potential structure refinement model to the experimental data, show the alternating shell-structure of anions around the cation, as anticipated. Spatial probability distributions reveal the main anion-to-cation features as in-plane interactions of anions with imidazolium ring hydrogens and cation-cation planar stacking. Interestingly, the presence of the polarised hydrogen-bond acceptor anion leads to increased anion-anion tail-tail structuring within each anion shell, indicating the onset of hydrophobic regions within the anion regions of the liquid.

Dose-dependent effects of the once-daily GLP-1 receptor agonist lixisenatide in patients with Type 2 diabetes inadequately controlled with metformin:a randomized, double-blind, placebo-controlled trial

To evaluate the dose-response relationship of lixisenatide (AVE0010), a glucagon-like peptide-1 (GLP-1) receptor agonist, in metformin-treated patients with Type 2 diabetes.

Modern-day clinical course of type 1 diabetes mellitus after 30 years' duration:the diabetes control and complications trial/epidemiology of diabetes interventions and complications and Pittsburgh epidemiology of diabetes complications experience (1983-2005)

Clinical treatment goals of type 1 diabetes mellitus (T1DM) have changed since the Diabetes Control and Complications Trial (DCCT) demonstrated reduced long-term complications with intensive diabetes therapy. There have been few longitudinal studies to describe the clinical course of T1DM in the age of intensive therapy. Our objective was to describe the current-day clinical course of T1DM.

Coated-platelet levels in patients with Type 1 and with Type 2 diabetes mellitus

Coated-platelet levels were quantified in 58 people with Type 1 diabetes, 90 with Type 2 diabetes, and 54 non-diabetic controls. In diabetes high coated-platelet levels were related to smoking and glucose control drugs, but not to glycaemia or other drugs. Prospective studies should evaluate coated-platelets and complications and drug effects.

In vivo glycated low-density lipoprotein is not more susceptible to oxidation than nonglycated low-density lipoprotein in type 1 diabetes

It has been suggested that low-density lipoprotein (LDL) modified by glycation may be more susceptible to oxidation and thus, enhance its atherogenicity. Using affinity chromatography, LDL glycated in vivo (G-LDL) and relatively nonglycated. (N-LDL) subfractions can be isolated from the same individual. The extent of and susceptibility to oxidation of N-LDL compared with G-LDL was determined in 15 type 1 diabetic patients. Total LDL was isolated and separated by boronate affinity chromatography into relatively glycated (G-) and nonglycated (N-) subfractions. The extent of glycation, glycoxidation, and lipoxidation, lipid soluble antioxidant content, susceptibility to in vitro oxidation, and nuclear magnetic resonance (NMR)-determined particle size and subclass distribution were determined for each subfraction. Glycation, (fructose-lysine) was higher in G-LDL versus N-LDL, (0.28 +/- 0.08 v 0.13 +/- 0.04 mmol/mol lysine, P <.0001). However, levels of glycoxidation/lipoxidation products and of antioxidants were similar or lower in G-LDL compared with N-LDL and were inversely correlated with fructose-lysine (FL) concentrations in G-LDL, but positively correlated in N-LDL. In vitro LDL (CuCl2) oxidation demonstrated a longer lag time for oxidation of G-LDL than N-LDL (50 +/- 0.16 v 37 +/- 0.15 min, P <.01), but there was no difference in the rate or extent of lipid oxidation, nor in any aspect of protein oxidation. Mean LDL particle size and subclass distribution did not differ between G-LDL and N-LDL. Thus, G-LDL from well-controlled type 1 diabetic patients is not more modified by oxidation, more susceptible to oxidation, or smaller than relatively N-LDL, suggesting alternative factors may contribute to the atherogenicity of LDL from type 1 diabetic patients.

Glycosylation of low density lipoprotein in patients with type 1 (insulin-dependent) diabetes:correlations with other parameters of glycaemic control

Glycosylation of low density lipoproteins obtained from 16 patients with Type 1 (insulin-dependent) diabetes and from 16 age-, sex-, and race-matched controls, was determined. The diabetic patients were normolipaemic and were in good or fair glycaemic control. Eleven patients performed home blood glucose monitoring. Glycosylation of low density lipoproteins in the diabetic patients was significantly higher (p less than 0.001) than in the control subjects, and was significantly correlated with haemoglobin A1c, (p less than 0.01), glycosylation of plasma proteins, (p less than 0.001), and mean home blood glucose, (p less than 0.01). This study confirms that, in diabetic patients, increased glycosylation of low density lipoprotein occurs to an extent which correlates closely with other commonly used indices of glycaemic control.

Effects of D- and L-glucose and mannitol on retinal capillary cells:Inhibition by nanomolar aminoguanidine

Hyperglycemia may contribute directly to pericyte loss and capillary leakage in early diabetic retinopathy. To elucidate relative contributions of glycation, glycoxidation, sugar autoxidation, osmotic stress and metabolic effects in glucose-mediated capillary damage, we tested the effects of D-glucose, L-glucose, mannitol and the potentially protective effects of aminoguanidine on cultured bovine retinal capillary pericytes and endothelial cells. Media (containing 5 mM D-glucose) were supplemented to increase the concentration of each sugar by 5, 10, or 20 mM. Subconfluent pericytes and endothelial cells were exposed to the supplemented media in the presence or absence of aminoguanidine (1 nM-100 µM) for three days. Cell counts, viability and protein were determined. For both cell types, all three sugars produced concentration-dependent decreases in cell counts and protein content (p

Grape-seed procyanidins modulate cellular membrane potential and nutrient-induced GLP-1 secretion in STC-1 cells

Grape-seed procyanidins (GSPE) modulate glucose homeostasis and it was suggested that GSPE may achieve this by enhancing the secretion of incretin hormones such as glucagon-like peptide-1 (GLP-1). Therefore, the aim of the present study is to examine in detail the effects of GSPE on intestinal endocrine cells (STC-1). GSPE was found to modulate plasma membrane potential in enteroendocrine cells, inducing depolarization at low concentrations (0.05 mg/L) and hyperpolarization at high concentrations (50 mg/L), and surprisingly this was also accompanied by suppressed GLP-1 secretion. Furthermore, how GSPE affects STC-1 cells under nutrient-stimulated conditions (i.e. glucose, linoleic acid and L-proline) was also explored, and we found that the higher GSPE concentration was effective in limiting membrane depolarization and reducing GLP-1 secretion. Next, it was also examined whether GSPE affected mitochondrial membrane potential, finding that this too is altered by GSPE, however this does not appear to explain the observed effects on plasma membrane potential and GLP-1 secretion. In conclusion, our results show that grape-seed procyanidins modulate cellular membrane potential and nutrient-induced enteroendocrine hormone secretion in STC-1 cells.

Genetic examination of SETD7 and SUV39H1/H2 methyltransferases and the risk of diabetes complications in patients with type 1 diabetes

Hyperglycemia plays a pivotal role in the development and progression of vascular complications, which are the major sources of morbidity and mortality in diabetes. Furthermore, these vascular complications often persist and progress despite improved glucose control, possibly as a result of prior episodes of hyperglycemia. Epigenetic modifications mediated by histone methyltransferases are associated with gene-activating events that promote enhanced expression of key proinflammatory molecules implicated in vascular injury. In this study, we investigated genetic polymorphisms of the SETD7, SUV39H1, and SUV39H2 methyltransferases as predictors of risk for micro- and macrovascular complications in type 1 diabetes.

Selective Targeting of Glucagon-Like Peptide-1 Signalling As a Novel Therapeutic Approach for Cardiovascular Disease in Diabetes

Glucagon-like peptide-1 (GLP-1) is an incretin hormone whose glucose-dependent insulinotropic actions have been harnessed as a novel therapy for glycaemic control in type 2 diabetes. Although it has been known for some time that the GLP-1 receptor is expressed in the cardiovascular system where it mediates important physiological actions, it is only recently that specific cardiovascular effects of GLP-1 in the setting of diabetes have been described. GLP-1 confers indirect benefits in cardiovascular disease (CVD) under both normal and hyperglycaemic conditions via reducing established risk factors, such as hypertension, dyslipidaemia and obesity, which are markedly increased in diabetes. Emerging evidence indicates that GLP-1 also exerts direct effects on specific aspects of diabetic CVD, such as endothelial dysfunction, inflammation, angiogenesis and adverse cardiac remodelling. However, the majority of studies have employed experimental models of diabetic CVD and information on the effects of GLP-1 in the clinical setting are limited although several large-scale trials are ongoing. It is clearly important to gain a detailed knowledge of the cardiovascular actions of GLP-1 in diabetes given the large number of patients currently receiving GLP-1 based therapies. This review will therefore discuss current understanding of the effects of GLP-1 on both cardiovascular risk factors in diabetes and direct actions on the heart and vasculature in this setting, and the evidence implicating specific targeting of GLP-1 as a novel therapy for CVD in diabetes.