Deprivation of the periplasmic chaperone SurA reduces virulence and restores antibiotic susceptibility of multidrug-resistant pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. Therefore, novel strategies for therapy are urgently required. The outer membrane composition of Gram-negative pathogens and especially of Pa restricts the efficacy of antibiotic entry into the cell and determines virulence. For efficient outer membrane protein biogenesis, the β-barrel assembly machinery (BAM) complex in the outer membrane and periplasmic chaperones like Skp and SurA are crucial. Previous studies indicated that the importance of individual proteins involved in outer membrane protein biogenesis may vary between different Gram-negative species. In addition, since multidrug-resistant Pa strains pose a serious global threat, the interference with both virulence and antibiotic resistance by disturbing outer membrane protein biogenesis might be a new strategy to cope with this challenge. Therefore, deletion mutants of the non-essential BAM complex components bamB and bamC, of the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects for both traits were associated with reduced levels of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the depletion of SurA in a multidrug-resistant clinical bloodstream isolate re-sensitized the strain to antibiotic treatment. From our data we conclude that SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and re-sensitizes multidrug-resistant strains to antibiotics

Regulation of Calcium-Permeable TRPV2 Channel by Insulin in Pancreatic β-Cells

OBJECTIVE—Calcium-permeable cation channel TRPV2 is expressed in pancreatic β-cells. We investigated regulation and function of TRPV2 in β-cells

Inactivation of class II PI3K-C2 alpha induces leptin resistance, age-dependent insulin resistance and obesity in male mice

AIMS/HYPOTHESIS: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice. METHODS: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a). RESULTS: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age. CONCLUSIONS/INTERPRETATION: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis. ACCESS TO RESEARCH MATERIALS: All reagents are available upon request

Gene Silencing of Phogrin Unveils Its Essential Role in Glucose-Responsive Pancreatic β-Cell Growth

OBJECTIVE—Phogrin and IA-2, autoantigens in insulin-dependent diabetes, have been shown to be involved in insulin secretion in pancreatic β-cells; however, implications at a molecular level are confusing from experiment to experiment. We analyzed biological functions of phogrin in β-cells by an RNA interference technique

Nephrin Is Expressed on the Surface of Insulin Vesicles and Facilitates Glucose-Stimulated Insulin Release

Nephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic beta-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in beta-cell function. Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin-transfected cells was used to study nephrin endocytosis. Nephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin-transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release. Our data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes

GABA Coordinates with Insulin in Regulating Secretory Function in Pancreatic INS-1 β-Cells

Pancreatic islet β-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABAARs). We and others recently reported that islet β-cells also express GABAARs and that activation of GABAARs increases insulin release. Here we investigate the effects of insulin on the GABA-GABAAR system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 µM) suppressed the GABA-induced current (IGABA) by 43%. Zinc-free insulin also suppressed IGABA to the same extent of inhibition by regular insulin. The inhibition of IGABA occurs within 30 seconds after application of insulin. The insulin-induced inhibition of IGABA persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABAARs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 µM, p<0.01) and insulin (1 µM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABAARs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABAAR signaling presenting a feedback mechanism for fine-tuning β-cell secretion

In Vitro Cellular Adaptations of Indicators of Longevity in Response to Treatment with Serum Collected from Humans on Calorie Restricted Diets

Calorie restriction (CR) produces several health benefits and increases lifespan in many species. Studies suggest that alternate-day fasting (ADF) and exercise can also provide these benefits. Whether CR results in lifespan extension in humans is not known and a direct investigation is not feasible. However, phenotypes observed in CR animals when compared to ad libitum fed (AL) animals, including increased stress resistance and changes in protein expression, can be simulated in cells cultured with media supplemented with blood serum from CR and AL animals. Two pilot studies were undertaken to examine the effects of ADF and CR on indicators of health and longevity in humans. In this study, we used sera collected from those studies to culture human hepatoma cells and assessed the effects on growth, stress resistance and gene expression. Cells cultured in serum collected at the end of the dieting period were compared to cells cultured in serum collected at baseline (before the dieting period). Cells cultured in serum from ADF participants, showed a 20% increase in Sirt1 protein which correlated with reduced triglyceride levels. ADF serum also induced a 9% decrease in proliferation and a 25% increase in heat resistance. Cells cultured in serum from CR participants induced an increase in Sirt1 protein levels by 17% and a 30% increase in PGC-1α mRNA levels. This first in vitro study utilizing human serum to examine effects on markers of health and longevity in cultured cells resulted in increased stress resistance and an up-regulation of genes proposed to be indicators of increased longevity. The use of this in vitro technique may be helpful for predicting the potential of CR, ADF and other dietary manipulations to affect markers of longevity in humans

Energy metabolism, altered proteins, sirtuins and ageing: converging mechanisms?

The predominant molecular symptom of ageing is the accumulation of altered gene products. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Genetics has revealed that ageing may be controlled by changes in intracellular NAD/NADH ratio regulating sirtuin activity. Physiological and other approaches indicate that mitochondria may also regulate ageing. A mechanism is proposed which links diet, exercise and mitochondria-dependent changes in NAD/NADH ratio to intracellular generation of altered proteins. It is suggested that ad libitum feeding conditions decrease NAD availability which also decreases metabolism of the triose phosphate glycolytic intermediates, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, which can spontaneously decompose into methylglyoxal (MG). MG is a highly toxic glycating agent and a major source of protein advanced-glycosylation end-products (AGEs). MG and AGEs can induce mitochondrial dysfunction and formation of reactive oxygen species (ROS), as well as affect gene expression and intracellular signalling. In dietary restriction–induced fasting, NADH would be oxidised and NAD regenerated via mitochondrial action. This would not only activate sirtuins and extend lifespan but also suppress MG formation. This proposal can also explain the apparent paradox whereby increased aerobic activity suppresses formation of glycoxidized proteins and extends lifespan. Variation in mitochondrial DNA composition and consequent mutation rate, arising from dietary-controlled differences in DNA precursor ratios, could also contribute to tissue differences in age-related mitochondrial dysfunction

Altered Insulin Receptor Signalling and β-Cell Cycle Dynamics in Type 2 Diabetes Mellitus

Insulin resistance, reduced β-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced β-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in β-cells – which also exhibits reduced β-cell mass, the β-cell-specific insulin receptor knockout (βIRKO). Freshly isolated islets and β-cell lines derived from βIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in βIRKO β-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in β-cell growth. These data provide evidence that human β- and α-cells can enter the cell-cycle, but proliferation of β-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in β-cell-cycle progression are attractive therapeutic targets to enhance β-cell regeneration in the treatment of T2DM