Palmitate induces insulin resistance in monocytes and increases expression of the integrin CD11b

Obesity and insulin resistance are important risk factors for atherosclerosis, and elevated level of plasma NEFA is a common feature in individuals with obesity and insulin resistance. Palmitate, one of the most abundant non-esterified SFA in plasma, has been reported to induce insulin resistance in adipose tissues and skeletal muscles and to cause an increased inflammatory response in monocytes. The present study investigated whether palmitate can induce insulin resistance in monocytes and its effect on monocyte adhesion molecular expression (CD11b). Insulin resistance was measured by in vitro uptake of insulin-stimulated 3H-labelled 2-deoxy-D-glucose into THP-1 cells, cell surface CD11b expression was measured by flow cytometry. The data showed that palmitate-induced insulin resistance in THP-1 monocytes was concentration and time dependent (Figure 1). The insulin-stimulated glucose uptake was significantly decreased in cells treated with 300 mM-palmitate compared with control cells (P<0.001) and was observed within 6 h, but was not a result of palmitate toxicity. There was no significant increase in caspase 3 activation (P>0.05). Treatment with 300 mM-palmitate for 24 h also caused a significant increase in surface CD11b expression in both U937 and THP-1 monocytic cell lines and human primary monocytes compared with the control (P<0.001). Both these effects were inhibited by co-incubation with Fumonisin B1, an inhibitor of de novo ceramide synthesis. In conclusion, these data show that palmitate, at physiological concentrations, can cause insulin resistance in monocytes and increase monocyte surface integrin CD11b expression, which is in part the result of the synthesis of ceramide.

Antibiotic and biocide resistance in Salmonella enterica and Escherichia coli 0157

Bacterial resistance to antibiotics and biocides is a prevalent problem, which may be exacerbated by the commonplace and often unnecessary inclusion of biocides into domestic products. Addition of antimicrobials, to domestic disinfectants has raised concern about promoting microbial resistance and potential cross-resistance to therapeutic antibiotics. This study investigated the potential for resistance in Salmonella enterica serovars Enteritidis, Typhimurium, Virchow and Escherichia call 0157 to commonly used biocides, to identify mechanisms underlying resistance and whether these provided cross-resistance to antibiotics. Salmonella enterica and E. coli 0157 strains were serially exposed to sub-inhibitory. concentrations of erythromycin (ERY), benzalkonium chloride (BKC), chlorhexidine hydrochloride (CHX)and triclosan (TLN). Once resistance was achieved permeability changes in the outer membrane, including LPS, cell surface charge and hydrophobicityand the presence of,an active efflux were investigated as possible resistance candidates. Thin layer chromatography (TLC) and Gas chromatography (GC) were carried out to examine fatty acid and lipid changes in E. coli 0157 isolates with reduced susceptibility to TLN. Cross-resistance was studied by the Stoke's method and standard microdilution assays. Examination of the outer membrane proteins and LPS did not reveal any significant changes between parent and resistant strains. The hydrophobicity of the cells increased as the cells were passaged and became less. susceptible. An active efflux system was the most likely mechanism of resistance in all strains tested and a fab1 mutation was associated with E. coli 0157 resistant to TLN isolates. In all isolates investigated the resistance was stable for over 30 passages in biocide-free media. A high degree of cross-resistance was obtained in TLN-resjstant Escherichia coli 0157 strains, which repeatedly exerted decreased susceptibility to various antimicrobials, including chloramphenicol, erythromycin, imipenem, tetracycline and trimethoprirn:, as well as to various biocides. The results of this laboratory-based investigation suggest that it is possible for microorganisms to become resistant to biocides when repeatedly exposed to sublethal concentrations. This may be especially the case in the domestic environment where administration of biocides is poorly controlled. Eventually it could lead to the undesirable situation of resident strains becoming resistant to disinfection and cross resistant to other antimicrobials.

Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.

Decreased efficiency of trypsinization of cells following photodynamic therapy:evaluation of a role for tissue transglutaminase

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cellular damage are to be fully understood. The relationship between sensitizer, fluence rate and the removal of cells by trypsinization was studied using the RIF-1 cell line. Following treatment of RIF-1 cells with pyridinium zinc (II) phthalocyanine (PPC), or polyhaematoporphyrin at 10 mW cm−2 (3 J cm−2), there was a significant number of cells that were not removed by trypsin incubation compared to controls. Decreasing the fluence rate from 10 to 2.5 mW cm−2 resulted in a two-fold increase in the number of cells attached to the substratum when PPC used as sensitizer; however, with 5,10,15,20 meso-tetra(hydroxyphenyl) chlorin (m-THPC) there was no resistance to trypsinization following treatment at either fluence rate. The results indicate that resistance of cells to trypsinization following PDT is likely to be both sensitizer and fluence rate dependent. Increased activity of the enzyme tissue-transglutaminase (tTGase) was observed following PPC-PDT, but not following m-THPC-PDT. Similar results were obtained using HT29 human colonic carcinoma and ECV304 human umbilical vein endothelial cell lines. Hamster fibrosarcoma cell (Met B) clones transfected with human tTGase also exhibited resistance to trypsinization following PPC-mediated photosensitization; however, a similar degree of resistance was observed in PDT-treated control Met B cells suggesting that tTGase activity alone was not involved in this process.

Glucosamine-induced insulin resistance in L6 muscle cells

Background: Glucosamine increases flux through the hexosamine pathway, causing insulin resistance and disturbances similar to diabetic glucose toxicity. Aim: This study examines the effect of glucosamine on glucose uptake by cultured L6 muscle cells as a model of insulin resistance. Methods: Glucose uptake by L6 myotubes was measured using the non-metabolized glucose analogue 2-deoxy-D-glucose after incubation with glucosamine for 4 and 24 h, with and without insulin and several other agents (metformin, peroxovanadium and D-pinitol) that improve glucose uptake in diabetic states. Results: After 4 h, high concentrations of glucosamine (5 × 10-3 and 10-2 M) reduced basal and insulin-stimulated glucose uptake by up to 50%. After 24 h, the effect of insulin was completely abolished by 10-2 M glucosamine and reduced over 50% by 5 × 10-3 M glucosamine. Lower concentrations of glucosamine did not significantly alter glucose uptake. The effect of glucosamine could not be attributed to cytotoxicity assessed by the Trypan Blue test. Metformin, peroxovanadium and D-pinitol, each of which increased glucose uptake by L6 cells, did not prevent the decrease in glucose uptake with glucosamine. Conclusion: Glucosamine decreased insulin-stimulated glucose uptake by L6 muscle cells, providing a potential model of insulin resistance with similarities to glucose toxicity. Insulin resistance induced by glucosamine was not reversed by three agents (metformin, peroxovanadium and D-pinitol) known to enhance or partially mimic the effects of insulin. © 2004 Blackwell Publishing Ltd.

Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents

The mechanisms by which bacteria resist killing by antibiotics and biocides are still poorly defined, although repeated exposure to sublethal concentrations of antibacterial agents undoubtedly contributes to their development. This study aimed both to investigate the potential of Salmonella enterica and Escherichia coli O157 for adaptive resistance to commonly used biocides and to determine any cross-resistance to antibiotics. Strains were repeatedly passaged in media containing increasing concentrations of a biocide or antibiotic until adaptive resistance was obtained. A wide panel of antimicrobial agents was then screened by using the adapted strain to determine cross-resistance, if any. Adaptive resistance was readily achieved for both S. enterica and E. coli O157. Cross-resistance in adaptively resistant S. enterica varied with the serotype; Salmonella enterica serovar Enteritidis expressed cross-resistance to chloramphenicol, whereas Salmonella enterica serovar Typhimurium expressed cross-resistance to chlorhexidine. Benzalkonium chloride-resistant Salmonella enterica serovar Virchow showed elevated resistance to chlorhexidine; however, chlorhexidine-resistant Salmonella serovar Virchow did not demonstrate reciprocal cross-resistance to benzalkonium chloride, suggesting specific rather than generic resistance mechanisms. E. coli O157 strains acquired high levels of resistance to triclosan after only two sublethal exposures and, when adapted, repeatedly demonstrated decreased susceptibilities to various antimicrobial agents, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. These observations raise concern over the indiscriminate and often inappropriate use of biocides, especially triclosan, in situations where they are unnecessary, whereby they may contribute to the development of microbial resistance mechanisms.