A Complex Extracellular Sphingomyelinase of Pseudomonas aeruginosa Inhibits Angiogenesis by Selective Cytotoxicity to Endothelial Cells

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy)

Purification of the pancreatic cholecystokinin receptor

We have previously shown that the pancreatic cholecystokinin (CCK) receptor can be solubilized in 1% digitonin. In this study, digitonin-solubilized CCK receptors from rat pancreas were purified using sequential affinity chromatography on ricin-II agarose and on AffiGel-CCK. Electrophoresis of the radioiodinated purified receptors on SDS-polyacrylamide gels followed by autoradiography revealed two proteins: a major band of Mr = 80,000-90,000, and a minor band of Mr = 55,000. Through the purification procedure, the receptors preserved their agonist specificity (CCK-8 Kd = 9.4 nM. The estimated purification was about 80,000 fold and consistent with the expected Bmax for a pure Mr = 80,000 protein binding one CCK molecule. This two-step purification procedure opens the possibility for molecular studies of the CCK receptor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28032/1/0000471.pd

The impact of salsalate treatment on serum levels of advanced glycation end products in type 2 diabetes.

OBJECTIVE Salsalate is a nonacetylated salicylate that lowers glucose levels in people with type 2 diabetes (T2D). Here we examined whether salsalate also lowered serum-protein-bound levels of early and advanced glycation end products (AGEs) that have been implicated in diabetic vascular complications. RESEARCH DESIGN AND METHODS Participants were from the Targeting Inflammation Using Salsalate for Type 2 Diabetes (TINSAL-T2D) study, which examined the impact of salsalate treatment on hemoglobin A1c (HbA1c) and a wide variety of other parameters. One hundred eighteen participants received salsalate, 3.5 g/day for 48 weeks, and 109 received placebo. Early glycation product levels (HbA1c and fructoselysine [measured as furosine]) and AGE levels (glyoxal and methylglyoxal hydroimidazolones [G-(1)H, MG-(1)H], carboxymethyllysine [CML], carboxyethyllysine [CEL], pentosidine) were measured in patient serum samples. RESULTS Forty-eight weeks of salsalate treatment lowered levels of HbA1c and serum furosine (P \u3c 0.001) and CML compared with placebo. The AGEs CEL and G-(1)H and MG-(1)H levels were unchanged, whereas pentosidine levels increased more than twofold (P \u3c 0.001). Among salsalate users, increases in adiponectin levels were associated with lower HbA1c levels during follow-up (P \u3c 0.001). Changes in renal and inflammation factor levels were not associated with changes in levels of early or late glycation factors. Pentosidine level changes were unrelated to changes in levels of renal function, inflammation, or cytokines. CONCLUSIONS Salsalate therapy was associated with a reduction in early but not late glycation end products. There was a paradoxical increase in serum pentosidine levels suggestive of an increase in oxidative stress or decreased clearance of pentosidine precursor

Dietary betaine supplementation increases Fgf21 levels to improve glucose homeostasis and reduce hepatic lipid accumulation in mice

Identifying markers of human insulin resistance may permit development of new approaches for treatment and prevention of type 2 diabetes. To this end, we analyzed the fasting plasma metabolome in metabolically characterized human volunteers across a spectrum of insulin resistance. We demonstrate that plasma betaine levels are reduced in insulin-resistant humans and correlate closely with insulin sensitivity. Moreover, betaine administration to mice with diet-induced obesity prevents the development of impaired glucose homeostasis, reduces hepatic lipid accumulation, increases white adipose oxidative capacity, and enhances whole-body energy expenditure. In parallel with these beneficial metabolic effects, betaine supplementation robustly increased hepatic and circulating fibroblast growth factor (Fgf)21 levels. Betaine administration failed to improve glucose homeostasis and liver fat content in Fgf21(-/-) mice, demonstrating that Fgf21 is necessary for betaine's beneficial effects. Together, these data indicate that dietary betaine increases Fgf21 levels to improve metabolic health in mice and suggest that betaine supplementation merits further investigation as a supplement for treatment or prevention of type 2 diabetes in humans

Detailed dimethylacetal and fatty acid composition of rumen content from lambs fed lucerne or concentrate supplemented with soybean oil

Articles in International JournalsLipid metabolism in the rumen is responsible for the complex fatty acid profile of rumen outflow compared with the dietary fatty acid composition, contributing to the lipid profile of ruminant products. A method for the detailed dimethylacetal and fatty acid analysis of rumen contents was developed and applied to rumen content collected from lambs fed lucerne or concentrate based diets supplemented with soybean oil. The methodological approach developed consisted on a basic/ acid direct transesterification followed by thin-layer chromatography to isolate fatty acid methyl esters from dimethylacetal, oxo- fatty acid and fatty acid dimethylesters. The dimethylacetal composition was quite similar to the fatty acid composition, presenting even-, odd- and branched-chain structures. Total and individual odd- and branched-chain dimethylacetals were mostly affected by basal diet. The presence of 18:1 dimethylacetals indicates that biohydrogenation intermediates might be incorporated in structural microbial lipids. Moreover, medium-chain fatty acid dimethylesters were identified for the first time in the rumen content despite their concentration being relatively low. The fatty acids containing 18 carbon-chain lengths comprise the majority of the fatty acids present in the rumen content, most of them being biohydrogenation intermediates of 18:2n26 and 18:3n23. Additionally, three oxo- fatty acids were identified in rumen samples, and 16-O-18:0 might be produced during biohydrogenation of the 18:3n23

Co-expressed immune and metabolic genes in visceral and subcutaneous adipose tissue from severely obese individuals are associated with plasma HDL and glucose levels: a microarray study

<p>Abstract</p> <p>Background</p> <p>Excessive accumulation of body fat, in particular in the visceral fat depot, is a major risk factor to develop a variety of diseases such as type 2 diabetes. The mechanisms underlying the increased risk of obese individuals to develop co-morbid diseases are largely unclear.</p> <p>We aimed to identify genes expressed in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) that are related to blood parameters involved in obesity co-morbidity, such as plasma lipid and glucose levels, and to compare gene expression between the fat depots.</p> <p>Methods</p> <p>Whole-transcriptome SAT and VAT gene expression levels were determined in 75 individuals with a BMI >35 kg/m². Modules of co-expressed genes likely to be functionally related were identified and correlated with BMI, plasma levels of glucose, insulin, HbA_1c, triglycerides, non-esterified fatty acids, ALAT, ASAT, C-reactive protein, and LDL- and HDL cholesterol.</p> <p>Results</p> <p>Of the approximately 70 modules identified in SAT and VAT, three SAT modules were inversely associated with plasma HDL-cholesterol levels, and a fourth module was inversely associated with both plasma glucose and plasma triglyceride levels (p < 5.33 × 10^-5). These modules were markedly enriched in immune and metabolic genes. In VAT, one module was associated with both BMI and insulin, and another with plasma glucose (p < 4.64 × 10^-5). This module was also enriched in inflammatory genes and showed a marked overlap in gene content with the SAT modules related to HDL. Several genes differentially expressed in SAT and VAT were identified.</p> <p>Conclusions</p> <p>In obese subjects, groups of co-expressed genes were identified that correlated with lipid and glucose metabolism parameters; they were enriched with immune genes. A number of genes were identified of which the expression in SAT correlated with plasma HDL cholesterol, while their expression in VAT correlated with plasma glucose. This underlines both the singular importance of these genes for lipid and glucose metabolism and the specific roles of these two fat depots in this respect.</p