Label-free CARS microscopy reveals similar triacylglycerol acyl chain length and saturation in myocellular lipid droplets of athletes and individuals with type 2 diabetes

Aims/hypothesis: Intramyocellular lipid (IMCL) content associates with development of insulin resistance, albeit not in insulinsensitive endurance-trained athletes (trained). Qualitative and spatial differences in muscle lipid composition may underlie this so-called athlete’s paradox. Here we studied triacylglycerol (TAG) composition of individual myocellular lipid droplets (LDs) in trained individuals and individuals with type 2 diabetes mellitus.Methods: Trained (˙V O2max 71.0 \ub1 1.6 ml O2 [kg lean body mass (LBM)]−1 min−1), normoglycaemic (fasting glucose 5.1 \ub1 0.1 mmol/l) individuals and untrained (V O2max 36.8 \ub1 1.5 ml O2 [kg LBM]−1 min−1) individuals with type 2 diabetes (fasting glucose 7.4 \ub1 0.5 mmol/l), with similar IMCL content (3.5 \ub1 0.7% vs 2.5 \ub1 0.3%, p = 0.241), but at opposite ends of the insulin sensitivity spectrum(glucose infusion rate 93.8 \ub1 6.6 vs 25.7 \ub1 5.3 μmol [kg LBM]−1 min−1 for trained individuals and those with type 2 diabetes, respectively) were included from our database in the present study. We applied in situ label-free broadbandcoherent anti-Stokes Raman scattering (CARS) microscopy to sections from skeletal muscle biopsies to measure TAG acyl chain length and saturation of myocellular LDs. This approach uniquely permits examination of individual LDs in their native environment, in a fibre-type-specific manner, taking into account LD size and subcellular location.Results: Despite a significant difference in insulin sensitivity, we observed remarkably similar acyl chain length and saturation in trained and type 2 diabetic individuals (chain length: 18.12 \ub1 0.61 vs 18.36 \ub1 0.43 number of carbons; saturation: 0.37 \ub1 0.05 vs 0.38 \ub1 0.06 number of C=C bonds). Longer acyl chains or higher saturation (lower C=C number) could be detected in subpopulations of LDs, i.e. large LDs (chain length: 18.11 \ub1 0.48 vs 18.63 \ub1 0.57 carbon number) and subsarcolemmal LDs (saturation: 0.34 \ub1 0.02 vs 0.36 \ub1 0.04 C=C number), which are more abundant in individuals with type 2 diabetes.Conclusions/interpretation: In contrast to reports of profound differences in the lipid composition of lipids extracted from skeletal muscle from trained and type 2 diabetic individuals, our in situ, LD-specific approach detected only modest differences in TAGcomposition in LD subpopulations, which were dependent on LD size and subcellular location. If, and to what extent, these modest differences can impact insulin sensitivity remains to be elucidated

Applied system analysis and technology of designing the software

An analogy is made between the stages of applied system analysis and the stages of software Engineerin

Is Endothelial Nitric Oxide Synthase a Moonlighting Protein Whose Day Job is Cholesterol Sulfate Synthesis? Implications for Cholesterol Transport, Diabetes and Cardiovascular Disease

Theoretical inferences, based on biophysical, biochemical, and biosemiotic considerations, are related here to the pathogenesis of cardiovascular disease, diabetes, and other degenerative conditions. We suggest that the “daytime” job of endothelial nitric oxide synthase (eNOS), when sunlight is available, is to catalyze sulfate production. There is a striking alignment between cell types that produce either cholesterol sulfate or sulfated polysaccharides and those that contain eNOS. The signaling gas, nitric oxide, a well-known product of eNOS, produces pathological effects not shared by hydrogen sulfide, a sulfur-based signaling gas. We propose that sulfate plays an essential role in HDL-A1 cholesterol trafficking and in sulfation of heparan sulfate proteoglycans (HSPGs), both critical to lysosomal recycling (or disposal) of cellular debris. HSPGs are also crucial in glucose metabolism, protecting against diabetes, and in maintaining blood colloidal suspension and capillary flow, through systems dependent on water-structuring properties of sulfate, an anionic kosmotrope. When sunlight exposure is insufficient, lipids accumulate in the atheroma in order to supply cholesterol and sulfate to the heart, using a process that depends upon inflammation. The inevitable conclusion is that dietary sulfur and adequate sunlight can help prevent heart disease, diabetes, and other disease conditions

A novel phosphoprotein analysis scheme for assessing changes in premalignant and malignant breast cell lines using 2-D liquid separations, protein microarrays, and tandem mass spectrometry

An analysis of phosphorylation changes that occur during cancer progression would provide insights into the molecular pathways responsible for a malignant phenotype. In this study we employed a novel coupling of 2-D liquid separations and protein microarray technology to reveal changes in phosphoprotein status between premalignant (AT1) and malignant (CA1a) cell lines derived from the human MCF10A breast cell lines. Intact proteins were first separated according to their p I and hydrophobicities, then arrayed on SuperAmine glass slides. Phosphoproteins were detected using the universal, inorganic phospho-sensor dye, Pro-Q Diamond. Using this dye, out of 140 spots that were positive for phosphorylation, a total of 85 differentially expressed spots were detected over a pH range of 7.2–4.0. Proteins were identified and their peptides sequenced by MS. The strategy enabled the identification of 75 differentially expressed phosphoproteins, from which 51 phosphorylation sites in 27 unique proteins were confirmed. Interestingly, the majority of differentially expressed phosphorylated proteins observed were nuclear proteins. Three regulators of apoptosis, Bad, Bax, and Acinus, were also differentially phosphorylated in the two cell lines. Further development of this strategy will facilitate an understanding of the mechanisms involved in malignancy progression and other disease-related phenotypes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61541/1/51_ftp.pd

The role of acid-base imbalance in statin-induced myotoxicity

Disturbances in acid-base balance, such as acidosis and alkalosis, have potential to alter the pharmacological and toxicological outcomes of statin therapy. Statins are commonly prescribed for elderly patients who have multiple co-morbidities such as diabetes mellitus, cardiovascular and renal diseases. These patients are at risk of developing acid-base imbalance. In the present study, the effect of disturbances in acid-base balance on the inter-conversion of simvastatin and pravastatin between lactone and hydroxy acid forms have been investigated in physiological buffers, human plasma and cell culture medium over pH ranging from 6.8 to 7.8. The effects of such inter-conversion on cellular uptake and myotoxicity of statins were assessed in vitro using C2C12 skeletal muscle cells under conditions relevant to acidosis, alkalosis and physiological pH. Results indicate that the conversion of the lactone forms of simvastatin and pravastatin to the corresponding hydroxy acid is strongly pH-dependent. At physiological and alkaline pH, substantial proportions of simvastatin lactone (~ 87% and 99%, respectively) and pravastatin lactone (~ 98% and 99%, respectively) were converted to the active hydroxy acid forms after 24 hours of incubation at 37 °C. At acidic pH, conversion occurs to a lower extent, resulting in greater proportion of statin remaining in the more lipophilic lactone form. However, pH alteration did not influence the conversion of the hydroxy acid forms of simvastatin and pravastatin to the corresponding lactones. Furthermore, acidosis has been shown to hinder the metabolism of the lactone form of statins by inhibiting hepatic microsomal enzyme activities. Lipophilic simvastatin lactone was found to be more cytotoxic to undifferentiated and differentiated skeletal muscle cells compared to more hydrophilic simvastatin hydroxy acid, pravastatin lactone and pravastatin hydroxy acid. Enhanced cytotoxicity of statins was observed under acidic conditions and is attributed to increased cellular uptake of the more lipophilic lactone or unionized hydroxy acid form. Consequently, our results suggest that co-morbidities associated with acid-base imbalance can play a substantial role in the development and potentiation of statin-induced myotoxicity

Non-traditional platinum compounds for improved accumulation, oral bioavailability, and tumor targeting

The five platinum anticancer compounds currently in clinical use conform to structure–activity relationships formulated (M. J. Cleare and J. D. Hoeschele, Bioinorg. Chem., 1973, 2, 187–210) shortly after the discovery that cis-diamminedichloroplatinum(II), cisplatin, has antitumor activity in mice. These compounds are neutral platinum(II) species with two am(m)ine ligands or one bidentate chelating diamine and two additional ligands that can be replaced by water through aquation reactions. The resulting cations ultimately form bifunctional adducts on DNA. Information about the chemistry of these platinum compounds and correlations of their structures with anticancer activity have provided guidance for the design of novel anticancer drug candidates based on the proposed mechanisms of action. This article discusses advances in the synthesis and evaluation of such non-traditional platinum compounds, including cationic and tumor-targeting constructs.National Cancer Institute (U.S.) (Grant CA34992

Mechanisms of Endothelial Dysfunction in Resistance Arteries from Patients with End-Stage Renal Disease

The study focuses on the mechanisms of endothelial dysfunction in the uremic milieu. Subcutaneous resistance arteries from 35 end-stage renal disease (ESRD) patients and 28 matched controls were studied ex-vivo. Basal and receptor-dependent effects of endothelium-derived factors, expression of endothelial NO synthase (eNOS), prerequisites for myoendothelial gap junctions (MEGJ), and associations between endothelium-dependent responses and plasma levels of endothelial dysfunction markers were assessed. The contribution of endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxation was impaired in uremic arteries after stimulation with bradykinin, but not acetylcholine, reflecting the agonist-specific differences. Diminished vasodilator influences of the endothelium on basal tone and enhanced plasma levels of asymmetrical dimethyl L-arginine (ADMA) suggest impairment in NO-mediated regulation of uremic arteries. eNOS expression and contribution of MEGJs to EDHF type responses were unaltered. Plasma levels of ADMA were negatively associated with endothelium-dependent responses in uremic arteries. Preserved responses of smooth muscle to pinacidil and NO-donor indicate alterations within the endothelium and tolerance of vasodilator mechanisms to the uremic retention products at the level of smooth muscle. We conclude that both EDHF and NO pathways that control resistance artery tone are impaired in the uremic milieu. For the first time, we validate the alterations in EDHF type responses linked to kinin receptors in ESRD patients. The association between plasma ADMA concentrations and endothelial function in uremic resistance vasculature may have diagnostic and future therapeutic implications

The status of platinum anticancer drugs in the clinic and in clinical trials

Since its approval in 1979 cisplatin has become an important component in chemotherapy regimes for the treatment of ovarian, testicular, lung and bladder cancers, as well as lymphomas, myelomas and melanoma. Unfortunately its continued use is greatly limited by severe dose limiting side effects and intrinsic or acquired drug resistance. Over the last 30 years, 23 other platinum-based drugs have entered clinical trials with only two (carboplatin and oxaliplatin) of these gaining international marketing approval, and another three (nedaplatin, lobaplatin and heptaplatin) gaining approval in individual nations. During this time there have been more failures than successes with the development of 14 drugs being halted during clinical trials. Currently there are four drugs in the various phases of clinical trial (satraplatin, picoplatin, LipoplatinTM and ProLindacTM). No new small molecule platinum drug has entered clinical trials since 1999 which is representative of a shift in focus away from drug design and towards drug delivery in the last decade. In this perspective article we update the status of platinum anticancer drugs currently approved for use, those undergoing clinical trials and those discontinued during clinical trials, and discuss the results in the context of where we believe the field will develop over the next decade