John S. Gounarides

Metabolic Signatures Uncover Distinct Targets in Molecular Subsets of Diffuse Large B Cell Lymphoma

Molecular signatures have identified several subsets of diffuse large B cell lymphoma (DLBCL) and rational targets within the B cell receptor (BCR) signaling axis. The OxPhos-DLBCL subset, which harbors the signature of genes involved in mitochondrial metabolism, is insensitive to inhibition of BCR survival signaling but is functionally undefined. We show that, compared with BCR-DLBCLs, OxPhos-DLBCLs display enhanced mitochondrial energy transduction, greater incorporation of nutrient-derived carbons into the tricarboxylic acid cycle, and increased glutathione levels. Moreover, perturbation of the fatty acid oxidation program and glutathione synthesis proved selectively toxic to this tumor subset. Our analysis provides evidence for distinct metabolic fingerprints and associated survival mechanisms in DLBCL and may have therapeutic implications.

Metabolic implications of dietary trans-fatty acids.

Dietary trans‐fatty acids are associated with increased risk of cardiovascular disease and have been implicated in the incidence of obesity and type 2 diabetes mellitus (T2DM). It is established that high‐fat saturated diets, relative to low‐fat diets, induce adiposity and whole‐body insulin resistance. Here, we test the hypothesis that markers of an obese, prediabetic state (fatty liver, visceral fat accumulation, insulin resistance) are also worsened with provision of a low‐fat diet containing elaidic acid (18:1t), the predominant trans‐fatty acid isomer found in the human food supply. Male 8‐week‐old Sprague–Dawley rats were fed a 10% trans‐fatty acid enriched (LF‐trans) diet for 8 weeks. At baseline, 3 and 6 weeks, in vivo magnetic resonance spectroscopy (1H‐MR) assessed intramyocellular lipid (IMCL) and intrahepatic lipid (IHL) content. Euglycemic–hyperinsulinemic clamps (week 8) determined whole‐body and tissue‐specific insulin sensitivity followed by high‐resolution ex vivo 1H‐NMR to assess tissue biochemistry. Rats fed the LF‐trans diet were in positive energy balance, largely explained by increased energy intake, and showed significantly increased visceral fat and liver lipid accumulation relative to the low‐fat control diet. Net glycogen synthesis was also increased in the LF‐trans group. A reduction in glucose disposal, independent of IMCL accumulation was observed in rats fed the LF‐trans diet, whereas in rats fed a 45% saturated fat (HF‐sat) diet, impaired glucose disposal corresponded to increased IMCLTA. Neither diet induced an increase in IMCLsoleus. These findings imply that trans‐fatty acids may alter nutrient handling in liver, adipose tissue, and skeletal muscle and that the mechanism by which trans‐fatty acids induce insulin resistance differs from diets enriched with saturated fats.

Nuclear magnetic resonance chromatography : applications of pulse field gradient diffusion NMR to mixture analysis and ligand-receptor interactions

Pulse field gradient (PFG) diffusion NMR spectroscopy is a non-invasive method for the spectroscopic separation and identification of compounds of interest from a mixture. Because it relies on differences in translational diffusion rates to resolve NMR signals from individual components, pulse field gradient NMR is a unique method for analyzing complex mixtures and for detecting intermolecular interactions. A number of multidimensional pulse field gradient NMR experiments have been developed to alleviate the overlap of NMR signals arising from a complex mixture and facilitate component identification. The applications of pulse field gradient NMR for mixture analysis and for the direct identification of high affinity ligands are reviewed.

NMR-based metabonomic studies on the biochemical effects of commonly used drug carrier vehicles in the rat.

The biochemical effects of a series of commonly used drug carrier vehicles were investigated using (1)H NMR spectroscopic and pattern recognition based metabonomic analysis. Animals were treated by oral gavage with six dosage vehicles: 0.5% (w/v) sodium carboxymethylcellulose/0.2% (v/v)tween; microemulsion (consisting of propylene glycol, ethanol, cremophor, and corn oil glycerides); labrafil [consisting of poly(ethylene glycol) 300 esterified with oleic acid] (30%)/corn oil (70%); 0.1 M sodium phosphate buffered water; poly(ethylene glycol) 300 and 0.5% methocel. Urine samples (n = 7) collected over a 96 h period post administration were analyzed using 600 MHz (1)H NMR spectroscopy, and principal components analysis of the spectral data was used to analyze these data. Of the six vehicles studied, three (labrafil/corn oil, PEG 300 and microemulsion) gave rise to strong vehicle-related signals in the (1)H NMR spectra of urine and were, therefore, deemed to be less suitable for NMR-based toxicity studies. To investigate any biochemical consequences of vehicle dosing, PCA was used to analyze spectral regions that did not contain vehicle-related signals, i.e., the NMR-detectable endogenous metabolite profile. PEG 300 and labrafil/corn oil induced changes in the biochemical composition of urine including increased concentrations of dicarboxylic acids, creatinine, taurine, and sugars, indicating that these vehicles were bioactive in their own right and that this might confound interpretation of biochemical effects of weakly toxic drugs dosed in these carriers. This study shows the importance of selecting appropriate vehicles for NMR-based metabonomic studies with a view to minimizing the possibility of vehicle resonances obscuring endogenous compound peaks. Furthermore, we have shown that at least two of the commonly used drug carrier vehicles caused metabolic perturbations in the urine profile. These alterations in the biochemical profile reflect vehicle-induced changes in the physiological status of the organism that may obscure the pharmacologic or toxicologic effects of drugs.

NMR Spectroscopy in β Cell Engineering and Islet Transplantation

Abstract: Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to β cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c‐myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl‐2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy. (2) Overnight incubation of islets and β cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR‐detectable markers using conventional 13C and 31P NMR spectroscopy of extracts. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high‐resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused β cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy.

High resolution MAS-NMR in combinatorial chemistry

High-resolution magic angle spinning (hr-MAS) NMR is a powerful tool for characterizing organic reactions on solid support. Because magic angle spinning reduces the line-broadening due to dipolar coupling and variations in bulk magnetic susceptibility, line widths approaching those obtained in solution-phase NMR can be obtained. The magic angle spinning method is amenable for use in conjunction with a variety of NMR-pulse sequences, making it possible to perform full-structure determinations and conformational analysis on compounds attached to a polymer support. Diffusion-weighted MAS-NMR methods such as SPEEDY (Spin-Echo-Enhanced Diffusion-Filtered Spectroscopy) can be used to remove unwanted signals from the solvent, residual reactants, and the polymer support from the MAS-NMR spectrum, leaving only those signals arising from the resin-bound product. This review will present the applications of high-resolution magic angle spinning NMR for use in combinatorial chemistry research.

Change in lactate production in Myc-transformed cells precedes apoptosis and can be inhibited by Bcl-2 overexpression

As a result of Myc‐dependent transcription of the LDH‐A gene, Myc‐transformed cells (Rat1‐Myc) exhibit increased lactate production rates (LPR) even under aerobic conditions (the Warburg effect). Recently, the increased susceptibility to stress‐induced apoptosis associated with Myc transfection has been linked to the overexpression of the LDH‐A gene. In this report we demonstrate that the overexpression of the anti‐apoptotic protein Bcl−2 in Rat1‐Myc cells (Rat1‐Myc‐Bcl−2) reduces the molar ratio of lactate production to glucose consumption (Y L/G). The Bcl−2 induced reduction in Y L/G may be associated with reduced expression of the LDH‐A gene, or a decrease in LDH‐A activity. Stimulation of apoptosis by staurosporine, a protein kinase C inhibitor, reduces the LPR in Rat1‐Myc cells in a dose‐dependent manner. The staurosporine effect on the LPR is rapid and precedes the execution phase of apoptosis as defined by caspase activation and PARP cleavage. This effect on LPR is completely blocked by Bcl‐2 overexpression. Serum starvation alone does not affect the LPR of Rat1‐Myc or Rat1‐Myc‐Bcl‐2 cells; however, the effect of staurosporine on the LPR of Rat1‐Myc cells is potentiated by serum starvation. These data demonstrate that Bcl‐2 overexpression reduces the Y L/G in Rat1‐Myc cells, perhaps via a reduction in the activity or expression of the LDH‐A gene, and this reduction may desensitize cells to some pro‐apoptotic stimuli. The reduction in LPR in response to staurosporine may be an early step in the induction of apoptosis in Rat1‐Myc cells. By abolishing the reduction in LPR, Bcl‐2 may protect Rat1‐Myc cells from staurosporine‐induced apoptosis. Moreover, the lack of effect by serum starvation on the LPR supports a model in which serum starvation induces apoptosis through a pathway distinct from that of the staurosporine and glucose‐dependent apoptotic pathway(s) in Myc‐transformed cells.

Direct bacterial killing in vitro by recombinant Nod2 is compromised by Crohn's disease-associated mutations.

BACKGROUND A homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR) domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohns disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations. METHODOLOGY/PRINCIPAL FINDINGS In this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohns-associated mutations. Wild-type, but not Crohns-associated, Nod2 LRR domains directly interacted with bacteria in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity. CONCLUSIONS/SIGNIFICANCE The lack of anti-bacterial activity demonstrated with Crohns-associated Nod2 mutations in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphisms with the disease.

On-resin macrocyclization of peptides via intramolecular SNAr reactions

On-resin macrocyclization via an SNAr reaction is employed in the synthesis of tocinoic acid analogs. Specifically, an N-terminal nitrofluorobenzene is attacked by a nucleophilic C-terminal sidechain. The remaining nitro group can be reduced and acylated. NMR is used to compare the conformation of the new macrocyclic peptides to tocinoic acid.

Effects of cevoglitazar, a dual PPARα/γ agonist, on ectopic fat deposition in fatty Zucker rats.

Aim:  By acting as both insulin sensitizers and lipid‐lowering agents, dual‐acting peroxisome proliferator‐activated receptors α/γ (PPARα/γ) agonists may be used to improve glucose tolerance in type 2 diabetic patients without inducing adiposity and body weight gain. Here, in an animal model of obesity and insulin resistance, the metabolic response to cevoglitazar, a dual PPARα/γ, was characterized using a combination of in vivo and ex vivo magnetic resonance methodologies and compared to treatment effects of fenofibrate, a PPARα agonist, and pioglitazone, a PPARγ agonist.