Development of UHPLC/Q-TOF Analysis Method to Screen Glycerin for Direct Detection of Process Contaminants 3-Monochloropropane-1,2-diol Esters (3-MCPDEs) and Glycidyl Esters (GEs).

The U.S. Food and Drug Administration's (FDA's) Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats since 2007. Renal failure accounted for 30% of reported cases. Jerky pet treats contain glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Glycidyl esters (GEs) and 3-monochloropropanediol esters (3-MCPDEs) are food contaminants that can form in glycerin during the refining process. 3-MCPDEs and GEs pose food safety concerns, as they can release free 3-MCPD and glycidol in vivo. Evidence from studies in animals shows that 3-MCPDEs are potential toxins with kidneys as their main target. As renal failure accounted for 30% of reported pet illnesses after the consumption of jerky pet treats containing glycerin, there is a need to develop a screening method to detect 3-MCPDEs and GEs in glycerin. We describe the development of an ultra-high-pressure liquid chromatography/quadrupole time-of-flight (UHPLC/Q-TOF) method for screening glycerin for MCPDEs and GEs. Glycerin was extracted and directly analyzed without a solid-phase extraction procedure. An exact mass database, developed in-house, of MCPDEs and GEs formed with common fatty acids was used in the screening.

Rapid, Selective, and Sensitive Method for Semitargeted Discovery of Congeneric Natural Products by Liquid Chromatography Tandem Mass Spectrometry.

Natural product congeners serve a useful role in the understanding of natural product biosynthesis and structure-activity relationships. A minor congener with superior activity, selectivity, and modifiable functional groups could serve as a more effective lead structure and replace even the original lead molecule that was used for medicinal chemistry modifications. Currently, no effective method exists to discover targeted congeners rapidly, specifically, and selectively from producing sources. Herein, a new method based on liquid-chromatography tandem-mass spectrometry combination is evaluated for targeted discovery of congeners of platensimycin and platencin from the extracts of Streptomyces platensis. By utilizing a precursor-ion searching protocol, tandem mass spectrometry not only confirmed the presence of known congeners but also provided unambiguous detection of many previously unknown congeners of platensimycin and platencin. This high-throughput and quantitative method can be rapidly and broadly applied for dereplication and congener discovery from a variety of producing sources, even when the targeted compounds are obscured by the presence of unrelated natural products.

Ketohexokinase knockout mice, a model for essential fructosuria, exhibit altered fructose metabolism and are protected from diet-induced metabolic defects.

Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, ß-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.

Using [2H]water to quantify the contribution of de novo palmitate synthesis in plasma: enabling back-to-back studies.

An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.

siRNA-mediated inhibition of SREBP cleavage-activating protein reduces dyslipidemia in spontaneously dysmetabolic rhesus monkeys.

BACKGROUND: SREBP cleavage-activating protein (SCAP) is a cholesterol binding endoplasmic reticulum (ER) membrane protein that is required to activate SREBP transcription factors. SREBPs regulate genes involved in lipid biosynthesis. They also influence lipid clearance by modulating the expression of LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Inhibiting SCAP decreases circulating PCSK9, triglycerides (TG), and LDL-cholesterol (LDL-C), both in vitro and in vivo. Type 2 diabetics with dyslipidemia are at high risk for cardiovascular diseases. These patients present a unique pathophysiological lipid profile characterized by moderately elevated LDL-C, elevated TG and reduced HDL-cholesterol (HDL-C). The spontaneous dysmetabolic rhesus monkey model (DysMet RhM) recapitulates this human dyslipidemia and therefore is an attractive preclinical model to evaluate SCAP inhibition as a therapy for this disease population. The objective to of this study was to assess the effect of SCAP inhibition on the lipid profile of DysMet RhM. METHOD: We assessed the effect of inhibiting hepatic SCAP on the lipid profile of DysMet RhM using an siRNA encapsulated lipid nanoparticle (siRNA-LNP). RESULTS: The SCAP siRNA-LNP significantly reduced LDL-C, PCSK9 and TG in DysMet RhM; LDL-C was reduced by ≥20%, circulating PCSK9 by 30-40% and TG by >25%. These changes by the SCAP siRNA-LNP agree with the predicted effect of SCAP inhibition and reduced SREBP tone on these endpoints. CONCLUSION: These data demonstrate that a SCAP siRNA-LNP improved the lipid profile in a clinically relevant preclinical disease model and provide evidence for SCAP inhibition as a therapy for diabetic dyslipidemic patients.

Application of time-of-flight mass spectrometry for screening of crude glycerins for toxic phorbol ester contaminants.

Since 2007, the U.S. Food and Drug Administration (FDA) has received numerous complaints of pet illnesses that may be related to the consumption of jerky pet treats. Many of those treats include glycerin as an ingredient. Glycerin can be made directly from oils such as palm seed oil, but can also be derived from the seed oil of toxic Jatropha plant during biodiesel production. If crude glycerin from biodiesel production from Jatropha curcas is used in the manufacture of animal feed, toxic tigliane diterpene phorbol esters (PEs), namely Jatropha factors (JFs), may be present and could lead to animal illnesses. Considering the numerous uses of glycerin in consumer products there is a need for a rapid method to screen crude glycerin for JF toxins and other PE contaminants. We describe the development of an ultra-high pressure liquid chromatography/quadrupole time of flight (UHPLC/Q-TOF) method for screening crude glycerin for PEs. An exact mass database, developed in-house, of previously identified PEs from Jatropha curcas as well as putative compounds was used to identify possible contaminants.

Correction: The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

[This corrects the article DOI: 10.1371/journal.pone.0164133.].

Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9, LDLR, and plasma lipids in mouse and rhesus monkey.

SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.

The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys.

OBJECTIVES: Platensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans. METHODS: We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor. RESULTS: The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice. CONCLUSIONS: These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic disorders.

Quantifying rates of glucose production in vivo following an intraperitoneal tracer bolus.

Aberrant regulation of glucose production makes a critical contribution to the impaired glycemic control that is observed in type 2 diabetes. Although isotopic tracer methods have proven to be informative in quantifying the magnitude of such alterations, it is presumed that one must rely on venous access to administer glucose tracers which therein presents obstacles for the routine application of tracer methods in rodent models. Since intraperitoneal injections are readily used to deliver glucose challenges and/or dose potential therapeutics, we hypothesized that this route could also be used to administer a glucose tracer. The ability to then reliably estimate glucose flux would require attention toward setting a schedule for collecting samples and choosing a distribution volume. For example, glucose production can be calculated by multiplying the fractional turnover rate by the pool size. We have taken a step-wise approach to examine the potential of using an intraperitoneal tracer administration in rat and mouse models. First, we compared the kinetics of [U-13C]glucose following either an intravenous or an intraperitoneal injection. Second, we tested whether the intraperitoneal method could detect a pharmacological manipulation of glucose production. Finally, we contrasted a potential application of the intraperitoneal method against the glucose-insulin clamp. We conclude that it is possible to 1) quantify glucose production using an intraperitoneal injection of tracer and 2) derive a "glucose production index" by coupling estimates of basal glucose production with measurements of fasting insulin concentration; this yields a proxy for clamp-derived assessments of insulin sensitivity of endogenous production.

Glucagon receptor antagonism induces increased cholesterol absorption.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

Effect of Error Propagation in Stable Isotope Tracer Studies: An Approach for Estimating Impact on Apparent Biochemical Flux.

Stable isotope tracers are widely used to quantify metabolic rates, and yet a limited number of studies have considered the impact of analytical error on estimates of flux. For example, when estimating the contribution of de novo lipogenesis, one typically measures a minimum of four isotope ratios, i.e., the precursor and product labeling pre- and posttracer administration. This seemingly simple problem has 1 correct solution and 80 erroneous outcomes. In this report, we outline a methodology for evaluating the effect of error propagation on apparent physiological endpoints. We demonstrate examples of how to evaluate the influence of analytical error in case studies concerning lipid and protein synthesis; we have focused on (2)H2O as a tracer and contrast different mass spectrometry platforms including GC-quadrupole-MS, GC-pyrolysis-IRMS, LC-quadrupole-MS, and high-resolution FT-ICR-MS. The method outlined herein can be used to determine how to minimize variations in the apparent biology by altering the dose and/or the type of tracer. Likewise, one can facilitate biological studies by estimating the reduction in the noise of an outcome that is expected for a given increase in the number of replicate injections.

Tracer-based estimates of protein flux in cases of incomplete product renewal: evidence and implications of heterogeneity in collagen turnover.

The synthesis of various molecules can be estimated by measuring the incorporation of a labeled precursor into a product of interest. Unfortunately, a central problem in many studies has been an inability to estimate the intracellular dilution of the precursor and therein correctly calculate the synthesis of the product; it is generally assumed that measuring the true product labeling is straightforward. We initiated a study to examine liver collagen synthesis and identified an apparent problem with assumptions regarding measurements of the product labeling. Since it is well known that collagen production is relatively slow, we relied on the use of [(2)H]H2O labeling (analogous to a primed infusion) and sampled animals over the course of 16 days. Although the water labeling (the precursor) remained stable and we observed the incorporation of labeled amino acids into collagen, the asymptotic protein labeling was considerably lower than what would be expected based on the precursor labeling. Although this observation is not necessarily surprising (i.e., one might expect that a substantial fraction of the collagen pool would appear "inert" or turn over at a very slow rate), its implications are of interest in certain areas. Herein, we discuss a novel situation in which tracers are used to quantify rates of flux under conditions where a product may not undergo complete replacement. We demonstrate how heterogeneity in the product pool can lead one to the wrong conclusions regarding estimates of flux, and we outline an approach that may help to minimize errors surrounding data interpretation.

The hepatitis C virus core protein inhibits adipose triglyceride lipase (ATGL)-mediated lipid mobilization and enhances the ATGL interaction with comparative gene identification 58 (CGI-58) and lipid droplets.

Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.

Circumambulatory movement of negative charge ("ring walk") during gas-phase dissociation of 2,3,4-trimethoxybenzoate anion.

A dramatic "ortho effect" was observed during gas-phase dissociation of ortho-, meta-, and para-methoxybenzoate anions. Upon activation under mass spectrometric collisional activation conditions, anions generated from all three isomers undergo a CO2 loss. Of the m/z 107 ions generated in this way, only the 1-dehydro-2-methoxybenzene anion from the ortho isomer underwent an exclusive formaldehyde loss. A peak for a formaldehyde loss in the spectra of 2,4-, 2,5-, and 2,6-dimethoxybenzoates and the absence of an analogous peak from 3,4- and 3,5-dimethoxy derivatives confirmed that this is a diagnostically useful ortho-isomer-specific phenomenon. Moreover, the spectrum from 2,3-dimethoxybenzoic acid showed peaks for two consecutive formaldehyde losses. The 1-dehydro-2,3,4-trimethoxybenzene anion (m/z 167) generated from 2,3,4-trimethoxybenzoate in this way endures three consecutive eliminations of formaldehyde units. For this, the negative charge, initially located on position 1, circumambulates to position 2, then to position 3, and finally to position 4 to form the final phenyl anion. The proposed stepwise fragmentation pathway, which resembles the well-known E1cB-elimination mechanism, is supported by tandem mass spectrometric observations made with 2-[(13)C(2)H3]methoxy-3-[(13)C]methoxy-4-methoxybenzoic acid, and ab initio calculations. In addition, the spectra of ions such as 1-dehydro-3,4-dimethoxybenzene anion show peaks for consecutive methyl radical losses, a feature that establishes the 1,2-relationship between the two methoxy groups.

Determination of low levels of 2H-labeling using high-resolution mass spectrometry: application in studies of lipid flux and beyond.

RATIONALE: The ability to measure low levels of (2)H-labeling is important in studies of metabolic flux, e.g. one can estimate lipid synthesis by administering (2)H2O and then measuring the incorporation of (2)H into fatty acids. Unfortunately, the analyses are complicated by the presence of more abundant naturally occurring stable isotopes, e.g. (13)C. Conventional approaches rely on coupling gas chromatographic separation of lipids with either quadrupole-mass spectrometry (q-MS) and/or pyrolysis-isotope ratio mass spectrometry (IRMS). The former is limited by high background labeling (primarily from (13)C) whereas the latter is not suitable for routine high-throughput analyses. METHODS: We have contrasted the use of continuous flow-pyrolysis-IRMS against high-resolution mass spectrometry (i.e. Qq-FT-ICR MS) for measuring the (2)H-enrichment of fatty acids and peptides. RESULTS: In contrast to IRMS, which requires ~30 min per analysis, it is possible to measure the (2)H-enrichment of palmitate via direct infusion high-resolution mass spectrometry (HRMS) in ~3 min per sample. In addition, Qq-FT-ICR MS enabled measurements of the (2)H-enrichment of peptides (which is not possible using IRMS). CONCLUSIONS: High-resolution mass spectrometry can be used to measure low levels of (2)H-labeling so we expect that this approach will enhance studies of metabolic flux that rely on (2)H-labeled tracers, e.g. (2)H2O. However, since the high-resolution analyses require greater amounts of a given analyte one potential limitation centers on the overall sensitivity. Presumably, future advances can overcome this barrier.

New methodologies for studying lipid synthesis and turnover: looking backwards to enable moving forwards.

Our ability to understand the pathogenesis of problems surrounding lipid accretion requires attention towards quantifying lipid kinetics. In addition, studies of metabolic flux should also help unravel mechanisms that lead to imbalances in inter-organ lipid trafficking which contribute to dyslipidemia and/or peripheral lipid accumulation (e.g. hepatic fat deposits). This review aims to outline the development and use of novel methods for studying lipid kinetics in vivo. Although our focus is directed towards some of the approaches that are currently reported in the literature, we include a discussion of the older literature in order to put "new" methods in better perspective and inform readers of valuable historical research. Presumably, future advances in understanding lipid dynamics will benefit from a careful consideration of the past efforts, where possible we have tried to identify seminal papers or those that provide clear data to emphasize essential points. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

Enhanced data-independent analysis of lipids using ion mobility-TOFMSE to unravel quantitative and qualitative information in human plasma.

RATIONALE: Lipids are involved in various biochemical and signaling pathways, cell structure and function, and the pathophysiology of many diseases. We took advantage of ion mobility spectrometry (IMS) in conjunction with ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry to gain quantitative and deeper qualitative structural insight within a single experiment. METHODS: Human plasma lipid extracts were analyzed using an Acquity UPLC system coupled to a Synapt G2-HDMS mass spectrometer system. The ion mobility gas employed was helium for the helium cell (150 mL/min) and nitrogen (80 mL/min) for the T-wave drift tube. The wave height for the T-wave cell was ramped in a linear fashion between 5-40 V. The mass spectra were acquired in an electrospray positive ionization mode. RESULTS: We resolved chromatographically co-eluting lipids further by ion mobility tube drift time and then subjected them to low- and high-energy fragmentation without pre-selecting respective precursor species. The fragment ions produced in a high-energy mode were aligned with their precursor ions in a low-energy mode. By aligning intact molecular spectra and fragment spectra for these lipids at a given ion mobility drift time and chromatographic retention time, we were able to obtain much cleaner fragment ion spectra for structural elucidation. For quantitative analysis we obtained a dynamic linear range from 0.002 to 2 µg/mL with and without an additional dimension of IMS. CONCLUSIONS: The additional dimension of IMS allowed us to perform quantitative and qualitative analysis within a single experiment in a relatively high-throughput manner thus providing deeper structural insights into lipids of biological interest and resulting in an information-rich dataset.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice.

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

Occurrence, distribution, dereplication and efficient discovery of thiazolyl peptides by sensitive-resistant pair screening.

Natural products have been major sources of antibacterial agents and remain very promising. Frequent rediscoveries of known compounds hampers progress of new discoveries and demands development and utilization of new methods for rapid biological and chemical dereplication. This paper describes an efficient approach for discovery of new thiazolyl peptides by sensitive-resistant pair screening and dereplication in a time and cost-effective manner at industrial scale. A highly effective library-based dereplication of thiazolyl peptides by high resolution fourier transform liquid chromatography mass spectrometry (HRFTLCMS) has been developed, which can detect and dereplicate very low levels of thiazolyl peptides particularly when combined with miniaturized high-throughput 96-well solid-phase extraction separation, and as well can be automated. Combination of sensitive (susceptible)-resistant pair screening, diversified screening collection and miniaturized high-throughput SPE and HRFTLCMS techniques were applied for discovery of new thiazolyl peptides. The combined approach allowed for identification of over 24 thiazolyl peptides represented by three of the five structural subgroups, including three novel compounds. In addition, it is possible for the first time to mechanistically group three structural subgroups of over 24 thiazolyl peptides. Furthermore, these studies helped to understand natural frequency of distribution of these compounds and helped in discovery of new producing strains of many thiazolyl compounds.