More information: http://www.ncbi.nlm.nih.gov/pubmed/21914785

Abstract

The lichen metabolite usnic acid (UA) has been promoted as a dietary supplement for weight loss, although cases of hepatotoxicity have been reported. Here we evaluated UA-associated hepatotoxicity in vitro using isolated rat hepatocytes. We measured cell viability and ATP content to evaluate UA induced cytotoxicity and applied (13)C isotopomer distribution measuring techniques to gain a better understanding of glucose metabolism during cytotoxicity. The cells were exposed to 0, 1, 5 or 10μM UA concentrations for 2, 6 or 24h. Aliquots of media were collected at the end of these time periods and the (13)C mass isotopomer distribution determined for CO(2), lactate, glucose and glutamate. The 1μM UA exposure did not appear to cause significant change in cell viability compared to controls. However, the 5 and 10μM UA concentrations significantly reduced cell viability as exposure time increased. Similar results were obtained for ATP depletion experiments. The 1 and 5μM UA doses suggest increased oxidative phosphorylation. Conversely, oxidative phosphorylation and gluconeogenesis were dramatically inhibited by 10μM UA. Augmented oxidative phosphorylation at the lower UA concentrations may be an adaptive response by the cells to compensate for diminished mitochondrial function.

Published by Elsevier Ltd.

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SiDMAP studies of two Amgen anti-diabetic agents (PDF Download)

Abstract:

Pharmacologic contributions of directly agonizing glucagon like peptide 1 (GLP-1) receptor or antagonizing glucagon receptor (GCGR) on energy state and glucose homeostasis were assessed in diet-induced obese (DIO) mice. Metabolic rate, respiratory quotient (RQ), hyperglycemic clamp, and gene expression were assessed using stable-isotope-based dynamic metabolic profiling (SiDMAP) studies of 13-labeled glucose during glucose tolerance test (GTT) in cohorts of DIO mice after a single administration of GLP-1 analog [GLP-1(23)] or anti-GCGR antibody (Ab.) GLP-1-(23) and GCGR Ab similarly improved GTT.

GLP-1-(23) decreased food intake and body weight trended lower. GCGR Ab modestly decreased food intake without significant effect on body weight. GLP-1-(23) and GCGR Ab decreased RQ with GLP-1, causing a greater effect. In a hyperglycemic clamp, GLP-1-(23) reduced hepatic glucose production (HGP), increased glucose infusion rate (GIR), increased glucose uptake in brown adipose tissue, and increased whole-body glucose turnover, glycolysis, and rate of glycogen synthesis. GCGR Ab slightly decreased HGP, increased GIR, and increased glucose uptake in the heart.

SiDMAP showed that GLP-1-(23) and GCGR Ab increased 13C lactate labeling from glucose, indicating that liver, muscle, and other organs were involved in the rapid disposal of glucose from plasma. GCGR Ab and GLP-1-(23) caused different changes in mRNA expression levels of glucose- and lipid metabolism-associated genes. The effect of GLP-1-(23) on energy state and glucose homeostasis was greater than GCGR Ab. Although GCGR antagonism is associated with increased circulating levels of GLP-1, most GLP-1-(23)-associated pharmacologic effects are more pronounced than GCGR Ab.

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Abstract

Diverse mechanisms of growth inhibition by luteolin, resveratrol, and quercetin in MIA PaCa-2 cells: a comparative glucose tracer study with the fatty acid synthase inhibitor C75

The rationale of this dose matching/dose escalating study was to compare a panel of flavonoids—luteolin, resveratrol, and quercetin—against the metabolite flux-controlling properties of a synthetic targeted fatty acid synthase inhibitor drug C75 on multiple macromolecule synthesis pathways in pancreatic tumor cells using [1,2-¹³C₂]-d-glucose as the single precursor metabolic tracer. MIA PaCa-2 pancreatic adenocarcinoma cells were cultured for 48 h in the presence of 0.1% DMSO (control), or 50 or 100 μM of each test compound, while intracellular glycogen, RNA ribose, palmitate and cholesterol as well as extra cellular ¹³CO₂, lactate and glutamate production patterns were measured using gas chromatography/mass spectrometry (GC/MS) and stable isotope-based dynamic metabolic profiling (SiDMAP). The use of 50% [1,2-¹³C₂]-d-glucose as tracer resulted in an average of 24 excess ¹³CO₂ molecules for each 1,000 CO₂ molecule in the culture media, which was decreased by 29 and 33% (P < 0.01) with 100 μM C75 and luteolin treatments, respectively. Extracellular tracer glucose-derived ¹³C-labeled lactate fractions (Σm) were between 45.52 and 47.49% in all cultures with a molar ratio of 2.47% M + 1/Σm lactate produced indirectly by direct oxidation of glucose in the pentose cycle in control cultures; treatment with 100 μM C75 and luteolin decreased this figure to 1.80 and 1.67%. The tracer glucose-derived ¹³C labeled fraction (Σm) of ribonucleotide ribose was 34.73% in controls, which was decreased to 20.58 and 8.45% with C75, 16.15 and 6.86% with luteolin, 27.66 and 19.25% with resveratrol, and 30.09 and 25.67% with quercetin, respectively. Luteolin effectively decreased nucleotide precursor synthesis pentose cycle flux primarily via the oxidative branch, where we observed a 41.74% flux (M + 1/Σm) in control cells, in comparison with only a 37.19%, 32.74%, or a 26.57%, 25.47% M + 1/Σm flux (P < 0.001) after 50 or 100 μM C75 or luteolin treatment. Intracellular de novo fatty acid palmitate (C16:0) synthesis was severely and equally blocked by C75 and luteolin treatments indicated by the 5.49% (control), 2.29 or 2.47% (C75) and 2.21 or 2.73% (luteolin) tracer glucose-derived ¹³C-labeled fractions, respectively. On the other hand there was a significant 192 and 159% (P < 0.001), and a 103 and 117% (P < 0.01) increase in tracer glucose-derived cholesterol after C75 or luteolin treatment. Only resveratrol and quercetin at 100 μM inhibited tracer glucose-derived glycogen labeling (Σm) and turnover by 34.8 and 23.8%, respectively. The flavonoid luteolin possesses equal efficacy to inhibit fatty acid palmitate de novo synthesis as well as nucleotide RNA ribose turnover via the oxidative branch of the pentose cycle in comparison with the targeted fatty acid synthase inhibitor synthetic compound C75. Luteolin is also effective in stringently controlling glucose entry and anaplerosis in the TCA cycle, while it promotes less glucose flux towards cholesterol synthesis than that of C75. In contrast, quercetin and resveratrol inhibit glycogen synthesis and turnover as their underlying mechanism of controlling tumor cell proliferation. Therefore the flavonoid luteolin controls fatty and nucleic acid syntheses as well as energy production with pharmacological strength, which can be explored as a non-toxic natural treatment modality for pancreatic cancer.

Full post: http://www.ncbi.nlm.nih.gov/pubmed/20647250

Abstract
Activated lymphocytes proliferate, secrete cytokines, and can make antibodies. Normally activated B and T cells meet the bioenergetic demand for these processes by up-regulating aerobic glycolysis. In contrast, several lines of evidence suggest that pathogenic lymphocytes in autoimmune diseases like lupus meet ATP demands through oxidative phosphorylation. Using (13)C-glucose as a stable tracer, we found that splenocytes from mice with lupus derive the same fraction of lactate from glucose as control animals, suggesting comparable levels of glycolysis and non-oxidative ATP production. However, lupus splenocytes increase glucose oxidation by 40% over healthy control animals. The ratio between pentose phosphate cycle (PPC) activity and glycolysis is the same for each group, indicating that increased glucose oxidation is due to increased activity of the TCA cycle in lupus splenocytes. Repetitive stimulation of cultured human T cells was used to model chronic lymphocyte activation, a phenotype associated with lupus. Chronically activated T cells rely primarily on oxidative metabolism for ATP synthesis suggesting that chronic antigen stimulation may be the basis for the metabolic findings observed in lupus mice. Identification of disease-related bioenergetic phenotypes should contribute to new diagnostic and therapeutic strategies for immune diseases.

PMID: 20647250 [PubMed - as supplied by publisher]

Tuesday, August 3, 2010
Fructose helps pancreatic cancer cells to multiply, UCLA study finds
WASHINGTON — Pancreatic tumor cells use fructose to divide and proliferate, U.S. researchers said Monday in a study that challenges the common wisdom that all sugars are the same.

Tumor cells fed both glucose and fructose used the two sugars in two different ways, the team at the University of California Los Angeles found.

They said their finding, published in the journal Cancer Research, might help explain other studies that have linked fructose intake with pancreatic cancer, one of the deadliest cancer types.

“These findings show that cancer cells can readily metabolize fructose to increase proliferation,” Anthony Heaney of UCLA’s Jonsson Cancer Center and colleagues wrote. “They have major significance for cancer patients given dietary refined fructose consumption, and indicate that efforts to reduce refined fructose intake or inhibit fructose-mediated actions may disrupt cancer growth.”

Americans take in large amounts of fructose, mainly in high-fructose corn syrup, a mix of fructose and glucose that is used in soft drinks, bread and a range of other foods. Politicians, regulators, health experts and the fructose industry have debated whether high-fructose corn syrup and other ingredients have been helping make Americans fatter and less healthy. Too much sugar of any kind not only adds pounds, but is also a key culprit in diabetes, heart disease and stroke, according to the American Heart Association. Several states, including New York and California, have weighed a tax on sweetened soft drinks, but the American Beverage Association has successfully opposed efforts to tax soda.

The industry has also argued that sugar is sugar. Heaney said his team found otherwise. They grew pancreatic cancer cells in lab dishes and fed them both glucose and fructose. Tumor cells thrive on sugar but they used the fructose to proliferate. “Importantly, fructose and glucose metabolism are quite different,” Heaney’s team wrote.

Los Angeles, California – July 21, 2010 – SiDMAP, LLC (www.sidmap.com), a privately held life-science company that provides metabolomics research services to the pharmaceutical and biotechnology industries and the academic biomedical research community worldwide, announced today results of a study conducted in collaboration with scientists at  Roche (Nutley, NJ), a leading pharmaceutical company, on a novel Roche drug for Type 2 diabetes.

The study, entitled “A Novel Approach for the Treatment of Type 2 Diabetes (T2D): Characterization of a Potent, Orally Active, Small Molecule Glycogen Synthase Activator,” was presented in Abstract 1389-P at the recently concluded American Diabetes Association (ADA), 70th Scientific Sessions, in Orlando, Florida.

Laszlo G. Boros, M.D., SiDMAP’s Chief Scientist, was SiDMAP’s lead investigator on the study. Andree R. Olivier, Ph.D., was lead investigator for Roche.

Using study methods designed by Dr. Boros in collaboration with Roche, the glycogen synthase activator GSA3 was administered to DIO mice (75 mg/kg), along with a stable isotope (non-emitting) glucose tracer. Subsequent laboratory analysis by SiDMAP of mouse tissue samples using the Company’s proprietary Stable Isotope Dynamic Metabolic Tracer technology demonstrated that GSA3 increased glycogen synthesis in mouse muscle and liver tissues, but the source of tracer-glucose in liver glycogen is primarily derived from an indirect pathway.

Dr. Boros said, “The purpose of the SiDMAP’s analysis of plasma and tissues from GSA3-treated DIO mice that had absorbed a stable isotope glucose tracer was to see if we could further validate Roche’s previous findings on the effects and mechanism of action of their potential T2D compound. The SiDMAP results were supportive of the previous Roche findings. The SiDMAP results also provided detailed pictures regarding glucose disposal mechanisms and system response using the highest scoring [U-13C6]-D-glucose tracer for TCA/pentose cycle and hepatic glucose production type clamp equivalent studies.”

As described in the study abstract, the SiDMAP findings, taken together with data on Roche GSA3 activator RO5289867, “provide evidence that direct pharmacological activation of GYS1 and GYS2 can lead to beneficial effects in whole body substrate metabolism and may be a viable approach for treating T2D and its co-morbidities.”

Dr. Olivier said, “The SiDMAP technology added value by giving Roche further confirmation and understanding of our compound’s mechanism of action, and enabled Roche to achieve its study objectives.”

According to Dr. Boros, “The precise and previously inaccessible flux map resulting from our in vivo study with Roche further support the use of Stable Isotope Dynamic Metabolic Profiling technology in the characterization of potential new drugs and their targets for Type 2 diabetes.”

The full text for Abstract 1389-P can be found at the following web address: http://ww2.aievolution.com/ada1001/index.cfm?do=cnt.page&pg=1009

About SiDMAP
SiDMAP, LLC is a metabolomics company that provides research services for drug discovery and development, biomedical research, and drug toxicology research. SiDMAP clients include global pharmaceutical companies, leading biotechnology companies, major academic research institutions and drug regulatory authorities.

Contact
Kate Hawkes, Manager, Business Development & Administration
SiDMAP, LLC
E-Mail Kate Hawkes, Phone:  310-478-1424, ext. 308

The new grant, funded by the National Institutes of Health / National Cancer Institute, provides support for metabolic profiling research in lung cancer. Shyam Biswal, Ph.D., Associate Professor at Johns Hopkins University, School of Medicine, in Baltimore, is Principal Investigator on the grant.
Entitled “Regulation of Tumorigenesis and Therapeutic Resistance by Nrf2 in Lung Cancer,” the grant will test the hypothesis that a gain in function of the gene transcription factor Nrf2 (Nuclear factor erythroid-2 related factor) promotes tumorigenesis in the presence of an oncogenic (cancer-causing) signal.

“SiDMAP opens new avenues for understanding and treating cancer by revealing the functional impact of known gene mutations on a complex hierarchy of metabolic reactions.” said Dr. Boros. He added, “SiDMAP is always glad to collaborate with top university investigators, such as our colleagues at Johns Hopkins, to accelerate research in the cancer field.”

Dr. Boros, a SiDMAP co-founder and Associate Professor at the UCLA School of Medicine, is a leading expert on the basic science and translational research applications of metabolomics, an evolving field of research focusing on metabolic changes associated with disease processes and response to drug treatment. Metabolomics studies are used by drug researchers to elucidate a drug’s mechanism of action (MOA) by identifying and measuring myriad metabolic changes that occur in cells and organs following drug administration. Metabolomics studies can also reveal early metabolic changes associated with increased potential for drug toxicity, and can be used to screen for cellular, animal model or patient phenotypes that respond more or less favorably to a particular drug. Metabolomics testing is also being used increasingly as a very precise tool for identifying novel drug targets, such as key enzymes that play critical roles in the function of metabolic pathways essential for the growth of cancer cells.

Dr. Boros and his research team at SiDMAP will collaborate with Dr. Biswal and his team at Johns Hopkins to determine whether a gain in Nrf2 function increases glucose metabolism via the pentose phosphate pathway and tricarboxlyic acid pathway—metabolic steps essential to tumorigenesis. The grant further seeks to determine if blocking Nrf2-dependent phosphate pathway enzymes inhibits growth of NSCLC cells and limits chemoresistance.

Use of SiDMAP’s proprietary stable-isotope dynamic metabolic profiling technology in the NIH/NCI-funded study at Johns Hopkins will help in understanding the regulation of lung tumorigenesis by a novel pathway and in developing a strategy for targeting this pathway to circumvent therapeutic resistance.

Contact

Kate Hawkes

Manager, Business Development & Administration
SiDMAP, LLC

Phone: 310-478-1424, ext. 308
khawkes@sidmap.com

Journal of Biological Chemistry 276: 37747-37753, 2001