Stable Isotope-based Dynamic Metabolic Profiling Research Articles

B30 Investigating Hepatic Dysfunction In The Httq111/+ Mouse With A Perturbagen-based Primary Hepatocyte System

<h3>Background</h3> We are interested in mapping and understanding phenotypes that precede the development of overt signs of Huntington’s disease, in hopes of identifying pathways with the potential to be disease-modifying. Notably, the Huntington’s disease mutation is associated with early metabolic alterations of unknown aetiology in both humans and various model systems. Our own data suggest that widespread metabolic alterations occur very early in B6. HttQ111/Q7 mice, which precisely recapitulate the genetics of human HD patients. <h3>Aims</h3> We aim to understand which tissues reveal the earliest transcriptional and metabolic changes in the B6. HttQ111/Q7 mice. <h3>Methods</h3> We are applying stable isotope-based dynamic metabolic profiling (SiDMAP) to cells and tissues to quantify metabolic flux, and RNA sequencing (RNASeq) to quantify transcript abundance. <h3>Results/outcome</h3> In a cross-tissue metabolic flux study of 90-day-old mice using U13C-glucose as a stable tracer, we observe that the most robust metabolic changes are observed in the liver. Transcriptomic analysis of matched samples with RNASeq reveals that the liver has more genotype-sensitive transcripts at this stage than does the striatum, the most vulnerable tissue in HD. Based on these <i>in vivo</i>results, we hypothesise that very early changes in fatty acid synthesis, mobilisation and/or catabolism are occuring in the liver of HttQ111/+ mice. To study this hypothesis in more mechanistic detail, we have established a system of primary hepatocytes isolated from 90 day old mice fed one of 3 diets from weaning as a lipid-relevant perturbagen: high fat (65% kcal/fat), medium fat (45% kcal/fat) and normal chow (10% kcal/fat). We reason that examining phenotypes in HttQ111/Q7 cells at steady states may not be as informative as investigating their dynamic responses to chemically defined perturbations. Our phenotypic analysis <i>in vitro</i>includes quantifying metabolic flux using multiple flux tracers (1,2–13C2-glucose and U13C-palmitate). We are also collecting transcriptomic data from matched samples using RNASeq, in hopes of identifying genotype-sensitive flux changes that are associated with coherent transcriptional alterations. <h3>Conclusions</h3> We conclude that early transcriptional and metabolic flux changes occur in the livers of B6. HttQ111/Q7 mice.

Metabolic Consequences of LDHA inhibition by Epigallocatechin Gallate and Oxamate in MIA PaCa-2 Pancreatic Cancer Cells.

Lactate dehydrogenase A (LDHA) is the enzyme that converts pyruvate to lactate and oxidizes the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD+. Several human cancers including the pancreas display elevated expression of LDHA. Because of its essential role in cancer metabolism, LDHA has been considered to be a potential target for cancer therapy. Recently, we have shown that a green tea extract significantly down-regulated LDHA in HPAF-II pancreatic cancer cells using global proteomics profiling. The present study is to investigate how EGCG, a major biological active constituent of green tea, targets the metabolism of human pancreatic adenocarcinoma MIA PaCa-2 cells. We compared the effect of EGCG to that of oxamate, an inhibitor of LDHA, on the multiple metabolic pathways as measured by extracellular lactate production, glucose consumption, as well as intracellular aspartate and glutamate production, fatty acid synthesis, acetyl-CoA, RNA ribose and deoxyribose. Specific metabolic pathways were studied using [1, 2-13C2]-d-glucose as the single precursor metabolic tracer. Isotope incorporations in metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS) and stable isotope-based dynamic metabolic profiling (SiDMAP). We found that the EGCG treatment of MIA PaCa-2 cells significantly reduced lactate production, anaerobic glycolysis, glucose consumption and glycolytic rate that are comparable to the inhibition of LDHA by oxamate treatment. Significant changes in intracellular glucose carbon re-distribution among major glucose-utilizing macromolecule biosynthesis pathways in response to EGCG and oxamate treatment were observed. The inhibition of LDHA by EGCG or oxamate impacts on various pathways of the cellular metabolic network and significantly modifies the cancer metabolic phenotype. These results suggest that phytochemical EGCG and LDHA inhibitor oxamate confer their anti-cancer activities by disrupting the balance of flux throughout the cellular metabolic network.

Pharmacological Targeting of Glucagon and Glucagon-Like Peptide 1 Receptors Has Different Effects on Energy State and Glucose Homeostasis in Diet-Induced Obese Mice

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 and respiratory quotient (RQ), hyperglycemic clamp, stable isotope-based dynamic metabolic profiling (SiDMAP) studies of (13)C-labeled glucose during glucose tolerance test (GTT) and gene expression were assessed 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 (13)C 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.

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-(13)C(2)]-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 (13)CO(2), 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-(13)C(2)]-d-glucose as tracer resulted in an average of 24 excess (13)CO(2) molecules for each 1,000 CO(2) 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 (13)C-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 (13)C 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 (13)C-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.

Dual modulation of both lipid oxidation and synthesis by peroxisome proliferator‐activated receptor‐γ coactivator‐1α and ‐1β in cultured myotubes

The peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family is a key regulator of mitochondrial function, and reduced mRNA expression may contribute to muscle lipid accumulation in obesity and type 2 diabetes. To characterize the effects of PGC-1 on lipid metabolism, we overexpressed PGC-1alpha and PGC-1beta in C2C12 myotubes using adenoviral vectors. Both PGC-1alpha and -1beta increased palmitate oxidation [31% (P<0.01) and 26% (P<0.05), respectively] despite reductions in cellular uptake [by 6% (P<0.05) and 21% (P<0.001)]. Moreover, PGC-1alpha and -1beta increased mRNA expression of genes regulating both lipid oxidation (e.g., CPT1b and ACADL/M) and synthesis (FAS, CS, ACC1/2, and DGAT1). To determine the net effect, we assessed lipid composition in PGC-1-expressing cells. Total lipid content decreased by 42% in palmitate-loaded serum-starved cells overexpressing PGC-1alpha (P<0.05). In contrast, in serum-replete cells, total lipid content was not significantly altered, but fatty acids C14:0, C16:0, C18:0, and C18:1 were increased 2- to 4-fold for PGC-1alpha/beta (P<0.05). Stable isotope-based dynamic metabolic profiling in serum-replete cells labeled with (13)C substrates revealed both increased de novo fatty acid synthesis from glucose and increased fatty acid synthesis by chain elongation with either PGC-1alpha or -1beta expression. These results indicate that PGC-1 can promote both lipid oxidation and synthesis, with net balance determined by the nutrient/hormonal environment.-Espinoza, D. O., Boros, L. G., Crunkhorn, S., Gami, H., Patti, M.-E. Dual Modulation of both lipid oxidation and synthesis by peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -1beta in cultured myotubes.

THE FATTY ACID SYNTHASE (FAS) INHIBITOR C75 AND SELECTED PHYTOCHEMICALS ARE EQUIPOTENT IN INHIBITING PROLIFERATION IN PANCREATIC CANCER CELLS VIA DIVERGENT METABOLIC PATHWAYS

The synthetic FAS inhibitor C75 and phytochemicals from food have been shown to reduce cancer cell proliferation and increase apoptosis. We evaluated the effect of inhibition of lipid synthesis pathways on proliferation, apoptosis, and metabolism in pancreatic cancer cells using stable isotope-based dynamic metabolic profiling (SiDMAP). MIA PaCa-2 cells were grown 48 h with 0-300 μM C75, luteolin, resveratrol, or quercetin (n = 3) and also in 0, 50, and 100 μM of FAS inhibitors plus [1,2-13C2]-D-glucose (50% enrichment, 180 mg/dL). C75 and each phytochemical induced a dose-dependent decrease in proliferation; luteolin was nearly as potent as C75. Luteolin was less proapoptotic than C75. SiDMAP showed intracellular palmitate de novo synthesis was equally blocked while cholesterol labeling was increased by C75 and luteolin treatments. Nucleotide RNA ribose turnover via the oxidative branch of the pentose cycle was also reduced more with luteolin than C75. Luteolin inhibited entry of glucose carbons into the TCA cycle. Overall luteolin inhibited fatty and nucleic acid synthesis as well as energy production with equal potency to C75. Luteolin had a more favorable profile of controlling acetate use for bypass fluxes such as cholesterol synthesis than C75. Therefore luteolin may act as a low-toxicity FAS inhibitor in pancreatic cancer. Funding: Hirshberg Foundation for Pancreatic Cancer Research and NIH 1P01AT003960.

1,2-13C2]-D-Glucose Profiles of the Serum, Liver, Pancreas, and DMBA-Induced Pancreatic Tumors of Rats

In vitro stable isotope glucose tracer studies indicate that undifferentiated cells of the pancreas use glucose primarily through the nonoxidative reactions of the pentose cycle for nucleic acid ribose synthesis, whereas normal or less transformed cells primarily use the oxidative branch of the cycle. The pancreatic heads of 4 groups (5/group) of male rats were implanted with time-release pellets designed to deliver placebo or 7,12-dimethylbenzanthracene (DMBA) at 11, 33, or 56 mg/d. Four weeks after pancreatic exposure to DMBA, [1,2-C2]-D-glucose tracer (1 g/kg) was injected intraperitoneally followed by sera collection at 1 and 2 hours and harvest of tumors, adjacent pancreatic tissue, and sera at 3 hours. Tumors (2-9 mm) were found across DMBA groups, with the largest in the high-dose group (> or =5 mm). Selective monitoring by gas chromatography-mass spectrometry of the doubly-labeled [1,2-C2]-D-ribose of RNA, which requires nonoxidative synthesis in the pentose cycle, showed a 2.8-, 2.9-, and 5.7-fold increase in pancreatic tumors. Liver and adjacent pancreas preferentially produced [1-C1]-D-ribose through the oxidative reactions of the cycle. Tumor-bearing animals also cleared and recycled tracer glucose at a faster rate. Simultaneous selective positional ion monitoring of C-labeled metabolites and their mass isotopomers in tissues and blood opens new avenues for the early detection and response to therapy testing of pancreatic cancer using GC-MS and/or magnetic resonance imaging-based methods. This study emphasizes the benefits of stable isotope-based dynamic metabolic profiling, when applied in vivo, and the several advantages it offers to positron emission tomography.

Detection of Resistance to Imatinib by Metabolic Profiling

Acquired resistance to imatinib mesylate is an increasing and continued challenge in the treatment of BCR-ABL tyrosine kinase positive leukemias as well as gastrointestinal stromal tumors. Stable isotope-based dynamic metabolic profiling (SIDMAP) studies conducted in parallel with the development and clinical testing of imatinib revealed that this targeted drug is most effective in controlling glucose transport, direct glucose oxidation for RNA ribose synthesis in the pentose cycle, as well as de novo long-chain fatty acid synthesis. Thus imatinib deprives transformed cells of the key substrate of macromolecule synthesis, malignant cell proliferation, and growth. Tracer-based magnetic resonance spectroscopy studies revealed a restitution of mitochondrial glucose metabolism and an increased energy state by reversing the Warburg effect, consistent with a subsequent decrease in anaerobic glycolysis. Recent in vitro SIDMAP studies that involved myeloid cells isolated from patients who developed resistance against imatinib indicated that non-oxidative ribose synthesis from glucose and decreased mitochondrial glucose oxidation are reliable metabolic signatures of drug resistance and disease progression. There is also evidence that imatinib-resistant cells utilize alternate substrates for macromolecule synthesis to overcome limited glucose transport controlled by imatinib. The main clinical implications involve early detection of imatinib resistance and the identification of new metabolic enzyme targets with the potential of overcoming drug resistance downstream of the various genetic and BCR-ABL-expression derived mechanisms. Metabolic profiling is an essential tool used to predict, clinically detect, and treat targeted drug resistance. This need arises from the fact that targeted drugs are narrowly conceived against genes and proteins but the metabolic network is inherently complex and flexible to activate alternative macromolecule synthesis pathways that targeted drugs fail to control.

Metabolic biomarker and kinase drug target discovery in cancer using stable isotope-based dynamic metabolic profiling (SIDMAP).

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and re-distribution among biosynthetic pathways. This results in a proliferating phenotype with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited de novo fatty acid synthesis and TCA cycle glucose oxidation. This primarily non-oxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment. Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-(13)C(2)]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

The Stable Isotope-based Dynamic Metabolic Profile of Butyrate-induced HT29 Cell Differentiation

Stable isotope-based dynamic metabolic profiling is applied in this paper to elucidate the mechanism by which butyrate induces cell differentiation in HT29 cells. We utilized butyrate-sensitive (HT29) cells incubated with [1,2-13C2]glucose or [1,2-13C2]butyrate as single tracers to observe the changes in metabolic fluxes in these cells. In HT29 cells, increasing concentrations of butyrate inhibited glucose uptake, glucose oxidation, and nucleic acid ribose synthesis in a dose-dependent fashion. Glucose carbon utilization for de novo fatty acid synthesis and tricarboxylic acid cycle flux was replaced by butyrate. We also demonstrated that these changes are not present in butyrate-resistant pancreatic adenocarcinoma MIA cells. The results suggest that the mechanism by which colon carcinoma cells acquire a differentiated phenotype is through a replacement of glucose for butyrate as the main carbon source for macromolecule biosynthesis and energy production. This provides a better understanding of cell differentiation through metabolic adaptive changes in response to butyrate in HT29 cells, demonstrating that variations in metabolic pathway substrate flow are powerful regulators of tumor cell proliferation and differentiation.