Shu Lim

Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose

We present a single-tracer method for the study of the pentose phosphate pathway (PPP) using [1,2-13C2]glucose and mass isotopomer analysis. The metabolism of [1,2-13C2]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions results in unique pentose and lactate isotopomers with either one or two13C substitutions. The distribution of these isotopomers was used to estimate parameters of the PPP using the model of Katz and Rognstad (J. Katz and R. Rognstad. Biochemistry 6: 2227-2247, 1967). Mass and position isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 30% enriched [1,2-13C2]glucose were determined using gas chromatography-mass spectrometry. After 24-72 h incubation, 1.9% of lactate molecules in the medium contained one 13C substitution ( m 1) and 10% contained two 13C substitutions ( m 2). A similar m 1-to- m 2ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose was 5.73 ± 0.52% of the glucose flux, which was identical to the value of PC (5.55 ± 0.73%) determined by separate incubations with [1-13C] and [6-13C]glucose.13C was found to be distributed in four ribose isotopomers ([1-13C]-, [5-13C]-, [1,2-13C2]-, and [4,5-13C2]ribose). The observed ribose isotopomer distribution was best matched with that provided from simulation by substituting 0.032 for TK and 0.85 for TA activity relative to glucose uptake into the model of Katz and Rognstad. The use of [1,2-13C2]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.

K- ras Codon-Specific Mutations Produce Distinctive Metabolic Phenotypes in Human Fibroblasts

Among K-ras mutations, codon 12 mutations have been identified as those conferring a more aggressive phenotype. This aggressiveness is primarily associated with slow proliferation but greatly increased resistance to apoptosis. Using transfected NIH3T3 fibroblasts with a mutated K-ras minigene either at codon 12 (K12) or at codon 13 (K13), and taking advantage of [1,2-13C2]glucose tracer labeling, we show that codon 12 mutant K-ras (K12)-transformed cells exhibit greatly increased glycolysis with only a slight increase in activity along pathways that produce nucleic acid and lipid synthesis precursors in the oxidative branch of the pentose phosphate pathway and via pyruvate dehydrogenase flux. K13 mutants display a modest increase in anaerobic glycolysis associated with a large increase in oxidative pentose phosphate pathway activity and pyruvate dehydrogenase flux. The distinctive differences in metabolic profiles of K12 and K13 codon mutated cells indicate that a strong correlation exists between the flow of glucose carbons towards either increased anaerobic glycolysis, and resistance to apoptosis (K12), or increased macromolecule synthesis, rapid proliferation, and increased sensitivity to apoptosis.

Genistein inhibits nonoxidative ribose synthesis in MIA pancreatic adenocarcinoma cells: a new mechanism of controlling tumor growth.

Genistein is a plant isoflavonoid bearing potent tumor growth–regulating characteristics. This effect of genistein has been attributed partially to its tyrosine kinase–regulating properties, resulting in cell-cycle arrest and limited angiogenesis. Genistein has been used in chemotherapy-resistant cases of advanced leukemia with promising results. Here we demonstrate that genistein primarily affects nucleic acid synthesis and glucose oxidation in tumor cells using the [1,2-13C2]glucose isotope as the single tracer and gas chromatography/mass spectrometry to follow various intracellular glucose metabolites. The ribose fraction of RNA demonstrated a rapid 4.6%, 16.4%, and 46.3% decrease in isotope uptake through the nonoxidative branch of the pentose cycle and a sharp 4.8%, 24.6%, and 48% decrease in 13CO2 release from glucose after 2, 20, and 200 &mgr;mol/L genistein treatment, respectively. Fatty acid synthesis and the 13C enrichment of acetyl units were not significantly affected by genistein treatment. De novo glycogen synthesis from media glucose was not detected in cultured MIA cells. It can be concluded from these studies that genistein controls tumor growth primarily through the regulation of glucose metabolism, specifically targeting glucose carbon incorporation into nucleic acid ribose through the nonoxidative steps of the pentose cycle, which represents a new paradigm for the antiproliferative action of a plant phytochemical.

Dynamic profiling of the glucose metabolic network in fasted rat hepatocytes using [1,2-13C2]glucose

Recent studies in metabolic profiling have underscored the importance of the concept of a metabolic network of pathways with special functional characteristics that differ from those of simple reaction sequences. The characterization of metabolic functions requires the simultaneous measurement of substrate fluxes of interconnecting pathways. Here we present a novel stable isotope method by which the forward and reverse fluxes of the futile cycles of the hepatic glucose metabolic network are simultaneously determined. Unlike previous radio-isotope methods, a single tracer [1,2-13C2]D-glucose and mass isotopomer analysis is used. Changes in fluxes of substrate cycles, in response to several gluconeogenic substrates, in isolated fasted hepatocytes from male Wistar rats were measured simultaneously. Incubation with these substrates resulted in a change in glucose-6-phosphatase/glucokinase and glycolytic/gluconeogenic flux ratios. Different net redistributions of intermediates in the glucose network were observed, resulting in distinct metabolic phenotypes of the fasted hepatocytes in response to each substrate condition. Our experimental observations show that the constraints of concentrations of shared intermediates, and enzyme kinetics of intersecting pathways of the metabolic network determine substrate redistribution throughout the network when it is perturbed. These results support the systems-biology notion that network analysis provides an integrated view of the physiological state. Interaction between metabolic intermediates and glycolytic/gluconeogenic pathways is a basic element of cross-talk in hepatocytes, and may explain some of the difficulties in genotype and phenotype correlation.

Fatty Acid Cycling in Human Hepatoma Cells and the Effects of Troglitazone

Fatty acid cycling by chain shortening/elongation in the peroxisomes is an important source of fatty acids for membrane lipid synthesis. Its role in the homeostasis of nonessential fatty acids is poorly understood. We report here a study on the cycling of saturated fatty acids and the effects of troglitazone in HepG2 cells in culture using [U-13C]stearate or [U-13C]oleate and mass isotopomer analysis. HepG2 cells were grown in the presence of 0.7 mmol/liter [U-13C]stearate or [U-13C]oleate, and in the presence and absence of 50 μm troglitazone for 72 h. Fatty acids extracted from cell pellets after saponification were analyzed by gas chromatography/mass spectrometry. Peroxisomal β-oxidation of uniformly 13C-labeled stearate (C18:0) and oleate (C18:1) resulted in chain shortening and produced uniformly labeled palmitate (C16:0) and palmitoleate (C16:1). In untreated cells, 16% of C16:0 was derived from C18:0 and 26% of C16:1 from C18:1 by chain shortening. Such contributions were significantly increased by troglitazone to 23.6 and 36.6%, respectively (p < 0.001). Desaturation of stearate contributed 67% of the oleate, while reduction of oleate contributed little to stearate (2%). The desaturation of C18:0 to C18:1 was not affected by troglitazone. Our results demonstrated a high degree of recycling of C18:0 and C18:1 to C16:0 and C16:1 through chain shortening and desaturation. Chain shortening was accompanied by chain elongation in the synthesis of other long chain fatty acids. Troglitazone specifically increased recycling by peroxisomal β-oxidation of C18 to C16 fatty acids, and the interconversion of long chain fatty acids was associated with reduced de novo lipogenesis.

Determination of protein synthesis in vivo using labeling from deuterated water and analysis of MALDI-TOF spectrum.

This paper describes a method of determining protein synthesis and turnover using in vivo labeling of protein with deuterated water and analysis of matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) spectrum. Protein synthesis is calculated using mass isotopomer distribution analysis instead of precursor to product amino acid enrichment ratio. During protein synthesis, the incorporation of deuterium from water changes the mass isotopomer distribution (isotope envelop) according to the number of deuterium atoms (0, 1, 2, 3, etc.) incorporated, and the distribution of the protein with 0, 1, 2, 3,... atoms of deuterium follows a binomial distribution. A mathematical algorithm by which the distribution of deuterium isotopomers can be extracted from the observed MALDI-TOF spectrum is presented. Since deuterium isotopomers are unique to newly synthesized proteins, the quantitation of their distribution provides a method for the quantitation of newly synthesized proteins. The combined use of postsource decay sequence identification and mass isotopomer distribution analysis makes the use of in vivo labeling with deuterated water a precise method to determine specific protein synthesis.