HepatoDyn: A Dynamic Model of Hepatocyte Metabolism That Integrates 13C Isotopomer Data.

The liver performs many essential metabolic functions, which can be studied using computational models of hepatocytes. Here we present HepatoDyn, a highly detailed dynamic model of hepatocyte metabolism. HepatoDyn includes a large metabolic network, highly detailed kinetic laws, and is capable of dynamically simulating the redox and energy metabolism of hepatocytes.

Overexpression of p53 Improves Blood Glucose Control in an Insulin Resistant Diabetic Mouse Model.

Objective: This paper aimed to assess the physiological effects of p53 on glucose homeostasis in vivo.

Methods: A recombinant adenoviral p53 (rAd-p53) vector was administered to insulin-resistant diabetic mice. Intraperitoneal glucose tolerance test was performed in all groups of mice.

Impaired expression of neuronal nitric oxide synthase in the gracile nucleus is involved in neuropathic changes in Zucker Diabetic Fatty rats with and without 2,5-hexanedione intoxication.

These studies examined the influence of 2,5-hexanedione (2,5-HD) intoxication on expression of neuronal nitric oxide synthase (nNOS) in the brainstem nuclei in Zucker Diabetic Fatty (ZDF) vs. lean control (LC) rats. Functional neuropathic changes were also investigated following axonal damage and impaired axonal transport induced by the treatment.

Asparagine depletion potentiates the cytotoxic effect of chemotherapy against brain tumors.

Unlabelled: Targeting amino acid metabolism has therapeutic implications for aggressive brain tumors. Asparagine is an amino acid that is synthesized by normal cells. However, some cancer cells lack asparagine synthetase (ASNS), the key enzyme for asparagine synthesis.

Inhibition of transketolase by oxythiamine altered dynamics of protein signals in pancreatic cancer cells.

Oxythiamine (OT), an analogue of anti-metabolite, can suppress the nonoxidative synthesis of ribose and induce cell apoptosis by causing a G1 phase arrest in vitro and in vivo. However, the molecular mechanism remains unclear yet. In the present study, a quantitative proteomic analysis using the modified SILAC method (mSILAC) was performed to determine the effect of metabolic inhibition on dynamic changes of protein expression in MIA PaCa-2 cancer cells treated with OT at various doses (0 μM, 5 μM, 50 μM and 500 μM) and time points (0 h, 12 h and 48 h).

Peroxisome proliferator-activated receptor gamma modulation and lipogenic response in adipocytes of small-for-gestational age offspring.

Background: Small-for-gestational age (SGA) at birth increases risk of development of adult obesity and insulin resistance. A model of SGA rat offspring has been shown to exhibit increased adipose tissue expression of a key adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), and increased fatty acid de novo synthesis during the nursing period, prior to onset of obesity. PPARγ agonists have been studied for potential use in the prevention of insulin resistance.

Inhibition of glycogen phosphorylation induces changes in cellular proteome and signaling pathways in MIA pancreatic cancer cells.

Objectives: Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells.

Methods: We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (5-chloro-1H-indole-2-carboxylic acid [1-(4-fuorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2 oxoethyl] amide) (25, 50, and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC (stable isotope labeling with amino acids in cell culture) method.

Effects of a novel cystine-based glutathione precursor on oxidative stress in vascular smooth muscle cells.

Chronic kidney disease (CKD) is associated with accelerated atherosclerosis and cardiovascular disease, which is largely mediated by oxidative stress. We investigated the effect of three glutathione (GSH) precursors: N-acetyl-cysteine (NAC), cystine as the physiological carrier of cysteine in GSH with added selenomethionine (F1), and NAC fortified with selenomethionine (F2) on oxidative stress induced by spermine (a uremic toxin) in cultured human aortic vascular smooth muscle cells (VSMC). VSMC were exposed to spermine (15 microM) with or without the given antioxidants (dose 50, 100, 200, and 500 microg/ml) or vehicle (control) and assessed for intracellular GSH levels, 4-hydroxy-trans-2-nonenal (4-HNE), and incorporation of 13C from glucose into alanine and protein.

Histone deacetylase inhibition results in a common metabolic profile associated with HT29 differentiation.

Cell differentiation is an orderly process that begins with modifications in gene expression. This process is regulated by the acetylation state of histones. Removal of the acetyl groups of histones by specific enzymes (histone deacetylases, HDAC) usually downregulates expression of genes that can cause cells to differentiate, and pharmacological inhibitors of these enzymes have been shown to induce differentiation in several colon cancer cell lines.

Proteomics in bone research.

Osteoporosis is prevalent among the elderly and is a major cause of bone fracture in this population. Bone integrity is maintained by the dynamic processes of bone resorption and bone formation (bone remodeling). Osteoporosis results when there is an imbalance of the two counteracting processes.

Inhibition of protein phosphorylation in MIA pancreatic cancer cells: confluence of metabolic and signaling pathways.

Oxythiamine (OT), a transketolase inhibitor, is known to inhibit pancreatic cancer cell proliferation. In this study, we investigated the effect of inhibition of the transketolase pathway on signaling pathways in MIA PaCa cancer cells using in-house proteomic techniques. We hypothesized that OT alter protein phosphorylation thus affecting cell cycle arrest and cell proliferation.

Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media.

Objectives: A new method of determining protein turnover by labeling protein with N amino acids was used in conjunction with serum-free cell culture to profile secreted proteins that are released by MIA PaCa-2 pancreatic cancer cells in culture.

Methods: MIA PaCa-2 cells were first cultured in Dulbecco modified Eagle medium (Gibco by Invitrogen, Carlsbad, Calif) with 10% fetal bovine serum, then in serum-free modified Eagle medium with or without 50% N algal amino acid mixture. The effect of oxythiamine chloride on secreteome was studied.

Quantitative proteomics and biomarker discovery in human cancer.

“Early diagnosis and prevention is a key factor in reducing the mortality and morbidity of cancer. However, currently available screening tools lack enough sensitivity for early diagnosis. It is important to develop noninvasive techniques and methods that can screen and identify asymptomatic patients who have cancer.

Characterization of the metabolic changes underlying growth factor angiogenic activation: identification of new potential therapeutic targets.

Angiogenesis is a fundamental process to normal and abnormal tissue growth and repair, which consists of recruiting endothelial cells toward an angiogenic stimulus. The cells subsequently proliferate and differentiate to form endothelial tubes and capillary-like structures. Little is known about the metabolic adaptation of endothelial cells through such a transformation.

Ethanol diversely alters palmitate, stearate, and oleate metabolism in the liver and pancreas of rats using the deuterium oxide single tracer.

Objective: To determine tissue-specific effects of alcohol on fatty acid synthesis and distribution as related to functional changes in triglyceride transport and membrane formation.

Methods: Tissue fatty acid profile and de novo lipogenesis were determined in adult male Wistar rats after 5 weeks of ethanol feeding using deuterated water and gas chromatography/mass spectrometry. Liver and pancreas fatty acid profiles and new synthesis fractions were compared with those from control rats on an isocaloric diet.

Quantitative proteomics: measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells.

Pancreatic cancer MIA PaCa cells were cultured in the presence and absence of (15)N amino acids mixture for 72 h. During protein synthesis, the incorporation of (15)N amino acids results in a new mass isotopomer distribution in protein, which is approximated by the concatenation of two binomial distributions of (13)C and (15)N. The fraction of protein synthesis (FSR) can thus be determined from the relative intensities of the "labeled" (new) and the "unlabeled" (old) spectra.

Compartmentalization of stearoyl-coenzyme A desaturase 1 activity in HepG2 cells.

Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the conversion of stearate (18:0) to oleate (18:1n-9) and of palmitate (16:0) to palmitoleate (16:1), which are key steps in triglyceride synthesis in the fatty acid metabolic network. This study investigated the role of SCD1 in fatty acid metabolism in HepG2 cells using SCD1 inhibitors and stable isotope tracers. HepG2 cells were cultured with [U-(13)C]stearate, [U-(13)C]palmitate, or [1,2-(13)C]acetate and (1) DMSO, (2) compound CGX0168 or CGX0290, or (3) trans-10,cis-12 conjugated linoleic acid (CLA).

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.

Nutrient-gene interaction: metabolic genotype-phenotype relationship.

The U.S. Department of Health and Human Services (DHHS)/USDA Dietary Guidelines for Americans is a science and population evidence-based guide on diet and physical activity, providing advice and recommendations to promote a healthier lifestyle and reduce the risk of chronic diseases, including cancer.

Coordination of peroxisomal beta-oxidation and fatty acid elongation in HepG2 cells.

A major product of mitochondrial and peroxisomal beta-oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal beta-oxidation of long chain fatty acids and the fate of its oxidation products are poorly understood and are the subjects of our research. In this report we describe a study of beta-oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-(13)C(18)]stearate or [U-(13)C(16)] palmitate.

Metabolic strategy of boar spermatozoa revealed by a metabolomic characterization.

Metabolomic characteristics in boar spermatozoa were studied using [1,2-(13)C(2)]glucose and mass isotopomer analysis. In boar spermatozoa, glycolysis was the main pathway of glucose utilization producing lactate/pyruvate, whereas no gluconeogenesis was seen. Slight glycogen synthesis through the direct pathway and some incorporation of pyruvate into the Krebs cycle also took place.

Defective RNA ribose synthesis in fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA).

Fibroblasts from patients with thiamine-responsive megaloblastic anemia (TRMA) syndrome with diabetes and deafness undergo apoptotic cell death in the absence of supplemental thiamine in their cultures. The basis of megaloblastosis in these patients has not been determined. Here we use the stable [1,2-13C2]glucose isotope-based dynamic metabolic profiling technique to demonstrate that defective high-affinity thiamine transport primarily affects the synthesis of nucleic acid ribose via the nonoxidative branch of the pentose cycle.