Expression Levels Of Metabolic Enzymes Research Articles

Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Identification of kynurenine and quinolinic acid as promising serum biomarkers for drug-induced interstitial lung diseases

BackgroundDrug-induced interstitial lung disease (DILD) is a lung injury caused by various types of drugs and is a serious problem in both clinical practice and drug development. Clinical management of the condition would be improved if there were DILD-specific biomarkers available; this study aimed to meet that need.MethodsBiomarker candidates were identified by non-targeted metabolomics focusing on hydrophilic molecules, and further validated by targeted approaches using the serum of acute DILD patients, DILD recovery patients, DILD-tolerant patients, patients with other related lung diseases, and healthy controls.ResultsSerum levels of kynurenine and quinolinic acid (and kynurenine/tryptophan ratio) were elevated significantly and specifically in acute DILD patients. The diagnostic potentials of these biomarkers were superior to those of conventional lung injury biomarkers, Krebs von den Lungen-6 and surfactant protein-D, in discriminating between acute DILD patients and patients with other lung diseases, including idiopathic interstitial pneumonia and lung diseases associated with connective tissue diseases. In addition to identifying and evaluating the biomarkers, our data showed that kynurenine/tryptophan ratios (an indicator of kynurenine pathway activation) were positively correlated with serum C-reactive protein concentrations in patients with DILD, suggesting the potential association between the generation of these biomarkers and inflammation. Our in vitro experiments demonstrated that macrophage differentiation and inflammatory stimulations typified by interferon gamma could activate the kynurenine pathway, resulting in enhanced kynurenine levels in the extracellular space in macrophage-like cell lines or lung endothelial cells. Extracellular quinolinic acid levels were elevated only in macrophage-like cells but not endothelial cells owing to the lower expression levels of metabolic enzymes converting kynurenine to quinolinic acid. These findings provide clues about the molecular mechanisms behind their specific elevation in the serum of acute DILD patients.ConclusionsThe serum concentrations of kynurenine and quinolinic acid as well as kynurenine/tryptophan ratios are promising and specific biomarkers for detecting and monitoring DILD and its recovery, which could facilitate accurate decisions for appropriate clinical management of patients with DILD.

Utility of human induced pluripotent stem cell-derived small intestinal epithelial cells for pharmacokinetic, toxicological, and immunological studies

The small intestine, which plays a crucial role in the absorption and metabolism of drugs and foods, serves as a target organ for drug-induced toxicity and immune interactions with functional foods and intestinal bacteria. Current alternative models of the human small intestine, such as Caco-2 cells and experimental animals, have limitations due to variations in the expression levels of metabolic enzymes, transporters, and receptors. This study presents investigations into the utility of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) for pharmacokinetic, toxicological, and immunological studies, respectively. While hiSIECs displayed small intestinal epithelial cell characteristics and barrier function, they demonstrated pharmacokinetic properties such as cytochrome P450 3A4/5 activity equivalent to human primary enterocytes and stable P-glycoprotein activity. These cells also demonstrated potential for assessing two forms of intestinal toxicity caused by anticancer drugs and gamma-secretase inhibitors, displaying immune responses mediated by toll-like and fatty acid receptors while serving as an inflammatory gut model through the addition of tumor necrosis factor alpha and interferon gamma. Overall, hiSIECs hold promise as an in vitro model for assessing pharmacokinetics, toxicity, and effects on the intestinal immunity of pharmaceuticals, functional foods, supplements, and intestinal bacteria.

High Dimensional Single-Cell Profiling Identifies Immune and Metabolic Heterogeneity in Immune Thrombocytopenia

Introduction Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by excessive platelet destruction and decreased platelet production. Ongoing efforts to characterize the pathophysiology of ITP have yielded certain insights, but the details of the immune and metabolic characteristics of the disease remain elusive. Tacrolimus has been demonstrated to be a promising therapeutic option for ITP patients in our previous report ( 63rd ASH, 2021), but the mechanisms are incompletely understood. Methods A total of 15 patients diagnosed with ITP and 8 healthy control (HC) individuals were enrolled between March and July 2023 in this study. Peripheral blood from ITP patients was collected at two time points: when admitted to the cohort without previous tacrolimus treatment and when receiving tacrolimus treatment for at least 28 days. We employed mass cytometry (cytometry by time-of-flight, CyTOF) with a panel of 27 phenotypic, 7 metabolic, and 8 functional markers to explore the immune landscape, metabolic characteristics, and phosphorylated signaling states at single-cell resolution. Additionally, a 10x Genomics platform was applied to perform single-cell RNA sequencing (scRNA-seq). Primary findings from our transcriptomic and proteomic analyses were further confirmed and extended by flow cytometry and RT-PCR. Results Using t-distributed stochastic neighbor embedding, a CyTOF panel of 42 markers partitioned the 32 clusters into 7 subsets (CD4+ T cells, CD8+ T cells, B cells, NK cells, monocytes/macrophages, dendritic cells, and γδ T cells). Quantification of immune cell distribution across cell clusters showed a relatively higher level of CD8+ T cells and lower CD4+:CD8+ T-cell ratios in the patients with ITP than in the HC individuals. Additionally, the proportion of NK cells was significantly lower in patients than in control individuals. With further investigation of CD4+ and CD8+ T cells, we observed a decrease of naïve T cells and Treg cells and skewed accumulation of Th1 cells and Tc1 cells in patients with ITP relative to HC individuals. We further investigated the metabolic and functional status of T cells with CyTOF. One-carbon metabolism (MTHFD2 and SHMT2) was found to be elevated in Th1 cells from ITP patients compared with those from HC individuals. Based on CyTOF analysis as well as flow cytometry validation, a subgroup of Th1 cells with elevated ERK1/2 phosphorylation and MTHFD2 expression was speculated to be associated with the pathogenesis of ITP. A longitudinal study demonstrated that the dysregulated immune subset exhibited HC-like expression after treatment with tacrolimus. To gain further insights into the molecular characteristics of immune cell subsets and metabolic status that are differentially expanded in ITP patients, we performed scRNA-seq analysis on peripheral blood mononuclear cell (PBMC) isolated from 5 ITP patients pre- or posttreatment with tacrolimus and 3 HC individuals. Similar to the CyTOF data, a relative increase in Th1 cells and memory T cells and a reduction in naïve T cells and Treg cells were found in the ITP patient group compared with the HC group. Among the 9 subclustered T-lineage cell types, the CD4+ CXCR3+ IFNγ+ Th1 cells with functional pathways such as TCR pathway activation, IL-17 signaling pathway, and MAPK signaling pathway draw our attention. Next, we examined the metabolic status of immune cells of ITP patients to elucidate the underlying mechanisms of dysfunction of immune cells. Our analysis suggested that aberrant metabolism including glucose metabolism, nucleotide metabolism, and one-carbon metabolism caused by Th1 cells were involved in the dysregulated immune response in ITP. Moreover, ITP patients had elevated levels of metabolic checkpoints (mTOR, HIF-1α, and c-Myc) and low expression of immune checkpoint regulators (CTLA4, TIM3, and LAG3) in Th1 cells. Patients who recovered from tacrolimus treatment had a lower proportion of Th1 cells and lower expression levels of metabolic enzymes than nonresponsive patients. Conclusion Using integrated single-cell proteomic and transcriptomic profiling, this study depicts the broad immune and metabolic landscape of PBMC from ITP patients. In particular, our data unveil that inflammatory Th1 cells play a critical role in the pathophysiology of ITP and suggest a novel therapeutic opportunity of tacrolimus towards reprogramming immune metabolism.

Ecological network analysis reveals cancer-dependent chaperone-client interaction structure and robustness

Cancer cells alter the expression levels of metabolic enzymes to fuel proliferation. The mitochondrion is a central hub of metabolic reprogramming, where chaperones service hundreds of clients, forming chaperone-client interaction networks. How network structure affects its robustness to chaperone targeting is key to developing cancer-specific drug therapy. However, few studies have assessed how structure and robustness vary across different cancer tissues. Here, using ecological network analysis, we reveal a non-random, hierarchical pattern whereby the cancer type modulates the chaperones’ ability to realize their potential client interactions. Despite the low similarity between the chaperone-client interaction networks, we highly accurately predict links in one cancer type based on another. Moreover, we identify groups of chaperones that interact with similar clients. Simulations of network robustness show that this group structure affects cancer-specific response to chaperone removal. Our results open the door for new hypotheses regarding the ecology and evolution of chaperone-client interaction networks and can inform cancer-specific drug development strategies.

The polyol pathway is an evolutionarily conserved system for sensing glucose uptake.

Cells must adjust the expression levels of metabolic enzymes in response to fluctuating nutrient supply. For glucose, such metabolic remodeling is highly dependent on a master transcription factor ChREBP/MondoA. However, it remains elusive how glucose fluctuations are sensed by ChREBP/MondoA despite the stability of major glycolytic pathways. Here, we show that in both flies and mice, ChREBP/MondoA activation in response to glucose ingestion involves an evolutionarily conserved glucose-metabolizing pathway: the polyol pathway. The polyol pathway converts glucose to fructose via sorbitol. It has been believed that this pathway is almost silent, and its activation in hyperglycemic conditions has deleterious effects on human health. We show that the polyol pathway regulates the glucose-responsive nuclear translocation of Mondo, a Drosophila homologue of ChREBP/MondoA, which directs gene expression for organismal growth and metabolism. Likewise, inhibition of the polyol pathway in mice impairs ChREBP's nuclear localization and reduces glucose tolerance. We propose that the polyol pathway is an evolutionarily conserved sensing system for glucose uptake that allows metabolic remodeling.

Generation of Human-Induced Pluripotent Stem Cell-Derived Functional Enterocyte-Like Cells for Pharmacokinetic Studies

SummaryWe aimed to establish an in vitro differentiation procedure to generate matured small intestinal cells mimicking human small intestine from human-induced pluripotent stem cells (iPSCs). We previously reported the efficient generation of CDX2-expressing intestinal progenitor cells from embryonic stem cells (ESCs) using 6-bromoindirubin-3′-oxime (BIO) and (3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester (DAPT) to treat definitive endodermal cells. Here, we demonstrate the generation of enterocyte-like cells by culturing human iPSC-derived intestinal progenitor cells on a collagen vitrigel membrane (CVM) and treating cells with a simple maturation medium containing BIO, DMSO, dexamethasone, and activated vitamin D3. Functional tests further confirmed that these iPSC-derived enterocyte-like cells exhibit P-gp- and BCRP-mediated efflux and cytochrome P450 3A4 (CYP3A4)-mediated metabolism. We concluded that hiPS cell-derived enterocyte-like cells can be used as a model for the evaluation of drug transport and metabolism studies in the human small intestine.

The diagnostic value of lower glucose consumption for IDH1 mutated gliomas on FDG-PET

BackgroundNon-invasive diagnosis of IDH1 mutation for gliomas has great clinical significance, and PET has natural advantage to detect metabolism, as IDH mutated gliomas share lower glucose consumption.MethodsClinical data of patients with gliomas and 18F-FDG PET were retrospectively reviewed. Receiver operating characteristic curve (ROC) analysis was conducted, and standard uptake value (SUV) was estimated in combination with grades or IDH1 mutation. The glucose consumption was investigated with U251 cells expressing wild-type or mutated IDH1 by glucose assay. Quantification of glucose was determined by HPLC in clinical tissues. Meanwhile, bioinformatics and western blot were applied to analyze the expression level of metabolic enzymes (e.g. HK1, PKM2, PC) in gliomas.ResultsSeventy-one glioma cases were enrolled, including 30 carrying IDH1 mutation. The sensitivity and specificity dependent on SUVmax (3.85) predicting IDH1 mutation reached 73.2 and 86.7%, respectively. The sensitivity and specificity of differentiating grades by SUVmax (3.1) were 92.3 and 64.4%, respectively. Glucose consumption of U251 IDH1 mutant cells (0.209 ± 0.0472 mg/ml) was obviously lower than IDH1wild-type cells (0.978 ± 0.0773 mg/ml, P = 0.0001) and astrocyte controls (0.335 ± 0.0592 mg/ml, P = 0.0451). Meanwhile, the glucose quantity in IDH1mutant glioma samples were significantly lower than those in IDH1 wild-type tissues (1.033 ± 1.19608 vs 6.361 ± 4.3909 mg/g, P = 0.0051). Silico analysis and western blot confirmed that HK1 and PKM2 in IDH1 wild-type gliomas were significantly higher than in IDH1 mutant group, while PC was significantly higher in IDH1 mutant gliomas.ConclusionSUVmax on PET can predict IDH1 mutation with adequate sensitivity and specificity, as is supported by reduced glucose consumption in IDH1 mutant gliomas.

A preliminary metatranscriptomic insight of eggshells conditioning on substrates metabolism during food wastes anaerobic fermentation

The anaerobic treatment of food wastes (FW) for resource recovery has been extensively studied. However, the information on the traits of functional genes and enzymes for substrates metabolisms and their associations with microbial community are little. In this study, the influences of eggshells conditioning on the substrates metabolism for volatile fatty acids production (VFAs) in the process of FW fermentation were investigated at genetic levels by using the metatranscriptomic approach. The obtained results suggested that the critical genes involved in the carbohydrate and protein metabolisms (i.e. pgmB, GPI, glsA, pyrB and etc.) were up-regulated in the eggshell-conditioned reactor, which were beneficial to the bioconversion of macromolecule organics during FW fermentation. Moreover, the functional genes related with the intermediate products metabolism (i.e. pyruvate acid, butanoate) also exhibited differential genetic expression levels, which resulted in the alteration of microbial metabolic pathways and contributed to the acetic and butyric acids accumulation. In addition, a preliminary association of microbial distribution and genetic expressions was analyzed. The distinct distribution of microbial community in different FW fermentation systems affected the corresponding microbial contribution to those genetic expression levels of metabolic enzymes involved in VFAs production. This study would provide new insights of the underlying mechanism of VFAs promotion in the eggshell-conditioned FW fermentation process from the perspectives of substrates metabolisms at genetic and functional traits.

Variability in metabolic cross-feeding behaviors in breast cancer and response to neoadjuvant chemotherapy.

e12645 Background: Success of neoadjuvant chemotherapy (NACT) varies by intrinsic subtype of a patient’s breast cancer (e.g., Normal-like, Luminal A/B, HER2-enriched, Triple Negative). The metabolic rate of a given tumor is implicated in response to chemotherapies, as many act by killing rapidly dividing cells. Metabolic dis-regulation opens avenues for cancer cells to exploit environmental niches arising in the tumor microenvironment (TME). Focusing on the role the TME plays in defining cancer behavior, we undertook a theoretical investigation to uncover how metabolic cross-feeding affects subtype behavior and patient response. Methods: An in silico analysis of cross-feeding variability arising from metabolic differences breast cancers was undertaken. TME community models consisting of fatty, glandular, and cancerous tissues’ metabolic behaviors were created. Expression levels of metabolic enzymes for 1222 community patients in TCGA BRCA were used to create models. Models were used in 3D simulations of tumors for 300 patients from the TCIA (Clark et al., J. Digital Imaging, 2013). Cross-feeding trends within each intrinsic subtype were analyzed. Results: Predicted TME metabolic capabilities were compared to literature: LA/LB had lower amino acid consumption than TNBC/HER2+ for asparagine, glutamine (Glu), tryptophan, phenylalanine (van Geldermolsen et al. Oncogone 2016; Furuya et al. Cancer Sci. 2012). Additionally, TNBC tumors produced Glu which was consumed by adipose tissue ( Cao et al. BMC Cancer, 2014), TNBC produced methionine and proline (Kanaan et al. Can. Gen. Prot. 2014), and TNBC/HER2+ cancers consumed high density lipoprotein produced by adipose (Balabum et al. Cancer Met. 2017). The simulations revealed novel cross-feeding behaviors. In general, TNBC/HER2+ relied on glucose as the primary energy source, while LA/LB relied on the amino acids alanine, glycine and Glu. TNBC/HER2+ cancers produced high levels of lactate which was consumed by adipose tissues. Uniquely, glycine produced by cancer was consumed by fat in TNBC cancers Several environmental niches were induced in the healthy tissue by the presence of the cancer; for example, ornithine was predicted to be cross-fed from fatty to glandular tissues in TNBC/HER2+ cancers, and glutamate and inosine from glandular to fatty tissues in HER2+ cancers. Conclusions: Metabolic niches provide opportunities for cancer subtypes. These results suggest that metabolic pathway usage can lead to difference in growth, cross-feeding, and drug efficacy.

A multiscale modelling approach to assess the impact of metabolic zonation and microperfusion on the hepatic carbohydrate metabolism.

The capacity of the liver to convert the metabolic input received from the incoming portal and arterial blood into the metabolic output of the outgoing venous blood has three major determinants: The intra-hepatic blood flow, the transport of metabolites between blood vessels (sinusoids) and hepatocytes and the metabolic capacity of hepatocytes. These determinants are not constant across the organ: Even in the normal organ, but much more pronounced in the fibrotic and cirrhotic liver, regional variability of the capillary blood pressure, tissue architecture and the expression level of metabolic enzymes (zonation) have been reported. Understanding how this variability may affect the regional metabolic capacity of the liver is important for the interpretation of functional liver tests and planning of pharmacological and surgical interventions. Here we present a mathematical model of the sinusoidal tissue unit (STU) that is composed of a single sinusoid surrounded by the space of Disse and a monolayer of hepatocytes. The total metabolic output of the liver (arterio-venous glucose difference) is obtained by integration across the metabolic output of a representative number of STUs. Application of the model to the hepatic glucose metabolism provided the following insights: (i) At portal glucose concentrations between 6–8 mM, an intra-sinusoidal glucose cycle may occur which is constituted by glucose producing periportal hepatocytes and glucose consuming pericentral hepatocytes, (ii) Regional variability of hepatic blood flow is higher than the corresponding regional variability of the metabolic output, (iii) a spatially resolved metabolic functiogram of the liver is constructed. Variations of tissue parameters are equally important as variations of enzyme activities for the control of the arterio-venous glucose difference.

Abstract 2967: Pancreatic cancer cell growth requires lipids released by tumor-induced stroma autophagy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is non-resectable in the majority of patients and highly resistant to chemotherapy, resulting in a poor survival. The tumor microenvironment and hypoxia are important modifiers of cancer progression in PDAC. Understanding the metabolic vulnerabilities of PDAC in the harsh tumor microenvironment may lead to novel therapeutic approaches with improved clinical efficacy. First, we found that PDAC cells showed beneficial effects of co-cultured stroma cells, but only under lipid-free serum conditions. To study the metabolic crosstalk between cancer cells and stroma in more detail, we performed an untargeted metabolomic screen of PDAC cells and fibroblasts co-cultured in normoxia and hypoxia, and performed RNA-seq profiling in parallel. We found that stromal cells are metabolically more responsive to co-culture than cancer cells. PDAC cells induce catabolic carbohydrate and protein metabolism in stromal cells, particularly in hypoxia. In contrast, 13C-based metabolic flux assays demonstrated that stromal cells display enhanced anabolic lipid metabolism in co-culture with PDAC cells. Furthermore, de novo synthesized 13C-labeled fatty acids in stromal cells were taken up by PDAC cells. In particular, PDAC cells showed extensive scavenging of lysophospholipids (lyso-PLs) from the culture medium, which was increased in co-culture under hypoxic conditions. These data were confirmed by analyzing portal vein plasma samples isolated from pancreatic cancer patients before and after surgery. In addition, we found metabolites and expression levels of metabolic enzymes from the glycerophospholipid pathway to be enriched in PDAC cells in co-culture and hypoxia. By using fibroblasts, human pancreatic stellate cells and patient-derived cancer-associated fibroblasts (CAFs), we demonstrate direct transfer of lyso-PLs from stromal to PDAC cells via lipid droplets. The transfer of lyso-PLs was abrogated by pharmacological inhibitors of autophagy, or by siRNA-mediated knockdown of autophagy genes in stromal and tumor cells. These data suggest that PDAC cells cause stroma cells to undergo autophagy, and reprogram stroma metabolism to obtain complex lipid species for their metabolic needs in the lipid-starved tumor microenvironment. Citation Format: Petrus R. De Jong, Sean-Luc Shanahan, Morgan A. Brand, Alejandro D. Campos, Anagha Srirangam, Nikolas Marino, Claudia P. Miller, Olga Zagnitko, Adam D. Richardson, David A. Scott, Brian P. James, Andrew P. Hodges, Ally Perlina, Alexey M. Eroshin, Randall French, Malene Hansen, Sally A. Litherland, Andrew M. Lowy, J. Pablo Arnoletti, Garth Powis. Pancreatic cancer cell growth requires lipids released by tumor-induced stroma autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2967. doi:10.1158/1538-7445.AM2017-2967

PPAR Agonists Promote the Differentiation of Porcine Bone Marrow Mesenchymal Stem Cells into the Adipogenic and Myogenic Lineages

Purpose: The aim of this work was to evaluate the effect of PPAR agonists on the differentiation and metabolic features of porcine mesenchymal stem cells induced to the adipogenic or myogenic lineages. Methods: Bone marrow MSCs from neonate pigs were isolated and identified by cell proliferation, cell surface markers or the gene expression of stem cells (CD44, CD90, CD105 or Oct4 and Nanog, respectively). Cells were differentiated into adipose or muscle cells and treated with the PPAR agonists; adipogenic and myogenic differentiation was promoted by adding these compounds. The expression of PPARγ (an adipose marker) and MyoD1 and MyHC (muscle markers), metabolic changes and expression levels of metabolic enzymes involved in glycolysis, lipogenesis, lipolysis and the pentose phosphate pathway were tested by qPCR. Results: MSCs from neonate pigs exhibited high proliferation and were positive for CD44, CD90 and CD105 markers and Oct4 and Nanog expression. The treatment that promoted the highest expression of PPARγ was 50 µ<smlcap>M</smlcap> of conjugated linoleic acid (CLA) c9 t11 (6.44 ± 0.69-fold, p ≤ 0.0001) in the adipose differentiation, and upregulation of HX2, ACCAα, ATGL, LPL and G6DP (p ≤ 0.0001) and downregulation of PFK and ACCAβ (p ≤ 0.0001) were found. For muscle differentiation, the best treatment was 50 µ<smlcap>M</smlcap> of CLA c10 t12 (59.72 ± 4.72-fold, p ≤ 0.0001), and metabolic changes were upregulation of PFK, ACCAβ, G6DP, CPT1 and PPARβ/δ (p ≤ 0.0001), but no effect was observed with HX2 and ACCAα (p ≥ 0.05). Conclusions: Our results suggest that differentiated cells exhibit a typical cell lineage metabolism and higher efficiencies both in anabolism and catabolism.

Abstract 3379: Metabolic influences of pancreatic tumor microenvironment on pancreatic cancer cell's metabolism

Abstract Pancreatic cancer, the most lethal of solid tumors, is associated with a five-year survival rate and high mortality. The lethality of this tumor stems from lack of early symptoms, inability for detection of cancerous pancreatic lesions, and a diagnosis window that is accompanied by tumor resistance and metastasis. The bulk of the tumor mass, the fibrotic stroma, has been deemed an active player in the initiation and progression of pancreatic ductal adinocarcinoma (PDAC). A number of studies have elucidated the interactions between stromal cells and pancreatic cancer cells (PCCs), and the extracellular matrix (ECM) and PCCs. However, the role of the tumor microenvironment on the metabolic machinery of PCCs remains an active field of investigation. In this study, we investigate the effect of pancreatic stellate cells (PSCs), cancer-associated fibroblasts (CAFs), with or without ECM components, in a two-dimensional or three-dimensional setting on the glycolytic and mitochondrial pathways of patient-derived PCCs. The modulation of the expression levels of metabolic enzymes by the tumor microenvironment was also investigated. The metabolic reprogramming induced in PCCs by normal and reactive pancreatic stroma was investigated by isotopomer flux analysis. Our results reveal that pancreatic reactive stroma differentially upregulates glutamine and arginine metabolism in PCCs. The insights obtained from our work will lead to the development of targeted therapies for stroma and pancreatic cancer cells. Citation Format: Joelle Baddour, Lifeng Yang, Juan C. Marini, Janusz Franco-Barraza, Edna Cukierman, Chaoxin Hu, Anirban Maitra, Deepak Nagrath. Metabolic influences of pancreatic tumor microenvironment on pancreatic cancer cell's metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3379. doi:10.1158/1538-7445.AM2014-3379

Stochasticity of metabolism and growth at the single-cell level

Elucidating the role of molecular stochasticity in cellular growth is central to understanding phenotypic heterogeneity and the stability of cellular proliferation. The inherent stochasticity of metabolic reaction events should have negligible effect, because of averaging over the many reaction events contributing to growth. Indeed, metabolism and growth are often considered to be constant for fixed conditions. Stochastic fluctuations in the expression level of metabolic enzymes could produce variations in the reactions they catalyse. However, whether such molecular fluctuations can affect growth is unclear, given the various stabilizing regulatory mechanisms, the slow adjustment of key cellular components such as ribosomes, and the secretion and buffering of excess metabolites. Here we use time-lapse microscopy to measure fluctuations in the instantaneous growth rate of single cells of Escherichia coli, and quantify time-resolved cross-correlations with the expression of lac genes and enzymes in central metabolism. We show that expression fluctuations of catabolically active enzymes can propagate and cause growth fluctuations, with transmission depending on the limitation of the enzyme to growth. Conversely, growth fluctuations propagate back to perturb expression. Accordingly, enzymes were found to transmit noise to other unrelated genes via growth. Homeostasis is promoted by a noise-cancelling mechanism that exploits fluctuations in the dilution of proteins by cell-volume expansion. The results indicate that molecular noise is propagated not only by regulatory proteins but also by metabolic reactions. They also suggest that cellular metabolism is inherently stochastic, and a generic source of phenotypic heterogeneity.

Influence of CLOCK on cytotoxicity induced by diethylnitrosamine in mouse primary hepatocytes

The Clock gene is a core clock factor that plays an essential role in generating circadian rhythms. In the present study, it was investigated whether the Clock gene affects the response to diethylnitrosamine (DEN)-induced cytotoxicity using mouse primary hepatocytes. DEN-induced cytotoxicity, after 24 h exposure, was caused by apoptosis in hepatocytes isolated from wild-type mouse. On the other hand, Clock mutant mouse ( Clk/ Clk) hepatocytes showed resistance to apoptosis. Because apoptosis is an important pathway for suppressing carcinogenesis after genomic DNA damage, the mechanisms that underlie resistance to DEN-induced apoptosis were examined in Clk/ Clk mouse hepatocytes. The mRNA levels of metabolic enzymes bioactivating DEN and apoptosis-inducing factors before DEN exposure were lower in Clk/ Clk cells than in wild-type cells. The accumulation of p53 and Ser15 phosphorylated p53 after 8 h DEN exposure was seen in wild-type cells but not in Clk/ Clk cells. Caspase-3/7 activity was elevated during 24 h DEN exposure in wild-type cells but not in Clk/ Clk cells. In addition, resistance to DEN-induced apoptosis in Clk/ Clk cells affected the cell viability. These studies suggested that the lower expression levels of metabolic enzymes bioactivating DEN and apoptosis inducing factors affected the resistance to DEN-induced apoptosis in Clk/ Clk cells, and the Clock gene plays an important role in cytotoxicity induced by DEN.

Coupling of Conjugating Enzymes and Efflux Transporters: Impact on Bioavailability and Drug Interactions

Abstract: Conjugating enzymes are traditionally recognized as one of the major biological barriers to the entry of xenobiotics/drugs into systemic circulation and represent one of the main pathways for their elimination. Similar to drugs that undergo extensive phase I metabolism, drugs that undergo extensive conjugation have poor bioavailability and are more prone to metabolism-based drug interactions. Previously, enterohepatic recycling is used to explain why certain xenobiotics have half-lives that are much longer than expected from intravenous injection studies. In addition, changes in expression levels of metabolic enzymes due to chemical induction or suppression are often recognized as the source of drug interaction or toxicity of pollutants and carcinogens. These traditional approaches, whereas yielding highly valuable information, fail to recognize the fact that many conjugates (especially hydrophilic ones) cannot permeate the cell membrane. In the present review, we will focus on the coupling process that involves both conjugating enzymes and efflux transporters. We will briefly review conjugating enzymes capable of producing highly hydrophilic metabolic products. The other focus of this review is on various transporters capable of moving negatively charged hydrophilic conjugates across the cellular membrane. Evidence will support the hypothesis that efficient coupling of the conjugating enzymes and efflux transporters enables enterohepatic recycling and enteric recycling processes. Termed as a "revolving door" theory, the hypothesis focuses on the role played by efflux transporter capable of modulating the cellular excretion of hydrophilic metabolites. Coupling process in intestine, liver and kidney will be discussed with an emphasis on the intestinal coupling process, since we have just begun to understand it. Biological consequence and new insights into how coupling process can impact bioavailability of xenobiotics, biological functions of drugs and carcinogens, and drug interactions will be discussed.