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Biotechnology and Bioengineering Feb 2019At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the...
At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.
Topics: Cell Proliferation; Glucose; HEK293 Cells; Humans; Lactic Acid; Metabolic Flux Analysis
PubMed: 30411322
DOI: 10.1002/bit.26858 -
The American Journal of Clinical... Dec 1986Metabolic responses to 20 days of overeating were examined in five healthy volunteers. Overfeeding caused a variable increase (1-18%) in basal metabolic rate but no...
Metabolic responses to 20 days of overeating were examined in five healthy volunteers. Overfeeding caused a variable increase (1-18%) in basal metabolic rate but no change in metabolic rate during light exercise. Postprandial resting metabolic rate was 8-40% higher (mean 18%) during overeating. The increase in oxygen consumption during a norepinephrine infusion was the same before (20 +/- 2%) and after (17 +/- 3%) overfeeding. Overfeeding elevated basal insulin concentrations in all subjects and increased the insulin response to intravenous glucose in four of five subjects. Overfeeding did not significantly alter mean serum T3 concentrations or erythrocyte 86Rb uptake (an index of Na+,K+-ATPase activity). These data do not confirm reports that overfeeding increases metabolic rate more during exercise than during rest. They also suggest that the increase in resting metabolic rate during overfeeding is not caused by increased responsiveness to norepinephrine or increased serum T3 concentrations.
Topics: Adult; Basal Metabolism; Blood Glucose; Energy Intake; Energy Metabolism; Erythrocytes; Feeding and Eating Disorders; Female; Glucose Tolerance Test; Humans; Hyperphagia; Insulin; Male; Norepinephrine; Thyroxine
PubMed: 3538842
DOI: 10.1093/ajcn/44.6.718 -
Methods in Molecular Biology (Clifton,... 2019Enhanced glutaminolysis and glycolysis are the two most remarkable biochemical features of cancer cell metabolism, reflecting increased utilization of glutamine and...
Enhanced glutaminolysis and glycolysis are the two most remarkable biochemical features of cancer cell metabolism, reflecting increased utilization of glutamine and glucose in proliferating cells. Most solid tumors often outgrow the blood supply, resulting in a tumor microenvironment characterized by the depletion of glutamine, glucose, and oxygen. Whereas mechanisms by which cancer cells sense and metabolically adapt to hypoxia have been well characterized with a variety of cancer types, mechanisms by which different types of tumor cells respond to a dynamic change of glutamine availability and the underlying importance remains to be characterized. Here we describe the protocol, which uses cultured Hep3B cells as a model in determining glutamine-dependent proliferation, metabolite rescuing, and cellular responses to glutamine depletion. These protocols may be modified to study the metabolic roles of glutamine in other types of tumor or non-tumor cells as well.
Topics: Adaptation, Biological; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Endoplasmic Reticulum Stress; Energy Metabolism; Gene Expression Regulation, Neoplastic; Glucose; Glutamine; Glycolysis; Humans; Metabolic Networks and Pathways; Neoplasms; Oxygen; Signal Transduction
PubMed: 30725468
DOI: 10.1007/978-1-4939-9027-6_22 -
Autophagy Jul 2008Autophagy plays a critical protective role maintaining energy homeostasis and protein and organelle quality control. These functions are particularly important in times... (Review)
Review
Autophagy plays a critical protective role maintaining energy homeostasis and protein and organelle quality control. These functions are particularly important in times of metabolic stress and in cells with high energy demand such as cancer cells. In emerging cancer cells, autophagy defect may cause failure of energy homeostasis and protein and organelle quality control, leading to the accumulation of cellular damage in metabolic stress. Some manifestations of this damage, such as activation of the DNA damage response and generation of genome instability may promote tumor initiation and drive cell-autonomous tumor progression. In addition, in solid tumors, autophagy localizes to regions that are metabolically stressed. Defects in autophagy impair the survival of tumor cells in these areas, which is associated with increased cell death and inflammation. The cytokine response from inflammation may promote tumor growth and accelerate cell non-autonomous tumor progression. The overreaching theme is that autophagy protects cells from damage accumulation under conditions of metabolic stress allowing efficient tolerance and recovery from stress, and that this is a critical and novel tumor suppression mechanism. The challenge now is to define the precise aspects of autophagy, including energy homeostasis and protein and organelle turnover, that are required for the proper management of metabolic stress that suppress tumorigenesis. Furthermore, we need to be able to identify human tumors with deficient autophagy, and to develop rational cancer therapies that take advantage of the altered metabolic state and stress responses inherent to this autophagy defect.
Topics: Animals; Autophagy; Humans; Neoplasms; Oxidative Stress
PubMed: 18326941
DOI: No ID Found -
Biomolecules Aug 2021Pregnane X Receptor (PXR) belongs to the nuclear receptors' superfamily and mainly functions as a xenobiotic sensor activated by a variety of ligands. PXR is widely... (Review)
Review
Pregnane X Receptor (PXR) belongs to the nuclear receptors' superfamily and mainly functions as a xenobiotic sensor activated by a variety of ligands. PXR is widely expressed in normal and malignant tissues. Drug metabolizing enzymes and transporters are also under PXR's regulation. Antineoplastic agents are of particular interest since cancer patients are characterized by significant intra-variability to treatment response and severe toxicities. Various PXR polymorphisms may alter the function of the protein and are linked with significant effects on the pharmacokinetics of chemotherapeutic agents and clinical outcome variability. The purpose of this review is to summarize the roles of PXR polymorphisms in the metabolism and pharmacokinetics of chemotherapeutic drugs. It is also expected that this review will highlight the importance of PXR polymorphisms in selection of chemotherapy, prediction of adverse effects and personalized medicine.
Topics: Acetylation; Antineoplastic Agents; Biotransformation; Gene Expression; Humans; Inactivation, Metabolic; Neoplasms; Phosphorylation; Polymorphism, Single Nucleotide; Precision Medicine; Pregnane X Receptor; Protein Domains; Protein Processing, Post-Translational; Sumoylation; Treatment Outcome; Ubiquitination
PubMed: 34439808
DOI: 10.3390/biom11081142 -
Frontiers in Immunology 2020The interplay between cellular stress and immune response can be variable and sometimes contradictory. The mechanisms by which stress-activated pathways regulate the... (Review)
Review
The interplay between cellular stress and immune response can be variable and sometimes contradictory. The mechanisms by which stress-activated pathways regulate the inflammatory response to a pathogen, in autoimmunity or during cancer progression remain unclear in many aspects, despite our recent knowledge of the signalling and transcriptional pathways involved in these diseases. In this context, over the last decade many studies demonstrated that cholesterol metabolism is an important checkpoint for immune homeostasis and cancer progression. Indeed, cholesterol is actively metabolized and can regulate, through its mobilization and/or production of active derivatives, many aspects of immunity and inflammation. Moreover, accumulation of cholesterol has been described in cancer cells, indicating metabolic addiction. The nuclear receptors liver-X-receptors (LXRs) are important regulators of intracellular cholesterol and lipids homeostasis. They have also key regulatory roles in immune response, as they can regulate inflammation, innate and adaptive immunity. Moreover, activation of LXRs has been reported to affect the proliferation and survival of different cancer cell types that show altered metabolic pathways and accumulation of cholesterol. In this minireview we will give an overview of the recent understandings about the mechanisms through which LXRs regulate inflammation, autoimmunity, and cancer, and the therapeutic potential for future treatment of these diseases through modulation of cholesterol metabolism.
Topics: Adaptive Immunity; Animals; Autoimmunity; Cholesterol; Humans; Immunity, Innate; Inflammation; Lipid Metabolism; Liver X Receptors; Metabolic Networks and Pathways; Neoplasms; Signal Transduction
PubMed: 33224146
DOI: 10.3389/fimmu.2020.584303 -
American Journal of Physiology. Heart... Apr 2013Ketone bodies are metabolized through evolutionarily conserved pathways that support bioenergetic homeostasis, particularly in brain, heart, and skeletal muscle when... (Review)
Review
Ketone bodies are metabolized through evolutionarily conserved pathways that support bioenergetic homeostasis, particularly in brain, heart, and skeletal muscle when carbohydrates are in short supply. The metabolism of ketone bodies interfaces with the tricarboxylic acid cycle, β-oxidation of fatty acids, de novo lipogenesis, sterol biosynthesis, glucose metabolism, the mitochondrial electron transport chain, hormonal signaling, intracellular signal transduction pathways, and the microbiome. Here we review the mechanisms through which ketone bodies are metabolized and how their signals are transmitted. We focus on the roles this metabolic pathway may play in cardiovascular disease states, the bioenergetic benefits of myocardial ketone body oxidation, and prospective interactions among ketone body metabolism, obesity, metabolic syndrome, and atherosclerosis. Ketone body metabolism is noninvasively quantifiable in humans and is responsive to nutritional interventions. Therefore, further investigation of this pathway in disease models and in humans may ultimately yield tailored diagnostic strategies and therapies for specific pathological states.
Topics: Animals; Cardiomyopathies; Cardiovascular Diseases; Coenzyme A-Transferases; Fatty Acids; Humans; Hydroxymethylglutaryl-CoA Synthase; Ketone Bodies; Lipogenesis; Liver; Mitochondria, Heart; Mitochondria, Liver; Myocardium; Obesity; Oxidation-Reduction; Signal Transduction
PubMed: 23396451
DOI: 10.1152/ajpheart.00646.2012 -
Drug Metabolism and Disposition: the... Dec 2015In vitro assays using liver subcellular fractions or suspended hepatocytes for characterizing the metabolism of drug candidates play an integral role in the optimization... (Review)
Review
In vitro assays using liver subcellular fractions or suspended hepatocytes for characterizing the metabolism of drug candidates play an integral role in the optimization strategy employed by medicinal chemists. However, conventional in vitro assays have limitations in their ability to predict clearance and generate metabolites for low-turnover (slowly metabolized) drug molecules. Due to a rapid loss in the activity of the drug-metabolizing enzymes, in vitro incubations are typically performed for a maximum of 1 hour with liver microsomes to 4 hours with suspended hepatocytes. Such incubations are insufficient to generate a robust metabolic response for compounds that are slowly metabolized. Thus, the challenge of accurately estimating low human clearance with confidence has emerged to be among the top challenges that drug metabolism scientists are confronted with today. In response, investigators have evaluated novel methodologies to extend incubation times and more sufficiently measure metabolism of low-turnover drugs. These methods include plated human hepatocytes in monoculture, and a novel in vitro methodology using a relay of sequential incubations with suspended cryopreserved hepatocytes. In addition, more complex in vitro cellular models, such as HepatoPac (Hepregen, Medford, MA), a micropatterned hepatocyte-fibroblast coculture system, and the HµREL (Beverley Hills, CA) hepatic coculture system, have been developed and characterized that demonstrate prolonged enzyme activity. In this review, the advantages and disadvantages of each of these in vitro methodologies as it relates to the prediction of clearance and metabolite identification will be described in an effort to provide drug metabolism scientists with the most up-to-date experimental options for dealing with the complex issue of low-turnover drug candidates.
Topics: Animals; Cells, Cultured; Coculture Techniques; Hepatocytes; Humans; Inactivation, Metabolic; Metabolic Clearance Rate; Microsomes, Liver; Pharmaceutical Preparations
PubMed: 26363026
DOI: 10.1124/dmd.115.066431 -
The Journal of Experimental Biology Mar 2024From bacteria to metazoans, higher density populations have lower per capita metabolic rates than lower density populations. The negative covariance between population...
From bacteria to metazoans, higher density populations have lower per capita metabolic rates than lower density populations. The negative covariance between population density and metabolic rate is thought to represent a form of adaptive metabolic plasticity. A relationship between density and metabolism was actually first noted 100 years ago, and was focused on spermatozoa; even then, it was postulated that adaptive plasticity drove this pattern. Since then, contemporary studies of sperm metabolism specifically assume that sperm concentration has no effect on metabolism and that sperm metabolic rates show no adaptive plasticity. We did a systematic review to estimate the relationship between sperm aerobic metabolism and sperm concentration, for 198 estimates spanning 49 species, from protostomes to humans from 88 studies. We found strong evidence that per capita metabolic rates are concentration dependent: both within and among species, sperm have lower metabolisms in dense ejaculates, but increase their metabolism when diluted. On average, a 10-fold decrease in sperm concentration increased per capita metabolic rate by 35%. Metabolic plasticity in sperm appears to be an adaptive response, whereby sperm maximize their chances of encountering eggs.
Topics: Humans; Male; Semen; Sperm Motility; Spermatozoa; Energy Metabolism
PubMed: 38380562
DOI: 10.1242/jeb.246674 -
Obesity Reviews : An Official Journal... Oct 2011Cells, tissues and organisms have the ability to rapidly switch substrate oxidation from carbohydrate to fat in response to changes in nutrient intake, and to changes in... (Review)
Review
Cells, tissues and organisms have the ability to rapidly switch substrate oxidation from carbohydrate to fat in response to changes in nutrient intake, and to changes in energy demands, environmental cues and internal signals. In healthy, metabolically normal individuals, substrate switching occurs rapidly and completely; in other words, substrate switching is 'flexible'. A growing body of evidence demonstrates that a blunted substrate switching from low- to high-fat oxidation exists in obese individuals, as well as in pre-obese and post-obese, and that this 'metabolic inflexibility' may be a genetically determined trait. A decreased fat oxidation can lead to a positive energy balance under conditions of high-fat feeding, due to depletion of glycogen stores that stimulates appetite and energy intake through glucostatic and glucogenostatic mechanisms, e.g. hepatic sensing of glycogen stores. Several genetic polymorphisms and single-nucleotide polymorphisms have been identified that are associated with low-fat oxidation rates and metabolic inflexibility, and genetic identification of susceptible individuals may lead to personalized prevention of weight gain using fat oxidation stimulants ('fat burners') in the future.
Topics: Dietary Fats; Energy Intake; Humans; Lipid Metabolism; Obesity; Oxidation-Reduction; Weight Gain
PubMed: 21692967
DOI: 10.1111/j.1467-789X.2011.00894.x