Name: extracellular glucose depletion as an indirect measure of glucose uptake in cells and tissues ex vivo
Uploaded: 2022-04-06T14:20:00
Description: Watch this Scientific Journal Video about Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo at JoVE.com

The project was supported by Ralph and Marian Falk Medical Research Catalyst Award and Kathleen Kelly Award. Other supports included the National Center for Research Resources UL1RR025755 and NCI P30CA16058 (OSUCCC), the NIH Roadmap for Medical Research. The content is solely the responsibility of the authors and does not represent the official views of the National Center for Research Resources or the NIH.

Animal studies were approved by the Institutional Animal Care and Use Committee of The Ohio State University (OSU, protocol 2007A0262-R4).
NOTE: All procedures must be done in a class II biosafety cabinet with the blower on and the lights off.
1. Preparation of materials
NOTE: All materials are listed in the Table of Materials.
<ol>
	<li>Prepare Medium 1, centrifugation Medium 2, and storage and working...

<ol>
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A meta-analysis.</a> Diabetes &amp; Metabolic Syndrome: Clinical Research &amp; Review. 14 (4), 535-545 (2020).</li><li>Lee, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+prognostic+impact+of+glucose+uptake+in+visceral+adipose+tissue+according+to+sex+in+patients+with+colorectal+cancer.">Different prognostic impact of glucose uptake in visceral adipose tissue according to sex in patients with colorectal cancer.</a> Scientific Reports. 11 (1), 21556 (2021).</li><li>Miner, M. W. 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The direct comparison of extracellular FD-glucose depletion with normalized intracellular glucose uptake in cells culture showed a high correlation, suggesting that extracellular glucose depletion could be a surrogate measurement for glucose uptake assessment. The measurement of extracellular FD-glucose can use a broad range of FD glucose concentrations, also 0.5-2.5 &#956;g FD-glucose/mL appear to provide the optimal range. Extracellular FD-glucose does not require normalization to cell number or protein concentrations ...

The demand for measuring glucose uptake rises together with the critical need to address an epidemic increase in a multitude of diseases dependent on glucose metabolism. Underlying mechanisms of degenerative metabolic diseases, neurological and cognitive disorders1, inflammatory2 and infectious diseases3, cancer4,5, as well as aging6, depend on glucose metabolism for energy and its storage, anabolic processes, protein, and gene modification, signaling, regulation of genes, and nucleic acids synthesis and replication7,8,9. Diabetes mellitus (DM) is directly related to malfunction of glucose uptake regulation. DM is a spectrum of chronic diseases such as type-1, -2, and -3 diabetes mellitus, gestational diabetes, maturity-onset diabetes of the young, and other types of this disease induced by environmental and/or genetic factors. In 2016, the first WHO Global report on diabetes demonstrated that the number of adults living with the most widespread DM has almost quadrupled since 1980 to 422 million adults10, and this number of DM patients has been rising exponentially for the last few decades. In 2019 alone, an estimate of 1.5 million deaths was directly caused by DM10. This dramatic upsurge is due to the rise in type-2 DM and the conditions driving it, including overweight and obesity10. The COVID-19 pandemic revealed a two-fold increase in mortality in patients with DM compared to the general population, suggesting the profound yet poorly understood role of glucose metabolism in the immune defense3. Prevention, early diagnosis, and treatment of DM, obesity, and other diseases require optimization of measurements of glucose uptake by different tissues, and identification of environmental11, nutritional12, endocrine13, genetic14, and epigenetic15 factors affecting glucose uptake.
In research, intracellular and/or tissue uptake of glucose is commonly measured by fluorescently-labeled glucose (FD-glucose) in vitro16,17,18 and in vivo19. FD-glucose became a preferred method compared to more precise methods using radioactively-labeled glucose20, analytical mass spectroscopy analysis21, metabolomics22, nuclear magnetic resonance methods23, and positron emission tomography/computed tomography (PET/CT)5,24. Unlike FD-glucose uptake, analytical methods requiring more biological material may involve a multi-step sample preparation, expensive instruments, and complex data analysis. Effective and inexpensive measurements of FD-glucose uptake in cell cultures have been utilized in proof-of-concept experiments and may require validation by other methods.
The basis of FD-glucose application for glucose uptake studies is the reduced metabolism of FD-glucose compared to endogenous glucose25. Nonetheless, both endogenous glucose and FD-glucose are dynamically distributed among all cellular compartments for use in anabolic, catabolic, and signaling processes. The compartmentalization and time-dependent processing25 of FD-glucose interfere with the fluorescence measurements, and represent the major limiting factors for the use of this assay in high-throughput screening experiments, kinetic analysis, 3D cell culture, co-cultures, and tissue explant experiments. Here, we provide data demonstrating a high correlation between the extracellular depletion of FD-glucose and its intracellular uptake, suggesting the extracellular depletion of FD-glucose as a surrogate measurement for intracellular glucose uptake. The measurement of extracellular depletion of glucose was applied to validate tissue-specific differences in glucose uptake in mice treated with insulin and an experimental drug18 to provide a proof-of-principle of this method.
The current protocol describes intracellular and extracellular (Figure 1) measurements of FD-glucose uptake in 3T3-L1 cells. Protocol sections 1-7 explain the culture and growth of cells for 48 h; cell starvation, stimulation, and baseline extracellular measurements; and post-stimulation measurements of extracellular FD-glucose and intracellular measurements of FD-glucose and protein. Protocol section 8 describes the ex vivo measurement of extracellular uptake of FD-glucose in tissues dissected from ob/ob mice in the presence and absence of insulin and&#160;amino acid compound 2 (AAC2) described elsewhere18.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3T3-L1 mouse fibroblasts</td>
			<td>ATCC</td>
			<td>CL-173</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Falcon</td>
			<td>353227</td>
			<td>Plastic ware</td>
		</tr>
		<tr>
			<td>B6.V-Lepob/J male mice</td>
			<td>Jackson Laboratory</td>
			<td>stock number 000632</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>BioTek Synergy H1 modular multimode microplate reader (Fisher Scientific, US)</td>
			<td>Fisher Scientific, US</td>
			<td>&#160;B-SHT</td>
			<td>Device</td>
		</tr>
		<tr>
			<td>Bovine serum</td>
			<td>Gibco/ThermoFisher</td>
			<td>161790-060</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Calf serum</td>
			<td>Gibco/ThermoFisher</td>
			<td>26010-066</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Cell incubator</td>
			<td>Forma</td>
			<td>Series II Water Jacket</td>
			<td>Device</td>
		</tr>
		<tr>
			<td>Diet (mouse/rat diet, irradiated)</td>
			<td>Envigo</td>
			<td>Teklad LM-485</td>
			<td>Diet</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma LifeScience</td>
			<td>D2650-100mL</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco/ThermoFisher</td>
			<td>&#160;11965-092</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500mL</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose)</td>
			<td>Sigma</td>
			<td>72987-1MG</td>
			<td>Assay</td>
		</tr>
		<tr>
			<td>Glucose-free and phenol red-free DMEM</td>
			<td>Gibco/ThermoFisher</td>
			<td>A14430-01</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Human insulin 10 mg/mL</td>
			<td>MilliporeSigma, Cat N 91077C</td>
			<td>Cat N 91077C</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Isoflurane, 5%</td>
			<td>Henry Schein</td>
			<td>NDC 11695-6776-2</td>
			<td>Anestaetic</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin (P/S)</td>
			<td>Gibco/ThermoFisher</td>
			<td>15140-122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Sigma-Aldrich</td>
			<td>DA537-500 mL</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Pierce bicinchoninic acid (BCA) protein assay</td>
			<td>ThermoFisher</td>
			<td>Cat N23225</td>
			<td>Assay</td>
		</tr>
		<tr>
			<td>Radioimmunoprecipitation assay lysis buffer</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-24948</td>
			<td>Assay</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco/ThermoFisher</td>
			<td>&#160;25300-054</td>
			<td>Cell culture</td>
		</tr>
	</tbody>
</table>

extracellular glucose depletion as an indirect measure of glucose uptake in cells and tissues ex vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Intracellular intake and extracellular glucose depletion were measured in 3T3-L1 preadipocytes, in response to different concentrations of FD-glucose (Figure&#160;2) with and without insulin stimulation. Figure&#160;2A demonstrates a dose-dependent increase in the intracellular uptake of FD-glucose, which was significantly increased in the presence of insulin. The concomitant decrease in extracellular FD-glucose in the same cells is shown in <strong class="xfig"...

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Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

Glucose uptake is the key to understanding health and disease. As glucose plays a pivotal role in metabolic and structural demands in the human body, as well as playing a vital role in regulation. The extracellular measurement of glucose allows for the development of high throughput assays for kinetics of glucose uptake in cells, tissues, and organs of different genetic background in response to nutrients or drugs.This assay would be a valuable tool for the discovery of therapeutics and nutrition for diabetes, cancer, degenerative diseases of the central nervous system, obesity, and all the diseases of glucose and metabolism. The assay needs an adjustment of time for starvation and stimulation that should be based on metabolic specifics for different tissues and cell cultures. Culture 3T3-L1 mouse fibroblasts by plating 10 to the 6 cells, diluted in 20 milliliters of medium 1 in 296 well plates.Plate 100 microliters of cell suspension per well, ensuring to maintain homogeneity of this suspension by mixing. Grow cells for 48 hours in a 37 degree Celsius incubator without changing media until about 70 to 80%confluency. Maintain cells confluent and fasted by culturing them in the same medium for 48 hours.Vary the incubation time from 24 to 72 hours, depending on the desired fasting catabolic state. Decant media from cells in the 96 well plates. Absorb the remaining fluid using sterile paper towels.Rinse the 96 well plate with 100 microliters of PBS per well, and absorb the remaining fluid with sterile paper towels. Add 100 microliters of glucose free DMEM per well, and incubate for 40 minutes. Adjust the incubation time depending on cell type stimuli, and desired level of fasting.Use eight replicates for each simulation condition. Therefore, prepare one milliliter for each simulation condition. Use seven simulation conditions without insulin.FD glucose of 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter, and 0 milligram per milliliter in 196 well plate. Use seven stimulation conditions with insulin. FD glucose of 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter, and 0 microgram per milliliter in another 96 well plate.To prepare stimulation conditions, dilute one microliter of five milligrams per milliliter FD glucose working solution, and 999 microliters of glucose free DMEM to obtain one milliliter of working stock solution. Using this working stock solution, prepare 1 to 2000, 1 to 5, 000, 1 to 10, 000, 1 to 25, 000, 1 to 50, 000, and 1 to 100, 000 delusions of FD glucose to obtain 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter in two milliliter tubes immediately before experiments in a biosafety cabinet without lights. Add one microliter of insulin to each of these conditions.After 40 minutes of incubation, decant the glucose free DMEM from each well in 96 well plates, and absorb the remaining fluid with sterile paper towels. Add 100 microliters each from the various treatment conditions described before, to wells along one column. And 100 microliters from the same treatment condition to the wells along the row in both 96 wall plates.Label the plates that utilized the conditions, with and without insulin. Add glucose free DMEM alone to the control wells. Incubate the plate for 40 minutes in the cell culture incubator in the dark.After the cells have been stimulated for 40 minutes. Transfer the stimulation media from both the plates into new 96 well plates, maintaining the same experimental layout. Wash the cells with PBS.Remove PBS and decant any remaining solution on sterile paper towels. Add a protease inhibitor to the radio immunoprecipitation assay buffer to protect proteins. Place plates containing radio immunoprecipitation assay in a shaker for 30 minutes.Using a microplate reader, measure fluorescence at excitation and emission wavelengths at 485 and 535 nanometers respectively. First in the medium containing extracellular FD glucose, and then the plate with radioimmunoprecipitation assay lized cells at the end of 30 minutes incubation. Perform BCA protein assay according to manufacturer's instructions.Quantify the protein concentrations in each well containing radio immunoprecipitation assay lized cells. Use 10 microliters of the radio immunoprecipitation assay homogenate, and measure protein concentrations, and triplicate to increase the accuracy of measurements in 96 well plates. Quantify protein based on protein standards analyzed together with samples on each 96 well plate.Incubate the harvested tissues or organs in a six well plate containing PBS for one minute. Handle each tissue or organ in a separate well. After one minute, place each tissue on a sterile paper towel to absorb PBS.Transfer tissues or organs in a separate six wall plate containing 4, 000 microliters of glucose free DMEM, and incubate for two minutes. After two minutes, remove and transfer the tissues into wells of a six well plate containing 0.29 Millimolar FD glucose working solution. Incubate the six well plates containing tissues in FD glucose working solution at 37 degrees Celsius.Collect 100 microliters of FD glucose working solution from each well after 0, 10, 20, 30, 40, 60, 90, and 120 minutes of incubation, and transfer collected 100 microliters of FD glucose working solution into 96 well plates to analyze the kinetics of extracellular FD glucose depletion. Shake before and after collection. Measure the fluorescence at excitation in emission wavelengths at 485 and 535 nanometer respectively using a microplate reader.Dose dependency in the intracellular uptake of FD glucose was significantly increased in the presence of insulin. The insulin stimulation led to significantly decreased extracellular FD glucose levels compared to the samples without insulin stimulation. Extracellular depletion of FD glucose can measure a change in glucose uptake with comparable accuracy as intracellular FD glucose uptake.The extracellular FD glucose was not depleted in non stimulated control visceral fat during 120 minutes of incubation. In contrast, pretreatment of mice with insulin or AAC2 before the dissection, led to significant depletion of extracellular FD glucose within a time interval of 30 minutes, and 60 minutes respectively. Extracellular FD glucose was not depleted in insulin stimulated liver explants, compared with non stimulated liver explants.Extracellular FD glucose was significantly decreased in the time dependent manner, and liver explants pretreated with AAC2.22%depletion of glucose was observed in the extracellular medium containing the non-treated brain after 60 minutes of incubation. The medium incubated with brains from insulin treated mice have shown a 5%linear decrease in FD glucose. AAC2 stimulated brains led to a profound rapid decrease to 67.4%of the extracellular FD glucose during the first 20 minutes.Substance involving washing are especially important for removing traces of glucose, blood, or serum within medium and, or organs, as it can interfere with the fluorescence reading of these mediums. After high throughput identification of compounds of metabolites in genes with glycemic properties, as well as the optimal conditions for their action in vivo experiments, could be validated with labeled FD glucose and PET scanning to confirm glucose accumulation in specific organs. This technique for discovery of glycemic action is both beneficial in deletion of numerous metabolites, therapeutics or their combinations, and highlights their actions in different organs.

extracellular-glucose-depletion-as-an-indirect-measure-glucose-uptake

Research

JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies &#8212; small, monomeric, high-affinity protein binders &#8212; that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters&#39; sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H+- and O2- sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming increasingly common. Ex vivo liver perfusions allow for a comprehensive analysis of liver metabolism using nuclear magnetic resonance (NMR), in nutritional conditions that can be rigorously controlled. As in silico simulations remain a primarily theoretical means of assessing hormone actions and the effects of pharmaceutical intervention, the perfused liver remains one of the most valuable test beds for understanding hepatic metabolism. As these studies guide basic insights into hepatic physiology, results must be accurate and reproducible. The greatest factor in the reproducibility of ex vivo hepatic perfusion is the quality of surgery. Therefore, we have introduced an organized and streamlined method to perform ex vivo mouse liver perfusions in the context of in situ NMR experiments. We also describe a unique application and discuss common issues encountered in these studies. The overall purpose is to provide an uncomplicated guide to a technique we have refined over several years that we deem the golden standard for obtaining reproducible results in hepatic resections and perfusions in the context of in situ NMR experiments. The distance to the center of the field for the magnet as well as the inaccessibility of the tissue to intervention during the NMR experiment makes our methods novel.

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Quantitative Image Analysis to Measure the mtDNA and Gene Expression Levels in the Transfected HeLa Cells

To begin the imaging of transfected HeLa cells select a sufficiently long exposure time for the individual fluorescence channels in live view mode based on the intensity histograms generated by the imaging software. Set the appropriate number of planes for imaging on the Z axis. Select the appropriate number of fields of view to display per well.To start the quantitative analysis of acquired images, perform background correction for all the images from all the fluorescence channels. Depending on the size of the analyzed objects, select the appropriate filter size. Next, start the image segmenting by creating a mask of the main object based on the ratio of the intensity of the fluorescent signal to the background for the channel corresponding to the cell nuclei.Create masks for the sub objects representing BrdU and mitochondrial DNA spots using the fluorescence channels appropriate for the given structures. Set the parameters and measure the intensity of the individual pixels within each mask for all the fluorescence channels. To obtain the total fluorescence intensities for the BrdU or mitochondrial DNA channel sum up the intensities of all the sub objects assigned to a given cell nucleus.To obtain the mean fluorescence intensity, divide the total fluorescence intensity by the sum of the area of the sub objects assigned to a given cell nucleus. The imaging results were verified by measuring the mitochondrial DNA and gene expression levels using quantitative real-time PCR. Treatment with dideoxycytidine or DDC resulted in a very strong decrease in the mitochondrial DNA levels.A weaker effect was observed in TFAM and TWINKLE helicase silencing, while the transfection of cells with mitochondrial DNA polymerase gamma or POLG siRNA had a modest effect on the mitochondrial DNA copy number. Quantifying the TFAM and TWINKLE helicase expression confirmed an efficient reduction in their mRNA expression to approximately 15 to 20%of the control levels. In POLG, the mRNA expression was reduced only to 30%which explains the unexpected modest effect on the mitochondrial DNA copy number.