NMR Study of Glucose and Fructose Metabolism in Glioblastoma Cells

¹³C NMR Study of Glucose and Fructose Metabolism in Glioblastoma Cells

CHAPTER 1

INTRODUCTION

1.1 Nuclear Magnetic Resonance

The quest for high resolution Nuclear Magnetic Resonance (NMR) has led researchers to utilize novel high magnetic field techniques^1,2. The importance of using a high magnetic field is necessary to tackle the inherent problem of low magnetic moments in nuclear spins^2,3. ¹³C is one of the nuclei with low magnetic moment which could be used for NMR purposes. The chemical shifts of ¹³C nuclei in organic molecules are spread out up to 200 ppm, enabling signal from each carbon in a compound be seen as a distinct peak³. Due to distinctive identification of carbon in each compound, there is low possibility of signal overlapping in NMR spectroscopies⁴.

¹³C NMR spectroscopy is a powerful technique for identifying and tracking biological compounds in a chemical reaction⁵. In studies of cancer, NMR spectroscopy is particularly useful because it has the specificity to identify and monitor specific biochemical changes in the cellular environment. Also, NMR spectroscopy isa unique tool for deciphering brain metabolismin vitro⁶.

The relatively weak signals obtained from the NMR spectroscopy enables probing sensitive physical systems, such as living systems, without significantly disturbing them. Considering the fast and accurate strategy that NMR offers for biochemical information collection at molecular levels, this technique is one of the most powerful methods for biochemical analysis, cancer cell studies, and biological fluids, etc⁷.

The NMR signal is proportional to the nuclear polarization P, which is governed by the Boltzmann distribution of nuclear spins on the Zeeman energy levels. In thermal equilibrium, for a spin-1/2 system, it is governed by a thermal population difference, determined by the Boltzmann factor:

                                P= tanh (γℏB₀/2kBT).

Here, P is the spin polarization, γ is its gyromagnetic ratio, B is the applied magnetic field, T is the temperature and ℏ and kB are the reduced Planck constant and the Boltzmann constant, respectively^1,3. As dictated by Boltzmann statistics, the relatively weak magnetic moments of nuclear spins lead to minute differences in the nuclear spin populations between the Zeeman energy levels at ambient conditions. Thus, the polarization level P, which is equal to the surplus number of nuclear spins residing in a particular Zeeman energy level, is on the order of ppm at achievable field strengths^8,9.

1.2 High Fructose Corn Syrup (HFCS)

     Artificial sugars and sweeteners are indispensable commodities in the modern diet; they are fairly inexpensive to manufacture and distribute, making it a favorable choice for food production companies to easily sell in mass quantities¹⁰. Arguably, the most popular of the artificial sweeteners is High Fructose Corn Syrup (HFCS).

HFCS, which is probably the most widely available of the artificial sugars, is a major source of energy that enters our metabolic pathways¹¹. HFCS found in numerous food products such as sodas to flavored waters, yogurt and ice cream are used excessively due to its low cost and versatility.

HFCS consists primarily of two monosaccharides or simple sugars, 40% glucose and 60% fructose ¹⁰. Fructose, a 6-carbon sugar, is commonly found in naturally occurring plant life such as fruits and organized insect complexes such as beehives. Glucose is what most medical patients with diabetes or similar complications would immediately recognize as “Blood Sugar”. In plant glucose is converted into starch for later energy consumption. In animals, glucose is the primary circulating sugar and combined with fructose creates common table sugar.

Even in the absence of underlying disease, HFCS is considered a dangerous substance when consumed in large amounts over a long period of time, imposing irreparable consequences upon one’s overall health and leading to diseases such as obesity, type 2 diabetes, and cancer, to name a few^10,11.

Several studies have been conducted on the repercussion of excessive sugar consumption in the realm of a precursors to cancers and diabetes; however, more recently scientists have closely observed another consequence of excessive sugar consumption, the exacerbation of cancerous tissues in the human body¹². As a particular example of such studies, metabolism of glioblastoma multiforme, also known as glioblastoma (GBM), has attracted a remarkable attention^13–15.

1.3 Glioblastoma

Glioblastoma Multiforme (GBM) is an aggressive type of the Central Nervous System (CNS) tumor that grows within brain tissue¹⁶.  GBM as well as other forms of cancer are capable of consuming sugars to extend their life span, and essentially acts as an energy source for survival.

This type of tumor is fast-growing, characterizing it as one of the most malignant of all primary brain tumor^15,16. GBM tumors are made up of many different types of cells which are not responsive to the same type of treatment, making it notoriously challenging to cure^12,14. That being said, the standard therapy incorporates surgery, radiotherapy, and adjuvant chemotherapy.

The surrounding microcellular environment of the GBM tissue is markedly acidic, due to the Warburg effect. The customary cycle of glycolysis is intercepted, over 80% of pyruvate from metabolized from glucose is converted to lactic acid. Lactic acid is a by-product of the process cells use to produce energy, which was considered to be an unimportant waste product for many years^17,18.

The success of the treatment, however, remains poor with a 12-15 range of months of the median survival time from diagnosis to the start of therapy¹⁹. According to the Surveillance, Epidemiology, and End Results (SEER), about 23,820 will be diagnosed with primary brain and other CNS tumors and about 17,760 people will be dead of this disease in the only United States²⁰. People with a cancerous brain or CNS tumor have the 5-year survival rate of approximately 32.9% from SEER 18 2009-2015^19–21. Hence, survival rates vary greatly depending on factors such as the type of brain or spinal cord tumor.

CHAPTER 2

CURRENT WORK AND PRELIMINARY DATA

 

2.1 Fructose and Glucose Metabolism in cancer

In this study, we have investigated the metabolism of [U-¹³C] glucose and [U-¹³C] fructose (see the chemical formula for these two sugars in Fig.1), in glioblastoma cells, specifically, the aggressive SfXl cell line. The experiments were performed in vitro. Cells were incubated with these ¹³C-labeled substrates. We have closely traced how SfXl brain cancer metabolizes both fructose and glucose.

A notable observation was that, in spite of having equal caloric content of fructose and glucose, they metabolized quite differently in brain cancer¹⁸.

Glucose enters cells by a transport mechanism (Glut-4) that is insulin dependent in most tissues. Once inside the cell, glucose is phosphorylated by glucokinase to become glucose-6-phosphate, from which the intracellular metabolism of glucose begins. Then, an isomerase converts glucose-6-phosphate into one of its isomers, fructose-6-phosphate. It gets catalyzed by the enzyme phosphofructokinase for producing fructose-1, 6-bisphosphate. The next step in glycolysis employs an enzyme, aldolase, to cleave fructose-1,6-bisphosphate into two three-carbon isomers: dihydroxyacetone-phosphate and glyceraldehyde-3-phosphate. After three more reactions, glycolysis ends with two pyruvate molecules¹¹ (Fig. 2).

In contrast to glucose, fructose enters cells via a Glut-5 transporter that does not depend on insulin. Also, glucose provides signals to the brain that fructose cannot provide because it is not transported into the brain. Fructose metabolism takes place in the liver, where fructose is converted to pyruvate, or under fasting conditions to glucose. It is then metabolized to intermediates of glycolysis^14,17.

For fructose to enter the pathways of intermediary metabolism, it must be phosphorylated by hexokinase or fructokinase. It then becomes fructose-1-phosphate and is cleaved by aldolase B to form dihydroxyacetone phosphate (DHAP) and glyceraldehyde, which is phosphorylated by ATP to form glyceraldehyde-3-phosphate. Both of them are intermediates of glycolysis. Alternatively, the fructose can

be converted to glucose by gluconeogenesis. Fructose metabolism parallels that of glycolysis. When it becomes pyruvate, it enters the Tricarboxylic Acid Cycle (TCA) and fatty acid synthesis^12,15(Fig.2). This is the reason why excess fructose can cause obesity, thereby affecting type 2 diabetes.

2.2 Differences between Cancer Cells and Normal Cells

Cancer cells differ from normal cells in many ways that allow them to grow out of control and become invasive. This is one reason that, unlike normal cells, cancer cells continue to divide uncontrollably. In addition, cancer cells can ignore signals that normally tell cells to stop dividing or that begin a process known as programmed cell death, or apoptosis. Cancer cells are also often able to evade the immune system, a network of organs, tissues, and specialized cells that protects the body from infections and other conditions¹⁶.

In glycolysis (normal cells), if oxygen is present, the two pyruvates, with help from the pyruvate dehydrogenaseenzyme complex, is converted to two Acetyl-CoA molecules. The acetyl-CoAs enter the mitochondrion where it fuels the TCA cycle. Each acetyl-CoA molecule goes through the TCA cycle. Energy is released in the form of ATP during aerobic oxidation of glucose to carbon dioxide and water, 36 moles of ATP are produced per glucose molecule. However, under anaerobic conditions glycolysis produces minimal energy in the form of ATP. This mode of glycolysis is not coupled to the TCA cycle and oxidative phosphorylation, the reaction producing lactic acid generates 2 moles of ATP per glucose molecule¹³.

Cancer cells exhibit aerobic glycolysis. This means that cancer cells derive most of their energy from glycolysis that is glucose is converted to lactate for energy followed by lactate fermentation, even when oxygen is available. This is termed the Warburg effect^14,22,23(Fig.3).

2.3 The Experiment and Methods

The following procedures were applied to both SfXl and HuH7 cell lines to prepare eight plates for each group of the experiment. All experiments were divided into four groups: i) [U-¹³C]  glucose with unlabeled fructose ii) [U-¹³C] glucose without unlabeled fructose iii) [U-¹³C] fructose with unlabeled glucose and iv) [U-¹³C] fructose without unlabeled glucose in a glucose-free DMEM with glutamine.

a)      Cell Culture:

Cells were seeded (see the confocal image in Fig. 4) at a density of

$5\times {10}^6\mathrm{ }$

live cells per milliliter into seven dishes (15 cm diameter), by 25 mL DMEM containing glucose and FBS. The number of live cells per milliliter was measured by the Trypan blue exclusion technique using a Countess automated cell counter (Thermo Scientific, Loughborough, UK).

Cells were carefully monitored under the EVOS FLoid Cell Imaging Station microscope (Thermo Fisher Scientific, Waltham, MA) to ensure not only that they grew uniformly but also minimize the likelihood of contamination. The cells were carried out for 24 hours in a water-saturated 5% CO₂/95% air atmosphere Panasonic CO₂ Incubator (Panasonic Healthcare Corporation of North American), at 37°C so as to allow the cells to adhere to the bottom of the plates; this is also done in order to improve the signal to noise ratio of the resulting spectra.

After 24 hours incubation time, the medium was removed and the plates were washed with 5 mL Dulbecco’s Phosphate Buffered Saline (PBS) (Sigma, St. Louis, MO). Fifteen milliliters of complete media, made from glucose-free DMEM with glutamine and doped with either 10 mM [U-¹³C] glucose (in the presence or absence of unlabeled fructose) or 10 mM [U-¹³C] fructose (in the presence or absence of unlabeled glucose), was individually administered to each of the seven cultured cells plate with at incubation times.

 Each of these plates was then incubated for a span of time ranging from 0.5 to 48 hours. One plate from each group was not doped with control ¹³C and additionally studied as a control.

Metabolites were extracted from cells and culture medium as detailed below:

After 48 hours had elapsed, the cells were harvested as follows: The medium was removed from each plate via pipette controller and stored in separate 50mL centrifuge tubes which were saved for the purposes of medium extraction. Perchloric acid (PCA) extraction of the cells was done and samples were then centrifuged (1828g for 15 min), and the supernatant fluid was stored at – 20°C until it was neutralized (6.5< pH < 8.0) with NaOH.

The cell extracts were lyophilized for 2 to 3 days, after which, each freeze-dried extract was re-suspended in 800 μL of D₂O doped with 10 mM ¹³C urea (Cambridge Isotopes Laboratories, Tewksbury, MA). The ¹³C urea was added to provide a reference in regard to both chemical shift and to determine relative concentrations. 400 µL aliquots of these D₂O solutions were placed in NMR tubes, purchased from Norell in 5mm outer diameter, for determination of ¹³C NMR spectra.

Carbon spectra were obtained using a Bruker Avance III HD 600 MHz system at The University of Texas at Dallas. These tubes were run overnight for a minimum of 2048 scans. Any living cells in the extracellular medium (which was kept before killing the cells with 12% PCA) were killed through the addition of 5 mL of 12% PCA. The media was then subjected to the same procedure for intracellular extracts that are killed with PCA. Extracellular medium extraction provides a greater amount of information than intracellular counterparts for lactate analysis and glucose consumption.

Data Analysis:

These spectra were initially analyzed using the software MestReNova (Mestrelab Research, S.L., A Coruña, and Spain). All other data analysis was performed using Igor Pro version 7.08 (Wavemetrics, Lake Oswego, OR).

b)      Western Blot:

Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells²⁴. Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest²⁵.

We performed a western blot procedure upon a 60-80% confluent plate of SfXl and HuH7 as detailed below: 

Cell lysates are the most common form of sample used for western blot. This should be done in a cold temperature with buffer containing added Protease Inhibitors (PI). After extracting the protein, it is very important to know the extract’s concentration. Calculation the concentration allows to measure the mass of the protein that is being loaded into each well by the relationship between concentration, mass, and volume. After determining the appropriate volume of the sample, we should load protein samples into the wells of the gel electrophoresis. Run the gel with low voltage (60 V) for separating gel; use higher voltage (140 V) for stacking gel.

After separating the protein mixture, it is transferred to a membrane by iBlot2. IBlot2 is an electric field oriented perpendicular to the surface of the gel, causing proteins to move out of the gel and onto the membrane. The membrane is placed between the gel surface and the positive electrode in a sandwich.

Blocking the membrane is a very important step of western blotting, as it prevents antibodies do not bind non-specifically to the membrane. A blocking solution made of TBS-T and roughly 5% nonfat dried milk will place the membrane in some of the blocking solution on a rocker for 1-2 hours at room temperature.

After blocking and washing, the blot will be incubated in a dilute solution of primary antibody on the shaker overnight at 4°C. The antibody is diluted in some of the TBS-T mixed with 5% bovine serum albumin (BSA). After washing the membrane, incubate the membrane in secondary antibody solution on the shaker for 1 hour. The membrane is ready for detection after washing it with TBS-T. In general, Western blot is a technique that is very useful for protein detection as it allows the user to quantify the protein expression as well.

a)      GCMS:

 

Gas Chromatography-Mass Spectrometer (GCMS) is a powerful method for the separation of organic and inorganic compounds. There are many advantages to using GCMS for compound analysis, including its ability to separate complex mixtures, to quantify analyses, and to determine trace levels of organic contamination²⁶.

The basic principle of MS utilizes the nature of ions. By accelerating an ion (an atom or molecule with an electrical charge) to a certain speed, and passing it through a magnetic field, the path of the ion will be deflected by the magnetic field. The amount of deflection depends on the intensity of the magnetic field and the mass number of the ion^26,27.

Ionized molecules are then accelerated through the instrument’s mass analyzer, which quite often is a quadrupole or ion trap. It is here that ions are separated based on their different mass-to-charge (m/z) ratios. The final steps of the process involve ion detection and analysis, with compound peaks appearing as a function of their m/z ratios. Peak heights, meanwhile, are proportional to the quantity of the corresponding compound. A complex sample will produce several different peaks, and the final readout will be a mass spectrum. Using computer libraries of mass spectra for different compounds, researchers can identify and quantitate unknown compounds and analyses²⁸. (See fig.6 for more details)

Fig. 6: The sample flows through the column and the compounds comprising the mixture of interest are separated by virtue of their relative interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase). The latter part of the column passes through a heated transfer line and ends at the entrance to ion source where compounds eluting from the column are converted to ions.

 

2.3 Results and Discussion     

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