This experiment investigated the uptake of glucose into mammalian Caco-2 and SHSY5Y cells. These cells have different structural and functional characteristics, the effects of which upon glucose uptake were compared and analysed. The cells were separately cultured, and incubated with radioactive glucose in two different conditions; with or without the presence of HgCl2 (which will be referred to as mercury), for various time periods. The glucose uptake was then measured in a scintillation counter and the concentration of glucose in Âµmol/mg protein in the cells was calculated by correlating the results with a calibration curve from a bovine serum albumin (BSA) assay which was used as a standard. Results showed that in the presence of mercury, glucose uptake is considerably reduced. At 30 minutes glucose uptake without mercury measured 1.99 x 10-1 Âµmol/mg protein in Caco-2 cells and 1.48 x 10-1 Âµmol/mg protein in SHSY5Y cells. These values decreased to 1.30 x 10-2 Âµmol/mg protein and 4.76 x 10-2 Âµmol/mg protein respectively in the presence of mercury. Mercury binds irreversibly to the transporter proteins (SGLT and GLUT) in the cell membranes altering their structure, rendering them non-functional hence inhibiting glucose uptake.
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Glucose is a key substance in mammals, it provides cells with the metabolic substrate required to generate energy in the form of ATP via oxidation shown below:
(6O2 + C6H12O6 + 38ADP rarrow.gif (63 bytes)38ATP + 6CO2 + 6H2O).
Glucose is obtained from the diet or as a product of gluconeogenesis in the liver and kidneys. Complex dietary carbohydrates are typically cleaved into smaller polysaccharides or monosaccharides by glycoside hydrolyse enzymes. The glucose can then undergo glycolysis to synthesize ATP (Garrett and Grisham, 2010).
Glucose must be transported across plasma membranes into the target cells in order to be utilized. The mode by which this transport occurs varies depending on the location. There are two principal transport mechanisms, facilitated diffusion and active uptake (primary and secondary), for which there are specific transporters. Six sodium- dependent glucose transporters SGLT1-SGLT6 (Wright, 2001) have been identified as well as facilitative glucose transporters GLUT1-GLUT12 and HMIT (Mueckler, 1994; Joost & Thorens,2001). An electrochemical gradient of sodium is set up by the sodium/potassium/ATP-ase pump which provides the energy for the transport of glucose against its concentration gradient, via SGLTs. These transporters are expressed in the luminal membrane of enterocytes in the small intestine and across the apical membrane of the proximal tubules in the kidneys (Wood and Tryahurn, 2003). These transporters are significantly more limited in expression in comparison to the facilitated glucose transporters which are found in most tissues, however the GLUT isoforms have more specific localisation, GLUT3 as an example is found exclusively in the brain. GLUTs "transport glucose down its concentration gradient" as stated by Lewin (2007).
The aim of this experiment was to investigate and measure the uptake of glucose into two types of mammalian cells; SYSY5Y cells and Caco-2 cells. SHSY5Y is a neuroblast clone of the neuroblastoma cell line SK-N-SH which "was established in 1970 from a metastatic bone tumour" as stated by the cell lines service. Caco-2 cells are an immortalized line of heterogeneous human epithelial colorectal adenocarcinoma cells.
Materials and Methods
In order to measure the glucose uptake of the cultured cells, the method as described previously (Uemura et al., 2005) was modified accordingly. Dulbecco's modified Eagle's medium (DMEM) was used to grow the cells (SHSY5Y and Caco-2). The medium was supplemented with 10% foetal calf serum, 100 units mL-1 penicillin, 100Âµg mL-1 streptomycin and 2mM glutamine. Stock cultures were grown in 75cm2 flasks and incubated at 37oC in a humidified atmosphere containing 5% CO2.The cells were divided into 6-well plates and grown until they reached confluence.
Transport and protein measurements
The cells were then washed twice with phosphate buffered saline (PBS) buffer and pre-incubated for two hours with 2mL of Krebs buffer (HEPES, 30mM; NaCl, 130 mM; KH2PO4, 4 mM; MgSO4, 1 mM; CaCl2, 1 mM; pH 7.4) at 37oC. At time zero, the buffer was extracted with a Gilson pipette and replaced with 2mL pre-warmed Krebs buffer containing 0.5 mM 2-DG and 0.02 Î¼Ci of [3H]-2-deoxyglucose ([3H]-2-DG) in wells 1-3 and Krebs buffer with the transport inhibitor 3mM mercuric chloride (HgCl2) in wells 4-6. The cultures were incubated for various time intervals of 5, 10, 15, 20, 30 and 40 minutes at 37oC. Uptake of [3H]-2-DG was terminated by adding 2mL of ice-cold PBS buffer containing 0.5mM 2-DG. Reagents were removed using a Gilson pipette and cells were washed by adding 2mL of ice-cold PBS buffer containing 0.5 mM 2-DG. Reagents were again removed and to digest the cells 2mL of cell lysis solution (0.5% Triton-X100 in 0.2M NaOH) were added. The plates were incubated at room temperature for 10 minutes on the plate shaker. To ensure cells had lysed, they were viewed under a microscope. The lysate was then used for two separate sections of the experiment.
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1. Glucose Uptake Assay
0.5mL samples of lysate were extracted from each well and transferred into 3 separate vials (for each well) using a clean tip for the Gilson pipette for each well. 10mL of scintillation cocktail was added using the automatic dispenser to each vial, these were then labelled on the lid accordingly. Vials were placed into the scintillation counter for readings to be obtained. 10ÂµL of each cell lysate (repeated in triplicate) were assayed in a 96-well plate, also containing 200ÂµL colour reagent (49 parts bichinchoninic acid and 1 part copper sulphate pentahydrate) per well and protein concentration was measured.
2. Protein BSA Assay
A BSA assay was set up in a 96-well plate to use as a standard protein concentration to then set up a calibration curve. 6 different volumes of 1 mg mL-1 BSA ranging from 0-10 ÂµL were pipetted into the plate so that the protein concentration increased across the rows. M-Q water was added accordingly to make the well volume up to 10 ÂµL, finally 200 ÂµL colour reagent was added into each well. Plate was covered and incubated at 37oC for 30 minutes. The plate was read at 570 nm in the automatic plate reader which provided data to input on excel which plotted a calibration curve of BSA concentration (mg mL-1) versus absorbance at 570 nm. Linear regression produced a straight line and equation that was used to estimate the amount of protein (mg) in each well for each cell type.
Results were processed using Microsoft Excel, paired T-tests were carried out to assess whether the results with and without mercury were statistically different and the p value gives an indication of its significance. If P<0.05 = significant, if P<0.01 = highly significant. SEM values were calculated for all results and error bars were displayed on the graphs using the appropriate values to display variability of means.
The results show glucose uptake in both cell types increased with increasing incubation time. The large difference seen in the uptake of glucose, in both cell types between the two different conditions are due to the inhibiting effects of mercury. It has been shown by Farmanfarmaian et al (1990) that "underÂ in vivoÂ orÂ in vitroÂ tissue incubation the bound mercury inhibits the absorption of nutrients such as amino acids and sugars." This is reflected in the results, shown clearly in Figure 1, when mercury is present in solution the uptake of glucose into both cell types is considerably reduced. The uptake in Caco-2 cells is higher and its uptake increases more with increasing time in comparison to SHSY5Y cells. The minimum glucose uptake is 5.78 x 10-2 Âµmol/mg protein at 5 minutes and increases to a maximum uptake of 3.76 x 10-1 Âµmol/mg protein at 40 minutes. Glucose uptake for Caco-2 cells averaged across all time points in the absence of mercury is 1.85 x 10-1 Âµmol/mg protein. Glucose uptake in SHSY5Y cells without mercury ranges from 2.36 x 10-2 Âµmol/mg protein at 5 minutes to 1.96 x 10-1 Âµmol/mg protein at 40 minutes, averaging at 8.02 x 10-2 Âµmol/mg protein taking into account all incubation times.
Figure 1: Glucose uptake in Caco-2 and SHSY5Y cells with and without mercury with increasing incubation time and error bars indicating SEM.
When mercury is present, the maximum glucose uptake for Caco-2 cells is reduced to 2.87 x 10-2 Âµmol/mg protein and 2.60 x 10-2 Âµmol/mg protein in SHSY5Y cells. The difference in glucose uptake between the absence and presence of mercury in Caco-2 and SHSY5Y cells is 0.1563 Âµmol/mg protein and 0.0542 Âµmol/mg protein respectively. These results are predominantly highly significant with several exceptions (see table 2 for data) for example each reading at 5 minutes is insignificant, as well as the reading at 10 minutes comparing the results between the cell types with mercury present (shown as N in the table).
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Table 1: SEM values for SHSY5Y and Caco-2 cells with and without mercury
Notably, glucose uptake still occurs in both cell types despite the presence of mercury (shown more clearly in figure 2). In both cell types after 5 minutes there is an initial decrease in glucose uptake and then an ambiguous pattern of uptake thereafter. The error bars are generally small, excluding the largest bar at 40 minutes in the Caco-2 cells (SEM=2.52 x 10-2) and there is no overlap of error bars between conditions.
Table 2: P values and significance of presence of mercury in Caco-2 and SHSY5Y cells and comparison between the two cells with mercury at each incubation time interval.
Caco-2 vs SHSY5Y (with HgCl2)
Figure 2: Glucose uptake in Caco-2 and SHSY5Y cells in the presence of mercury at each incubation time interval and error bars indicating SEM.
Some results which were perhaps unusual are the glucose uptake readings for Caco-2 cells at 20 and 30 minutes (without mercury) and SHSY5Y cells with mercury at 30 minutes where the graph increases and then decreases again at 40 minutes.
As illustrated in the results, the uptake of glucose in Caco-2 and SHSY5Y differs and this is due to the expression of various transporters in their respective cell membranes. Caco-2 cells express GLUT1, 2, 5 and 12 as well as SGLT1. Increased glucose uptake in Caco-2 cells is largely due to the expression of these sodium-dependent glucose transporters which actively transport glucose up its concentration gradient (see introduction). Furthermore, SGLT1 and GLUT 1 are high affinity glucose transporters consequently these factors result in higher uptake than SHSY5Y cells. These neuronal-like cells express only GLUT transporters 1, 3, 4, 6, 8 and HMIT. These all transport glucose via facilitated diffusion which is a passive process and can only transport glucose down its concentration gradient.
To differentiate the intracellular metabolic effects from the membrane effects on glucose uptake, mercury was used as a transport inhibitor. When mercury was present in the solution, a dramatic reduction in glucose uptake resulted in both cells. Mercury can denature proteins "by binding to carboxylate and, in particular sulfhydryl groups in proteins." As described by Meisenberg and Simmons (2006).In this instance, the mercury irreversibly bound to the protein transporters, disrupting the disulphide bonds which altered the tertiary structure of the molecule resulting in denaturation. Once the GLUT and SGLT transporters were denatured the uptake of glucose was inhibited hence the lower uptake level shown in the results above. Some uptake however was shown to still occur (see figure 2) and because both transporters have been blocked by mercury this is most likely due to the passage of glucose across the membrane via passive diffusion. This is a limited process because glucose is a hydrophilic molecule and therefore cannot pass readily through the membrane. Alternatively perhaps mercury was not present in sufficient concentrations to block all of the membrane transporter proteins, therefore some remaining functional. The inconstant level of glucose uptake shown by both cells in the presence of mercury is perhaps due to dynamic concentration gradients across the membrane and possibly unequal volumes of mercury in each well due to experimental/human error. There are studies that concur with these results such as work by Farmanfarmaian et al (1989) but also conflicting evidence that suggests mercury may act to increase glucose transport in other mammalian cells (Barnes and Kircher, 2005). Therefore further testing is necessary to confirm findings.
A protein assay was included to measure the number of cells, a conversion factor allowed for fair comparison of radioactive glucose in the protein. The results lacking significance could be due to the short amount of time (5, 10 minutes) that had elapsed, after this point all the results were significant (Table1). It was expected that the graph showing glucose uptake without mercury would increase with time and then level off , this is not evident in these results but if the incubation times were increased then the graph would eventually reach maximum uptake. The size of the error bars show that there is quite small deviation from the mean in the results collected, with some exceptions due to anomalous results. For example the reading at 40 minutes in Caco-2 cells had a maximum value of 6.05 x 10-1 Âµmol/mg protein. This could be corrected by increasing the number of samples to provide accurate measurements. It appears that the result at 20 minutes in Caco-2 cells without mercury is anomalous. The original data shows an unusually high uptake of glucose (2.57 x 10-2 Âµmol/mg protein) which could be due to human or experimental error such as contamination. If selective transporter blockers were used this could discriminate more clearly between the different modes of uptake.
When their expression of glucose transporters is affected several diseases can result, for example; De Vivo Disease associated with a deficiency in GLUT1. There is also important ongoing research into therapeutic strategies for obesity and diabetes which is a growing global crisis. SGLT blockers are being developed which increase glucose lost through urine, these are currently undergoing clinical trials.
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