This experiment investigated the consumption of glucose into mammalian Caco-2 and SHSY5Y cells. These cells have different structural and functional features, the effects of which upon glucose uptake were compared and analysed. The cells were individually cultured, and incubated with radioactive glucose in two different conditions ; with or without the presence of HgCl2 ( which will be referred to as quicksilver ) , for assorted clip periods. The glucose consumption was so measured in a scintillation counter and the concentration of glucose in Aµmol/mg protein in the cells was calculated by correlating the consequences with a standardization curve from a bovine serum albumen ( BSA ) assay which was used as a criterion. Consequences showed that in the presence of quicksilver, glucose consumption is well reduced. At 30 proceedingss glucose uptake without quicksilver measured 1.99 ten 10-1 Aµmol/mg protein in Caco-2 cells and 1.48 ten 10-1 Aµmol/mg protein in SHSY5Y cells. These values decreased to 1.30 ten 10-2 Aµmol/mg protein and 4.76 ten 10-2 Aµmol/mg protein severally in the presence of quicksilver. Mercury binds irreversibly to the transporter proteins ( SGLT and GLUT ) in the cell membranes changing their construction, rendering them non-functional hence suppressing glucose consumption.
Glucose is a cardinal substance in mammals, it provides cells with the metabolic substrate required to bring forth energy in the signifier of ATP via oxidization shown below:
( 6O2 + C6H12O6 + 38ADP rarrow.gif ( 63 bytes ) 38ATP + 6CO2 + 6H2O ) .
Glucose is obtained from the diet or as a merchandise of gluconeogenesis in the liver and kidneys. Complex dietetic saccharides are typically cleaved into smaller polyoses or monosaccharoses by glycoside hydrolyse enzymes. The glucose can so undergo glycolysis to synthesise ATP ( Garrett and Grisham, 2010 ) .
Glucose must be transported across plasma membranes into the mark cells in order to be utilized. The manner by which this conveyance occurs varies depending on the location. There are two chief conveyance mechanisms, facilitated diffusion and active consumption ( primary and secondary ) , for which there are specific transporters. Six sodium- dependant glucose transporters SGLT1-SGLT6 ( Wright, 2001 ) have been identified every bit good as facilitative glucose transporters GLUT1-GLUT12 and HMIT ( Mueckler, 1994 ; Joost & A ; Thorens,2001 ) . An electrochemical gradient of Na is set up by the sodium/potassium/ATP-ase pump which provides the energy for the conveyance of glucose against its concentration gradient, via SGLTs. These transporters are expressed in the luminal membrane of enterocytes in the little bowel and across the apical membrane of the proximal tubules in the kidneys ( Wood and Tryahurn, 2003 ) . These transporters are significantly more limited in look in comparing to the facilitated glucose transporters which are found in most tissues, nevertheless the GLUT isoforms have more specific localization of function, GLUT3 as an illustration is found entirely in the encephalon. GLUTs “ conveyance glucose down its concentration gradient ” as stated by Lewin ( 2007 ) .
The purpose of this experiment was to look into and mensurate the consumption of glucose into two types of mammalian cells ; SYSY5Y cells and Caco-2 cells. SHSY5Y is a neuroblast ringer of the neuroblastoma cell line SK-N-SH which “ was established in 1970 from a metastatic bone tumor ” as stated by the cell lines service. Caco-2 cells are an immortalized line of heterogenous human epithelial colorectal glandular cancer cells.
Materials and Methods
In order to mensurate the glucose consumption of the civilized cells, the method as described antecedently ( Uemura et al. , 2005 ) was modified consequently. Dulbecco ‘s modified Eagle ‘s medium ( DMEM ) was used to turn the cells ( SHSY5Y and Caco-2 ) . The medium was supplemented with 10 % fetal calf serum, 100 units mL-1 penicillin, 100Aµg mL-1 streptomycin and 2mM glutamine. Stock civilizations were grown in 75cm2 flasks and incubated at 37oC in a humidified ambiance incorporating 5 % CO2.The cells were divided into 6-well home bases and grown until they reached meeting.
Conveyance and protein measurings
The cells were so washed twice with phosphate buffered saline ( PBS ) buffer and pre-incubated for two hours with 2mL of Krebs buffer ( HEPES, 30mM ; NaCl, 130 millimeter ; KH2PO4, 4 millimeter ; MgSO4, 1 millimeter ; CaCl2, 1 millimeter ; pH 7.4 ) at 37oC. At clip zero, the buffer was extracted with a Gilson pipette and replaced with 2mL pre-warmed Krebs buffer incorporating 0.5 mM 2-DG and 0.02 I?Ci of [ 3H ] -2-deoxyglucose ( [ 3H ] -2-DG ) in Wellss 1-3 and Krebs buffer with the conveyance inhibitor 3mM mercurous chloride ( HgCl2 ) in Wellss 4-6. The civilizations were incubated for assorted clip intervals of 5, 10, 15, 20, 30 and 40 proceedingss at 37oC. Consumption of [ 3H ] -2-DG was terminated by adding 2mL of ice-cold PBS buffer incorporating 0.5mM 2-DG. Reagents were removed utilizing a Gilson pipette and cells were washed by adding 2mL of ice-cold PBS buffer incorporating 0.5 mM 2-DG. Reagents were once more removed and to digest the cells 2mL of cell lysis solution ( 0.5 % Triton-X100 in 0.2M NaOH ) were added. The home bases were incubated at room temperature for 10 proceedingss on the home base shaker. To guarantee cells had lysed, they were viewed under a microscope. The lysate was so used for two separate subdivisions of the experiment.
1. Glucose Uptake Assay
0.5mL samples of lysate were extracted from each well and transferred into 3 separate phials ( for each well ) utilizing a clean tip for the Gilson pipette for each well. 10mL of scintillation cocktail was added utilizing the automatic dispenser to each phial, these were so labelled on the lid consequently. Phials were placed into the scintillation counter for readings to be obtained. 10AµL of each cell lysate ( repeated in triplicate ) were assayed in a 96-well home base, besides incorporating 200AµL color reagent ( 49 parts bichinchoninic acid and 1 portion Cu sulfate pentahydrate ) per good and protein concentration was measured.
2. Protein BSA Assay
A BSA check was set up in a 96-well home base to utilize as a standard protein concentration to so put up a standardization curve. 6 different volumes of 1 mg mL-1 BSA runing from 0-10 AµL were pipetted into the home base so that the protein concentration increased across the rows. M-Q H2O was added consequently to do the well volume up to 10 AµL, eventually 200 AµL coloring material reagent was added into each well. Plate was covered and incubated at 37oC for 30 proceedingss. The home base was read at 570 nanometer in the automatic home base reader which provided informations to input on excel which plotted a standardization curve of BSA concentration ( mg mL-1 ) versus optical density at 570 nanometer. Linear arrested development produced a consecutive line and equation that was used to gauge the sum of protein ( milligram ) in each well for each cell type.
Consequences were processed utilizing Microsoft Excel, paired T-tests were carried out to measure whether the consequences with and without quicksilver were statistically different and the p value gives an indicant of its significance. If P & lt ; 0.05 = important, if P & lt ; 0.01 = extremely important. SEM values were calculated for all consequences and mistake bars were displayed on the graphs utilizing the appropriate values to expose variableness of agencies.
The consequences show glucose consumption in both cell types increased with increasing incubation clip. The big difference seen in the consumption of glucose, in both cell types between the two different conditions are due to the inhibiting effects of quicksilver. It has been shown by Farmanfarmaian et Al ( 1990 ) that “ underA in vivoA orA in vitroA tissue incubation the bound quicksilver inhibits the soaking up of foods such as aminic acids and sugars. ” This is reflected in the consequences, shown clearly in Figure 1, when quicksilver is present in solution the consumption of glucose into both cell types is well reduced. The consumption in Caco-2 cells is higher and its uptake additions more with increasing clip in comparing to SHSY5Y cells. The minimal glucose consumption is 5.78 ten 10-2 Aµmol/mg protein at 5 proceedingss and increases to a maximal consumption of 3.76 ten 10-1 Aµmol/mg protein at 40 proceedingss. Glucose uptake for Caco-2 cells averaged across all clip points in the absence of quicksilver is 1.85 ten 10-1 Aµmol/mg protein. Glucose consumption in SHSY5Y cells without quicksilver scopes from 2.36 ten 10-2 Aµmol/mg protein at 5 proceedingss to 1.96 ten 10-1 Aµmol/mg protein at 40 proceedingss, averaging at 8.02 ten 10-2 Aµmol/mg protein taking into history all incubation times.
Figure 1: Glucose consumption in Caco-2 and SHSY5Y cells with and without quicksilver with increasing incubation clip and mistake bars bespeaking SEM.
When quicksilver is present, the maximal glucose consumption for Caco-2 cells is reduced to 2.87 ten 10-2 Aµmol/mg protein and 2.60 ten 10-2 Aµmol/mg protein in SHSY5Y cells. The difference in glucose consumption between the absence and presence of quicksilver in Caco-2 and SHSY5Y cells is 0.1563 Aµmol/mg protein and 0.0542 Aµmol/mg protein severally. These consequences are preponderantly extremely important with several exclusions ( see table 2 for informations ) for illustration each reading at 5 proceedingss is undistinguished, every bit good as the reading at 10 proceedingss comparing the consequences between the cell types with quicksilver nowadays ( shown as N in the tabular array ) .
Table 1: SEM values for SHSY5Y and Caco-2 cells with and without quicksilver
Notably, glucose consumption still occurs in both cell types despite the presence of quicksilver ( shown more clearly in figure 2 ) . In both cell types after 5 proceedingss there is an initial lessening in glucose consumption and so an equivocal form of uptake thereafter. The mistake bars are by and large little, excepting the largest saloon at 40 proceedingss in the Caco-2 cells ( SEM=2.52 x 10-2 ) and there is no convergence of mistake bars between conditions.
Table 2: Phosphorus values and significance of presence of quicksilver in Caco-2 and SHSY5Y cells and comparing between the two cells with quicksilver at each incubation clip interval.
Caco-2 V SHSY5Y ( with HgCl2 )
Figure 2: Glucose consumption in Caco-2 and SHSY5Y cells in the presence of quicksilver at each incubation clip interval and mistake bars bespeaking SEM.
Some consequences which were possibly unusual are the glucose uptake readings for Caco-2 cells at 20 and 30 proceedingss ( without quicksilver ) and SHSY5Y cells with quicksilver at 30 proceedingss where the graph additions and so decreases once more at 40 proceedingss.
As illustrated in the consequences, the consumption of glucose in Caco-2 and SHSY5Y differs and this is due to the look of assorted transporters in their respective cell membranes. Caco-2 cells express GLUT1, 2, 5 and 12 every bit good as SGLT1. Increased glucose consumption in Caco-2 cells is mostly due to the look of these sodium-dependent glucose transporters which actively transport glucose up its concentration gradient ( see debut ) . Furthermore, SGLT1 and GLUT 1 are high affinity glucose transporters accordingly these factors result in higher consumption than SHSY5Y cells. These neuronal-like cells express merely GLUT transporters 1, 3, 4, 6, 8 and HMIT. These all transport glucose via facilitated diffusion which is a inactive procedure and can merely transport glucose down its concentration gradient.
To distinguish the intracellular metabolic effects from the membrane effects on glucose consumption, quicksilver was used as a conveyance inhibitor. When quicksilver was present in the solution, a dramatic decrease in glucose consumption resulted in both cells. Mercury can denature proteins “ by adhering to carboxylate and, in peculiar sulfhydryl groups in proteins. ” As described by Meisenberg and Simmons ( 2006 ) .In this case, the quicksilver irreversibly bound to the protein transporters, interrupting the disulphide bonds which altered the third construction of the molecule ensuing in denaturation. Once the GLUT and SGLT transporters were denatured the consumption of glucose was inhibited therefore the lower uptake degree shown in the consequences above. Some uptake nevertheless was shown to still happen ( see figure 2 ) and because both transporters have been blocked by quicksilver this is most likely due to the transition of glucose across the membrane via inactive diffusion. This is a limited procedure because glucose is a hydrophilic molecule and hence can non go through readily through the membrane. Alternatively possibly quicksilver was non present in sufficient concentrations to barricade all of the membrane transporter proteins, hence some staying functional. The inconstant degree of glucose uptake shown by both cells in the presence of quicksilver is possibly due to dynamic concentration gradients across the membrane and perchance unequal volumes of quicksilver in each well due to experimental/human mistake. There are surveies that concur with these consequences such as work by Farmanfarmaian et Al ( 1989 ) but besides conflicting grounds that suggests quicksilver may move to increase glucose conveyance in other mammalian cells ( Barnes and Kircher, 2005 ) . Therefore farther testing is necessary to corroborate findings.
A protein check was included to mensurate the figure of cells, a transition factor allowed for just comparing of radioactive glucose in the protein. The consequences missing significance could be due to the short sum of clip ( 5, 10 proceedingss ) that had elapsed, after this point all the consequences were important ( Table1 ) . It was expected that the graph demoing glucose consumption without quicksilver would increase with clip and so level off, this is non apparent in these consequences but if the incubation times were increased so the graph would finally make maximal consumption. The size of the mistake bars show that there is rather little divergence from the mean in the consequences collected, with some exclusions due to anomalous consequences. For illustration the reading at 40 proceedingss in Caco-2 cells had a maximal value of 6.05 ten 10-1 Aµmol/mg protein. This could be corrected by increasing the figure of samples to supply accurate measurings. It appears that the consequence at 20 proceedingss in Caco-2 cells without quicksilver is anomalous. The original information shows an remarkably high consumption of glucose ( 2.57 x 10-2 Aµmol/mg protein ) which could be due to human or experimental mistake such as taint. If selective transporter blockers were used this could know apart more clearly between the different manners of consumption.
When their look of glucose transporters is affected several diseases can ensue, for illustration ; De Vivo Disease associated with a lack in GLUT1. There is besides of import ongoing research into curative schemes for fleshiness and diabetes which is a turning planetary crisis. SGLT blockers are being developed which increase glucose lost through piss, these are presently undergoing clinical tests.
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