The polyol pathway is a two step metabolic pathway in which glucose is first reduced to sorbitol by Aldose Reductase (AR). The sorbitol is further converted to fructose by the enzyme Sorbitol Dehydrogenase (SDH). In normal cells the glucose enters the glycolysis pathway and unused amount of glucose enters the polyol pathway. The polyol pathway becomes active when there is more amount of glucose present in the cells. But in hyperglycaemic state there is more amount of glucose entering the polyol pathway which further leads to many health complications such as diabetes and also retina and kidney damage. So a need arises for completely understanding the significance of the Polyol pathway and then detecting the presence of Aldose Reductase. The findings reported here indicate clearly the activity of NADPH and also confirm the presence of Aldose Reductase in the Polyol Pathway.
Key Words: Polyol pathway, Aldose Reductase, NADPH.
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Recombinant Protein Expression is a very important technology for the production of Biopharmaceuticals. The expression of recombinant proteins in different host systems has always been a major feature and challenge in the Biotechnology industry (Baldi, L et al 2006). The most important criteria for selecting the host system is that the cell line must have the stability of expression. There are several expression systems but the majority of the recombinant proteins are produced in either Mammalian cells or in E.Coli (Dickson, A.J. et al, 2003).
Mammalian cells are the most common preferred hosts for recombinant protein production because of their stable gene expression, correct protein folding, high levels of protein expression, glycosylation patterns and post translational modifications (Wurm, F.M. 2009). There are various mammalian expression systems namely Chinese hamster ovary (CHO) cells, Nonsecreting (NSO) myeloma cells, Baby hamster kidney cells (BHK) and PER.C6 Human embryonic retinal cell line (Dickson, A.J. et al, 2003). The Chinese hamster ovary cells are the most important cell lines for the production of recombinant proteins (Hacker, L.D 2009). .
Glucose is utilised by the cells for energy. The majority of the glucose in the body enters the normal Glycolysis pathway (Fig 1) whereas the unused glucose enters the Polyol Pathway (Fig 1). The Polyol pathway also called as the sorbitol or Aldose Reductase pathway is thus one minor pathway of glucose metabolism. The polyol pathway is a two step enzymatic pathway. In the first step glucose is first reduced to sorbitol using nicotinamide adenine dinucleotide phosphate (NADPH). This conversion of glucose to sorbitol is the rate determining step of the pathway (Kinoshita and Nishimura, 1988). The first and the rate limiting enzyme of this pathway is Aldose Reductase (AR).
Further in the second step sorbitol is oxidised to fructose using nicotinamide adenine dinucleotide (NAD+) and the enzyme facilitating this conversion is sorbitol dehydrogenase (SDH).
Sorbitol which is an alcohol that is strongly hydrophilic diffuses through the cell membrane at a much slower rate than glucose and fructose. As a result it tends to accumulate intracellularly and thus causes an increase in the osmotic pressure. The increase in osmotic pressure causes the cells to undergo apoptosis. The polyol pathway tends to become active when the intracellular glucose levels are being elevated. When the glucose levels in the blood are normal this conversion will not cause any problem as AR has low affinity for glucose at normal concentrations. However in hyperglycemic state there is a rise in the affinity levels of AR for glucose. This rise leads to the accumulation of sorbitol and eventually leads to the activation of the Polyol pathway.
Figure 1: Glycolysis and Polyol Pathway. G 6 P: Glucose-6-phosphate, F 6 P: Fructose-6-phosphate, F D P: Fructos1,6 diphosphate, GA3P: Glyceraldehyde 3 phosphate, DHAP: Dihydro acetone phosphate
The polyol pathway appears to play a major role in diabetic complications that lead to the damage of the nervous tissue and also to the retina and the kidney. Although it is known for quite some time that the polyol pathway leads to major health problem such as diabetes inhibiting the pathway started only after a primary role was assigned to Aldose Reductase (the first enzyme in the polyol pathway). In order to understand the role of Aldose reductase and to validate the hypothesis Samuel, L.T. and Harding, B.W had set up an enzymatic assay for Aldose Reductase. The search for a suitable extract for enzymatic assay was on and was found that rat eye lens and ischemic hearts gave positive results for the Aldose Reductase assay. Thus the detection of Aldose Reductase activity in the polyol pathway and then inhibiting the pathway will halt the unused glucose in the cells to enter the polyol pathway. Thus there will be an increase in the amount of glucose entering the glycolysis pathway and eventually more amount of energy released. Inhibiting the first step of the polyol pathway is important as the conversion of glucose to sorbitol can be stopped, which marks the end of many health complications.
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In order to completely understand the first step of the polyol pathway I have carried out experiments in order to study the expression and detection of Aldose Reductase in a new mammalian cell line.
2. Materials and Methods:
2.1 Cell line and Growth medium:
In this study a new cell line namely LB01 from Lonza Biologics has been used. The cell line was grown in CD CHO (Invitrogen) medium. The CD CHO is a chemically defined protein free medium. The cell lines were grown as suspension cultures in this medium. Methionine Sulfoximine (MSX) was added to the medium to culture the cell lines. 25µM of MSX was added in order to maintain a selective pressure. LB01 cells at 0.2 - 106 cell/ml were cultured in a 50ml PETG Sterile flask and maintained in a Unitron shaking incubator at 37°C and 100rpm.
2.2 Determination of Cell viability:
The cell number and viability were measured by Trypan blue exclusion. 20µl of 1% (w/v) trypan blue was mixed with 20µl of the cell suspension and then loaded onto the haemocytometer. The number of live and dead cells were counted by using light microscopy so that the total and the viable cell numbers could be compared.
2.3 Growth Analysis and Sub culturing:
LB01 cells were grown as suspension cultures and the cell density was measured for up to 14 days in order to analyse the growth pattern of the cells. The cells were routinely sub cultured during the mid-exponential phase of batch culture that is usually every three to four days.
2.4 Protein Extraction for Enzymatic Assay:
5ml of the cells during the day 5 of their growth phase were taken in a 10ml BD Falcon tube. The tubes were centrifuged at 10,000 - g for 20 min at 4°C. The resulting pellet was washed twice with 3ml of PBS. The pellet was again centrifuged so as to remove any PBS that is remaining. 200µl of the extraction buffer was then added to the pellet. The extraction buffer consisted of 25Mm Tris (pH 7.4), 50mM Nacl, Aprotinin (10µg/ml), Leupeptin (10µg/ml) and PMSF (0.57mM). The pellet along with the extraction buffer was vortexed. The sample was then freeze-thawed in Liquid Nitrogen for 4 times and then centrifuged at 10,000 - g for 20 min at 4°C. The supernatant was collected and stored in 20µl aliquots at -80°C.
2.5 Enzymatic Assay:
For the enzymatic assay, each 1ml cuvette contained 0.15M Potassium Phosphate buffer (0.33ml), 0.7M Glucose 9 (0.1ml), 10mM NADPH (10µl), cell extract, and water (0.55ml). The absorbance was measured in a spectrophotometer at 340nm. The decrease in absorption was measured every 2 min for the first 14 min and the decrease in the absorbance values was taken as the measure of NADPH oxidized.
2.6 Protein extraction for Western Blot:
2ml of the cells at a concentration of 5 - 106 cells/ml are taken in falcon tube and centrifuged at 10,000 - g for 20 min at 4°C. The resulting pellet was washed with PBS twice and recentrifuged to remove any PBS that is remaining. The pellet was resuspended in RIPA buffer (300µl), Aprotinin (3µl), Leupeptin (3µl) and PMSF (3.5µl). The sample mixture was then incubated on ice for 30 min. Following incubation the sample was centrifuged at 10,000 - g for 20 min at 4°C. The supernatant was collected and stored as 25µl aliquots at -80°C.
2.7 Biorad Protein Assay:
The Biorad assay for determining the protein concentration was performed in a 96 well plate. The assay was performed according to the normal laboratory protocol and the absorbance was measured on a plate reader at 570nm.
2.8 Western Blot:
The western blot technique was carried out as per the normal protocol. A 12.5% separating and stacking gel was used and once the gel was completely set it was placed on the running stand. The protein samples were prepared accordingly with the sample buffer and 30µl of the protein sample was loaded onto the gel. The protein samples were loaded onto the gel in a specific order along with the standard protein marker. The gel was run at 60v until the dye front has just crossed the stacking layer and then the current was further increased to 200v and run for 40 min. Once the gel has run completely the gel was transferred onto the nitrocellulose membrane on a trans-blot apparatus. The gel was transferred at 15v for 30 min. The membrane was further stained with Ponsceau stain and then blocked with 3% Milk solution for a minimum of 1hour. The membrane was later incubated with the primary antibody and then secondary antibody. The Aldose Reductase (FL-316) from Santa Cruz Biotechnology was used as the primary antibody and was diluted at a range of 1:1000. Anti-rabbit IgG HRP was used as a secondary antibody. The JAR cell lysate from Santa Cruz Biotechnology was used as a positive control for the detection of the protein. The membranes were at last subjected to Enhanced Chemiluminescence (ECL) for 2 mins and for 5 mins respectively.
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3.1 Growth Curve of LB01 cells:
LBO1 cells were grown as suspension cultures and the growth rate of the LB01 cell line had been measured for a period of 14 days. Each day the cells were counted by trypan blue exclusion using a light microscope from which the viable cell density was calculated. It has been observed that the cells grow at an exponential phase till they reach their maximum viable cell density on Day 7 after which they enter the decline phase. The viable cell density decreases gradually after day 7 and at a certain point of time (Day 13) it eventually becomes zero. It is thus highly recommended that the sub culturing of the cells is done on day 4 or day 5 as the cells are in the mid-exponential phase of batch culture. The viable cell density values thus obtained have been represented graphically (Fig. 2). The percentage viability of the LB01 cell line are also represented by Figure 3.
Figure 2: Growth curve of LB01 cell line. The viable cell density of the cell line was calculated by trypan blue exclusion.
Figure 3: Percentage of Viable cells
3.2 Enzymatic Assay Analysis:
The enzymatic assay was set up as described previously in section 2.4. The change in NADPH value was spectrophotometrically monitored in the presence of glucose as well as in the absence of glucose. In order to analyse the rate of decrease of NADPH three concentrations of the cell extract (5µl, 10µl and 20µl) were added to the assay. The rate of decrease in the values of NADPH in the presence of glucose is more when compared to the values without glucose. This decrease in values clearly states that NADPH reduces glucose to sorbitol in the polyol pathway.
Table 1 (A): Enzyme activity values with 5µl of the extract in the presence and in the absence of Glucose
In the presence of Glucose
In the absence of Glucose
Table 1. (B): Enzyme activity values with 10µl of the extract in the presence and absence of Glucose
In the presence of Glucose
In the absence of Glucose
Table 1 (C): Enzyme activity values with 20µl of the extract in the presence and absence of Glucose
In the presence of Glucose
In the absence of Glucose
3.4 Western Blot:
To examine the expression of Aldose Reductase Western Blot analysis was carried out with the enzyme extracted as described in section 2.6. The Biorad Protein assay was first performed and the amount of protein sample to be loaded onto the gel was determined. The protein concentration was 8.12µg/µl in LB01 cell extract. Aldose Reductase (FL-316) was used as the primary antibody for detecting Aldose Reductase protein in LB01 cells. Anti rabbit IgG HRP was used as the secondary antibody and after Enhanced Chemiluminescence (ECL) a band of 37kDa which confirms the presence of Aldose Reductase was obtained. The membrane was subjected to 2mins and 5 mins exposure to ECL. The bands obtained (Fig. 4(a) and 4(b)) clearly indicate the presence of Aldose Reductase. It has also been observed that there is a slight darker band in the film that is exposed to ECL for 5 mins than when compared to 2 mins exposure of the film.
1 2 3 4 5 6
Figure 4 (a): 2 mins exposure to ECL.
1 2 3 4 5 6
Figure 4 (b): 5 mins exposure to ECL.
Lanes 1: Protein Marker, Lane 2: JAR cell lysate (Positive control), Lane 3: Cell line A, Lane 4: Cell line B, Lane 5: Cell line C, Lane 6: LB01 Cell line
Aldose Reductase is a member of the aldo-keto reductase family. The aldose reductase has broad substrate specificity. The experiments that have been done clearly demonstrate that there is a positive correlation between NADPH and Aldose Reductase as there is a decrease in the absorbance values.. It is confirmed that glucose is reduced to sorbitol with the oxidation of NADPH TO NADP+ and facilitated by Aldose Reductase Enzyme. Consequently though there is a decrease in absorbance of NADPH values in the enzymatic assay the rate of decrease is not constant. The reason may be that the enzyme present in the cells may be not sufficient for the assay. Previous studies of Aldose Reductase experiments showed a gradual rate of decrease in NADPH absorbance that was proportional to the amount of extract added but these were performed with Rats lens (Saraswat, M. Et al, 2008). Other studies on ischemic rat hearts (Hwang, C. Y., et al. 2001) have also resulted in positive results for the aldose reductase assay. These studies give us an idea that there is definitely certain amount of enzyme present in the extract but this might probably not be enough for the enzyme assay. The decrease in absorbance values affirms the conversion of NADPH to NADP+.
In order to move forward and firmly confirm the presence of Aldose Reductase in LB01 cells a western blot analysis was performed. The positive results from western blot analysis, that is the appearance of the band at 37kDa confirms the presence of Aldose Reductase in the cell extract. JAR Cell lysate (positive control) which was also loaded onto the gel also gives us an affirmation of the Aldose reductase protein.
Studies by Hers (1957, 1960a,b) have shown that Aldose Reductase was present in the seminal vesicles that is responsible for the conversion of Glucose to sorbitol. I was able to extend Hers observation with a new cell line and confirm the presence of Aldose Reductase. In these cell lines no direct relationship could be determined between the rate of decrease in NADPH values and the amount of enzyme extract that was added to the assay. However, the experiments clearly demonstrated positive results with the detection of Aldose reductase by western blot analysis to confirm the presence of the protein.
Since the presence of Aldose Reductase is confirmed it is possible to further research on inhibiting the Aldose Reductase activity. There are several Aldose reductase inhibitors available such as Tolrestat, Ponalrestat, Zenarestat, Sorbinal and Alrestatin. Most of the aldose reductase inhibitors are currently not available due to undesirable side effects. However Tolrestat is said to have shown positive results in experimental models of renal medullary cells (Yancey, P. H. Et al).
Based on the experimental results the enzyme Aldose Reductase is responsible for the reduction of glucose to sorbitol in the Polyol Pathway. It is thus possible to inhibit this pathway by using Aldose Reductase inhibitors so that the unused glucose does not enter the Polyol pathway. If the amount of glucose entering the polyol pathway is reduced then there will be more amount of energy entering the Glycolysis pathway and eventually more amount of energy released. Inhibiting this pathway will also lead to a decrease in diabetic health complications.
The experimental work has been carried out under the supervision of Professor Alan Dickson at the University of Manchester. I am very thankful to my Project Supervisor Professor Alan Dickson for giving me an opportunity to do this project and also for all the valuable guidance and support that he had given me. I would like to also thank my course director Dr. Anil Day for the guidance on my project. I also thank my family and friends for their help during my project period.