INTRODUCTION

 

Types of Diabetes

One of the more common diseases affecting the world today is diabetes. There are two different kinds of diabetes, diabetes insipidus and diabetes mellitus. Diabetes insipidus is a pathological endocrine condition characterized by extreme thirst and excessive production of very dilute urine. The essential feature of the disorder appears to be a lack of antidiuretic hormone (vasopressin) or a blocking of its action. Diabetes mellitus is the more commonly known form of the disease, which results from some complication with the body’s dealing with glucose.

There are two major types of diabetes mellitus. Type I diabetes mellitus is caused by a lack of insulin production. Type II diabetes mellitus, or late-onset diabetes, is the most common form of the disease. About 90% of all people with diabetes have Type II diabetes mellitus. This form of diabetes results from sluggish insulin production, tissue resistance to the secreted insulin, or any malfunction in carbohydrate metabolism. It is estimated that by the time a person is 60, there is a 75% chance of contracting Type II diabetes. By age 75, the chances of contracting Type II diabetes increases to 80%.

While the exact causes of diabetes remain obscure, certain facts are evident. Beta cell damage in Type I diabetes is the result of a process called autoimmunity, in which a diabetic person's immune system creates antibodies that destroy their own beta cells. Type II diabetes, on the other hand, is linked to heredity and obesity, notably upper-body obesity--which refers to a waist-to-hip circumference ratio of more than 85:100.

As of now there are only ways to manage the disease. Though popular thought is that diabetes has been put under control and is not fatal, death from diabetes can only be postponed. Eventually the level of glucose in the blood corrodes the blood vessels resulting in serious cardiovascular complications. In addition, corrosion of the tiny capillaries in the eyes results in diabetic retinopathy, which leads to blindness. Diabetics are also subject to kidney diseases and frequent infection. Untreated diabetes leads to ketosis, the accumulation of ketones, products of fat breakdown, in the blood; this is followed by acidosis (accumulation of acid in the blood) with nausea and vomiting. As the toxic products continue to build up, the patient goes into diabetic coma.

 

Available Treatments

There are currently few ways to treat diabetes. For those who lack the ability to produce insulin, treatment can involve the administration of synthetic insulin. For those who have a resistance to insulin, a possible treatment is through the administration of insulin mimetics. These formulations penetrate the cell membrane without the aid of receptors and initiate the same reactions that the binding of insulin to the insulin receptor normally would. Insulin mimetics can also be administered to patients in which the insulin receptor is faulty or mutated, since mimetics do not require binding to a receptor to be effective. Since these mimetics are usually metals, it is important to test for and analyze the specific mimetic’s therapeutic effects versus its possible toxic effects. As of now, selenium and vanadium compounds are believed to be non-toxic and are able to aid in glucose absorption. Cadmium compounds, however, are found to be toxic.

Despite their value in treating many instances of diabetes, insulin mimetics are not helpful in Type II diabetes where receptor mutation is not the problem. In this case, a method must be found to mimic the “missing link” in the metabolic cycle.

 

Glucose and Carbohydrate Metabolism

In essence, diabetes is an excess of glucose in the bloodstream. It is this characteristic that leads to all of the problems that branch off the contraction of this disease. However isolating the exact step of bodily interaction with glucose that is malfunctioning or absent is very difficult. Even after this step is isolated, there is still the task of fixing this step in order to put the metabolic pathway in working condition.

This experiment deals with one such step of the pentose-phosphate pathway.

The pentose-phosphate pathway (Figure 1), the series of actions comprising a metabolic pathway which branches off glycolysis, is one of the pathways that a glucose molecule may traverse when entering a cell. This series of actions occur in the cell cytoplasm and are initiated with the binding of insulin to its receptor on the given cell membrane. The pentose-phosphate pathway is driven by a number of enzymes. When glucose 6-phosphate enters the pathway, it is converted to 6-phosphogluconolactone via the enzyme glucose 6-phosphate dehydrogenase (G6PDH).

It has been previously shown that glucose-induced synthesis of this enzyme occurs transcriptionally. The purposes of this experiment are to determine the relationship between the amount of glucose present in the active cell and the amount of transcripted

G6PDH and to narrow the region of the transcripting gene promoter that is related to the glucose response.

HYPOTHESES AND OBJECTIVES

 

This will be done by examining the effects of glucose on the entire 935 base-pair G6PDH promoter, as well as examining the effects of glucose on subsets of this promoter, namely the last 187 base pairs of the promoter and the last 635 base pairs of the promoter (Figure 2). The G6PDH promoter contains a series of base-pairs (an E-box) common in many glucose-responsive enzymes. Therefore, it is hypothesized that G6PDH transcription increases with the amount of glucose present within the cell. This E-box is also contained within the 635 base pair section of the 935 base pair G6PDH promoter. Therefore, it is also expected that the 635 base-pair section will respond to glucose. However, the 187-base pair section does not contain this E-box and therefore it is hypothesized that the 187 base pair section of the promoter will not respond to glucose.

 

Significance of the Study

Examining the effects of glucose on G6PDH will lead to a better understanding of both the enzyme and the pathway it is a part of. Since pathways responsible for carbohydrate metabolism in some way involve glucose, determining the glucose-responsiveness of G6PDH may also shed light on the effects of glucose on many other enzymes that are involved in carbohydrate metabolism, whether they are located in the same pathway as G6PDH or not. In addition, isolating the glucose responsive region of the G6PDH promoter will open the door for possible “genetic therapies” for patients with diabetes related to G6PDH malfunction or deficiency. Currently, there is no remedy for such a mutation.

 

EXPERIMENTAL

 

Figure 2. Rat G6PDH Promoter DNA Sequence. The transcription start site is denoted by a +1.

Sequence homology to the human gene is shown by the dashed underline and mouse homology

is shown by the solid underline. Adapted from Rank, et al (1994) Biochimica et Biophysica Acta,

1217, 90-92. The entire sequence is the 935 base pair promoter used for cell transfection. The subsets

of the promoter used for cell transfection are shown as follows: Blue: 187 base pair section. Red+Blue:

635 base-pair section.

 

Materials

 

On the day before the Sprague-Dawley rat was sacrificed, 130 plates were coated with commercially obtained rat-tail collagen and were placed under UV light overnight. In addition, digestion buffer is used immediately after the dissection on the liver tissue and Trypan blue dye is used to dye the cells to determine cell viability. For the purposes of transfection, the 935 base-pair, 635 base-pair, and 187 base-pair plasmid constructs were obtained. In addition, the cells must be exposed to luciferin in order for the G6PDH Promoter Luciferase Construct to be effective. Lipofectin is also needed to carry out the lipid-mediated transfection.

 

Methods

Immediately after the liver was excised, it is placed in a beaker containing enough digestion buffer to immerse the liver.

The liver was then rinsed under a hood over sterile gauze using digestion buffer. The tissue was pressed through sterile 250 to 275 micron mesh sieves in order to begin the isolation of the individual hepatocytes. The cells were then centrifuged and the supernatant liquor was discarded. Just enough media was added to the hepatocyte pellet to immerse the cells. The cell pellet was then pipetted to disperse the hepatocytes. The cell suspension was then centrifuged again and the media was extracted, leaving the hepatocyte pellet.

The cells were then re-suspended in 5mM Waymouth’s media with BSA. Trypan blue dye was then added to approximately 25 mL of the solution. The cell/stain solution was then plated and the cell viability determined. This is done by finding the area of the plate with the greatest number of blue-dyed cells (dead cells) and by counting the number of viable and non-viable cells in the given area (4 nanometer by 4 nanometer square). This was done using a hemocytometer.

Optimum cell dispersion on the plate would be a single layer of hepatocytes, with minimal space in between cells. Based on the dispersion of the stained cells, the non-stained cells were plated on three different plates at three different test concentrations. Plating at higher cell concentrations would result in more crowded cell dispersion. Plating at lower concentrations would allow for greater space between cells. Based on the dispersion of the cells on the test plates, the optimum concentration at which to plate the rest of the cells was determined, and the cells were plated at that concentration. The optimum plating concentration ranged from 200 to 400 mL of cells for every 4 mL of final solution plated. The culture dishes were then transferred to a 37°C incubator in an atmosphere of 5% CO2: 95% air.

After allowing an attachment time of at least 4 hours, the plates were rinsed with 37°C 5mM Waymouth’s serum free media in order to remove the BSA. The old media was removed, and fresh serum free media was added. This media also contains luciferin (30mL luciferin for 4mL of plated media). The luciferin diffuses into the hepatocytes. The plates were then returned to the incubation chamber overnight.

The cells can then be transfected with the various plasmid constructs—935 base pair, 635 base pair and 187 base pair. The plasmid construct is shown in Figure 3. 24 plates were set aside for transfection (8 for each plasmid construct). Transfection is

 

 

 

 

 

 

 

 

 

 

 

Fig 3. The G6PDH/Luciferase Plasmid Construct. The hepatocytes are transfected with this DNA construct which

 responds to a glucose stimulus by increasing the transcription of luciferase (Luc) through the G6PDH promoter (935 bp).

The enzyme luciferase, if reacted with luciferin, produces scintillations that can be measured with a scintillation counter

as counts per minute (CPM).

 

 

effected under a sterile hood. Each plasmid solution is first suspended in lipofectin for 10 minutes after which the plasmid/lipofectin solution was mixed with 5mM Waymouth’s media. The media was then set to rest to help the lipid to surround the plasmid construct. About 9 minutes into this period, the media on the plated cells was decanted. One minute later, the 5mM Waymouth’s media containing the plasmid was added to the cells. The plates were then returned to the incubator for 6 hours, after which the media was changed.

By this time the plasmid would have entered the cells via the lipid-mediated transfection. The 5mM Waymouth’s media was then decanted and washed with the new solution (which differs for each plate) before adding the fresh media. A different glucose concentration—5, 10, 20, or 30mM—of serum free Waymouth’s media was added to every two of the 8 plates for each plasmid construct. The plates are then returned to the incubator for another 18 to 20 hours.

Finally, the cells were scraped, lysed, and measured for luciferase counts and total protein content. A scintillation counter is used for measuring the luciferase counts and total protein content is measured using the Lowry protein assay (Lowry et. al., 1951).

RESULTS AND ANALYSIS

 

Basis

If the G6PDH promoter is activated by glucose, then the G6PDH Promoter Luciferase Plasmid construct would transcript the Luciferase protein. Therefore, the amount of transcribed Luciferase is directly proportional to G6PDH transcription in response to the given glucose concentration. However, the amount of Luciferase in a sample cannot be measured, though the total amount of protein in the sample can be measured using a Lowry protein assay. However, since the cell contains luciferin, any luciferase produced would make the sample fluoresce, since luciferase would react with the luciferin in the hepatocyte. The fluorescence of each sample can be attributed solely to the amount of luciferase transcribed by the plasmid. Therefore, to bring the various values of each sample to a common unit, a value that can be solely accredited to the amount of Luciferase in each sample, Luciferase activity was measured as the number of scintillations given off by the sample (counts per minute) over the total amount of protein in the sample (mg).

Since it also hypothesized that increased sugar concentration would result in a greater amount of transcribed luciferase, luciferase activity is measured over a range of sugar concentrations between 5mM and 30mM glucose. The results are compared with 5mM glucose concentration as the control value. This media glucose concentration is used as the base rather than using media void of glucose as the control value because the hepatocytes would die if they are not provided with enough glucose to make ATP.

Since the luciferase activity of a given glucose concentration can be many times over that of the control, the results are represented as the fold increase of luciferase activity in comparison with the luciferase activity of cells in 5mM glucose.

 

Data Handling

 

To achieve homogeneity of the data and eliminate outliers in the results, the data was subjected to a Q-test. For a given sample, a Q-critical value can be looked up from a reference table based on the number of data points for a given set and the mean of that set of numbers. In addition, a Q-value can be calculated for any given data point. The Q-value for a data point is equal to the difference between that data point and its nearest neighbor divided by the difference between that data point and its farthest neighbor. If a data point’s calculated Q-value is greater that the Q-critical value for the set, then the data point is an outlier and can be removed from the data set.

The results were analyzed using a Student’s t-test. The Luciferase activity for each glucose concentration was compared to the luciferase activity for 5mM glucose concentration. Data was deemed significant for p-values of 0.05 or less.

 

Effect of Glucose on Luciferase Activity

935 Base Pair Construct Only the 20mM glucose concentration luciferase activity was found to be significantly greater than the 5mM glucose luciferase activity in the 935 base-pair construct. (Figure 4 and Table 1) In fact, glucose activity for the entire promoter appears to peak around a concentration of 20mM glucose. However, the luciferase activity appears to increase as the amount of glucose applied to the cell increases from 5mM to 20mM  glucose  concentration.  Additional  experiments are to be

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4. The Effects of Glucose on the 935 Base Pair G6PDH Luciferase Palsmid Construct.

Abbreviaion and Symbols: Glc-glucose; star-indicates statistical significance over the value for

 5mM glucose by Student’s t-test, p<0.05

 

 

Treatment	2/4/01	2/11/01	2/18/01	2/25/01	3/4/01	3/11/01	Mean	Std. Error
5mM Glc	1	1	1	1	1	1	1	-
10mM Glc	0.19	2.28	0.91	0.33	0.82	1.79	1.05	±0.34
20mM Glc	3.23	1.92	3.54	1.22	0.51	1.65	2.01	±0.48
30mM Glc	2.46	2.17	34.4	12.1	0.1	1.73	1.62	±0.53

 

 

 

 

Table 1. 935G6PDH Promoter Section Luciferase Activity in Relation to Glucose Levels in Waymouth's Media. The data presented show the fold increase in

luciferase activity of 935 bp construct cells when compared to the luciferase activity of 935 bp construct cells immersed in Waymouth’s media containing

5mM of glucose. The treatment is the varying glucose concentration of the Waymouth’s media that the cells were immersed in. Luciferase activity (CPM/µg)

was measured by calculating the scintillation of luciferin (counts per minute) over the amount of protein in the cell(µg). Abbreviations: Glu-glucose, Values

in white: These data points were eliminated by Q-test.

 

 

 

run on either side of 20mM glucose concentration to verify the existence of any possible peak in the glucose response for the promoter.

635 Base Pair Construct None of the values for the 635 base pair construct are significantly greater than the 5mM glucose concentration (Figure 5 and Table 2). Since, the number of experimental runs for this construct is less than those for the other plasmid constructs, more runs are needed to achieve a higher level of statistical significance. However, the trend shows that, as the concentration of glucose in the media increases, luciferase activity also increases. This increase is particularly striking between 20mM and 30mM glucose concentrations, even though data variance is high. In addition, the trend of the data for the 635 and 935 base-pair constructs is similar, meaning that both constructs react similarly to the presence of glucose. Therefore, it appears that the 635 base pair section does respond to glucose.

187 Base Pair Construct In the 187 base pair construct, both the 10mM and 20mM glucose concentration luciferase activities were found to be significantly greater than the 5mM glucose luciferase activity (Figure 6 and Table 3). Therefore, this section does respond to glucose. However, the amount of transcribed glucose appears to peak at 10mM glucose concentration. The original hypothesis that the 187 base-pair section of the promoter will not respond to glucose has not come out to be true. However, the glucose response of the 187 base-pair has a different response pattern than the 635 or 935 base pair plasmid constructs. Though the 10mM and 20mM glucose concentrations both show statistically significant increases in luciferase activity when compared with 5mM glucose   concentration, the 20 mM  glucose  shows  activity  much  less  than  that of  the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5. The Effects of Glucose on the 635 Base Pair G6PDH Luciferase Plasmid Construct. Abbreviation: Glc-glucose

 

 

 

 

Treatment	2/4/01	2/11/01	2/18/01	2/25/01	3/4/01	3/11/01	Mean	Std. Error
5mM Glc	1	1	1	1	1	1	1	-
10mM Glc	0.12	0.8	1.53	7.41	1.41	43.2	1.05	±0.27
20mM Glc	0.20	0.84	2.83	12.24	1.04	85.61	1.19	±0.44
30mM Glc	30.34	0.46	8.92	44.76	0.9	51.76	2.81	±2.04

 

 

 

 

 

 

 

 

Table 2. 635G6PDH Promoter Section Luciferase Activity in Relation to Glucose Levels in Waymouth's Media. The data presented shows the fold increase in luciferase activity of 635 bp construct cells when compared to the luciferase activity of 635 bp construct cells immersed in Waymouth’s media containing 5mM of glucose. The treatment is the varying glucose concentration of the Waymouth’s media that the cells were immersed in. Luciferase activity (CPM/µg) was measured by calculating the scintillation of luciferin (counts per minute) over the amount of protein in the cell(µg). Abbreviations: Glc-glucose, Values in white or blue: These data points were eliminated by Q-test.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6. The Effects of Glucose on the 187 Base Pair G6PDH Luciferase Plasmid Construct. Abbreviation and Symbols: Glc-glucose; star indicates statistical significance over the value for 5mM glucose by Student’s t-test, p<0.05

 

 

 

 

 

 

 

 

 

 

 

 

Treatment	2/4/01	2/11/01	2/18/01	2/25/01	3/4/01	3/11/01	Mean	Std. Error
5mM Glc	1	1	1	1	1	1	1	-
10mM Glc	2.24	5.1	25.2	8.73	5.23	1.46	4.55	±1.29
20mM Glc	42.10	2.91	15.7	2.84	2.54	0.5	2.76	±0.11
30mM Glc	21.70	1.05	7.74	1.61	1.4	0.98	1.26	±0.15

 

 

 

 

 

 

 

 

 

 

 

Table 3. 187G6PDH Promoter Section Luciferase Activity in Relation to Glucose Levels in Waymouth's Media. The data presented show the fold increase in luciferase activity of 187 bp construct cells when compared to the luciferase activity of 187 bp construct cells immersed in Waymouth’s media containing 5mM of glucose. The treatment is the varying glucose concentration of the Waymouth’s media that the cells were immersed in. Luciferase activity (CPM/µg) was measured by calculating the scintillation of luciferin (counts per minute) over the amount of protein in the cell(µg). Abbreviations: Glc-glucose, Values in white and blue: These data points were eliminated by Q-test.

 

 

 

10mM glucose. The amount of transcribed G6PDH for glucose concentrations on either side of  10mM glucose concentration needs to be measured to show that a peak in glucose response does exist.

 The glucose response for the 187 base pair may be due to indirect glucose activation. For example, the 187 base-pair construct contains a SP-1 bonding site, a protein that is known to be glucose responsive.

CONCLUSIONS

 

For the 935 base pair construct (the entire G6PDH promoter), the amount of transcribed G6PDH increases as the amount of glucose applied to the cell increases from 5mM to 20mM glucose concentration. Transcription for the entire promoter appears to peak around a concentration of 20mM glucose. Additional experiments are to be run on either side of 20mM glucose concentration to verify the existence of any possible peak in the glucose response for the promoter

The trend for the 635 base pair construct shows that, as the concentration of glucose in the media increases, luciferase activity also increases The increase is particularly striking between 20mM and 30mM glucose concentrations, even though data variance is high. In addition, the trend of the data for the 635 and 935 base-pair constructs is similar. Therefore, both constructs react similarly to the presence of glucose

For the 187 base pair construct, the amount of transcribed glucose appears to peak at 10mM glucose concentration, though both 10 and 20mM concentrations lead to statistically significant increases. However, the 187 base-pair section has a statistically different response pattern from the entire promoter and the 635 base-pair section. If the 187 base-pair section is mediated by a different element, the element and its relationship to the amount of glucose in the cell need to be determined.

SUMMARY

 

            The glucose response of the gene promoter of the G6PDH enzyme was observed. Being a glucose-catabolizing enzyme, malfunction in G6PDH transcription can result in a wide array of diseases, most commonly diabetes. A 635-base pair section of the G6PDH promoter was found to be definitively glucose responsive, a phenomenon that has never been observed for this enzyme. Similar findings in other enzymes may lead to a wide array of gene therapies for those who suffer from Type II diabetes.

ACKNOWLEDGMENTS

I thank Dr. Susan Stapleton, Associate Professor of Biochemistry at Western Michigan University, for agreeing to be my mentor for this project and providing the facilities and guidance for its execution. I also thank Ms. Daryl Arkwright, a doctoral student working under Dr. Stapleton, for instructing me on experimental procedures and on instrumental analysis.

I would also like to thank Dr. John Goudie for piloting me through the entire project, helping me to meet various deadlines, and for encouraging me to compete in the Regional Science Fair. I would also like to thank him for instructing me extensively in scientific methods and for taking me to various seminars to meet active scientists.

I also want to thank my parents, Dr. Raja and Lakshmi Aravamuthan, for their constant encouragement and for their spending many a late night with me. I owe an additional debt of gratitude to my parents for taking care of all the logistics involved in getting my project display to the science fair.

Finally, I want to take this opportunity to thank the organizers and prime movers of the Junior Science and Humanities Symposium who have provided this forum for youngsters like me to present our work, learn from that of others, and learn from the experts in the field.

REFERENCES

 

Costa Rosa, L. F. B. P., Cury, Y., & Cury R. (1992). Effects of insulin, glucocorticoids            and thyroid hormones on the activities of key enzymes of glycolysis,   glutaminolysis, the pentose-phosphate pathway and the Krebs cycle in rat             macrophages. Journal of Endocrinology, 135, no. 2, 213-219.

 

Jeevy, A., Xi, J. (1997). The glucose response of glucokinase with AP1 receptor sites. Journal of Biological Chemistry, 1387, 309-312

 

Koehler, A., Bahns, S., Van Noorden, C. J. F. (1998). Determination of Kinetic           Properties of G6PDH and PGDH and the Expression of PCNA during Liver         Carcinogenesis in Coastal Flounder. Marine Environmental Research, 46, no             1/5, 179.v

 

Lowry, O.H., Rosebrough N.J., Farr, A.L., & Randall, R.J. (1951). Protein      Measurement with the folin phenol reagent. Journal of Biological Chemistry,            193, 265-275

 

Rank, K.B., Harris, P.K., Ginsberg, L.C. & Stapleton, S.R. (1994). Isolation and         sequence of a rat glucose-6-phosphate dehydrogenase promoter. Biochimica et        Biophysica Acta, 1217, 90-92.