METHODSMETHODS

Effects of glucose alterations on MnSOD and CuZnSOD expression in human retinal

pigmented epithelial cellsKatie M. Hertz

Mentor: Dr. Kaltreider

Department of Biology, York College of Pennsylvania

RESULTSRESULTS

CONCLUSIONSCONCLUSIONS

This study concludes that SOD gene expression increased under different alterations of glucose. Inhat et al. (2007) showed that SOD protein levels and protein activity in ARPE cells were significantly lower in a 24-hr oscillating 5, 30mM treatment compared to continuous 5mM. Together these findings suggest that in diabetics with complications a defect in SOD may not exist at the gene level but at the protein level.

The 30mM (2 weeks) to 5mM (1 week) treatment group suggests cell memory; after week 3 MnSOD continued to increase 2.5 fold rather than drop back to baseline.

Results suggest that the breakdown of superoxide predominately occurs in the mitochodria by MnSOD; however, the spike in CuZnSOD expression after the week 3 of treatment suggests that some superoxide leaks into the cytoplasm.

REFERENCESREFERENCESAmerican Diabetes Association (ADA). Conditions and Treatments. Available from: <http://www.diabetes.org/type-1-diabetes/treatment-conditions.jsp>. Accessed 2008 January 27.

Baynes, J.W. and Thorpe, S.R.. 1999. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes [serial online] 48: 1-9.

Brownlee, M. 2005. The pathobiology of diabetic complications: a unifying mechanism. Diabetes [serial online] 54:1615-1625.

Ceriello, A., della Russo, P., Amstad, P., and Cerutti, P. 1996. High glucose induces antioxidant enzymes in human endothelial cells in culture: evidence linking hyperglycemia and oxidative stress. Diabetes [serial online] 45: 471-77.

Hodgkinson, A.D., Bartlett, T., Oates, P.J., Millward, B.A., and Demaine, A.G. 2003. The response of antioxidant genes to hyperglycemia is abnormal in patients with type 1 diabetes and diabetic nephropathy. Diabetes [serial online] 52: 846-851.

Ihnat, M., Kaltreider, R., Thorpe, J.E., Green, D., Kamat, C., Leeper, M., Shanner, A., Warnke, L., Piconi, L., and Ceriello, A. 2007. Attenuated superoxide dismutase induction in retinal cells in response to intermittent high versus continuous glucose. American Journal of Biochemistry and Biotechnology 3(1): 16-23.

Pagaonkar, V.A., Leverenz, V.R., Dang, L., Chen, S.,Pelliccia, S. and Giblin, F.J. 2004. Thioredoxin reductase may be essential for the normal growth of hyperbaric oxygen-treated human lens epithelial cells. Experimental Eye Research 79(6): 847–857.

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS I would especially like to thank my mentor Dr. Kaltreider for all of his time, insight, and guidance over the past few years as I worked on this project. I would also like to acknowledge the entire York College Biology Faculty for all of their support and the knowledge they bestowed upon me over the past four years.

Isolate RNA

RT0.5µg RNA

Electrophoresis2% agarose gel10min @ 200VGelstar Orange

DensitometryFluorochem Imager

Based on Integrated Density Value

Quantify RNA

Cell Culture Week 1

Isolate RNA Isolate RNA

OBJECTIVE

To determine the effects of alterations of glucose concentrations on MnSOD and CuZnSOD mRNA expression in human retinal pigment epithelial (RPE) cells.

228 175

5mM

5m

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30 to

5m

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5 to

30m

M

5 to

30m

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30 to

5m

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Cell Culture ARPE-19 cells

Split 1:4 every 3-4 days in T25 flasks

Glucose Treatments Controls:

−5mM−30mM

Experimental:−5 to 30mM daily oscillation−5mM (2 weeks) to 30mM (1 week)−30mM (2 weeks) to 5mM (1 week)

Cell Culture Week 3

Cell Culture Week 2

INTRODUCTIONINTRODUCTION

20.8 million people in the U.S. have diabetes according to the American Diabetes Association (ADA). Diabetes is marked by hyperglycemia and oxidative stress (Baynes and Thorpe 1999), and many diabetics develop complications such as retinopathy, a leading cause of blindness in adults. Diabetes control and early detection are currently the best ways to prevent tissue damage (ADA) although the exact molecular mechanism leading to damage is still unknown.

Brownlee (2005) proposed a “unifying mechanism” whereby hyperglycemia causes an excess of reactive oxygen species (ROS),

such as superoxide anion (O2-), to be produced

by the electron transport chain during cellular respiration.

Superoxide dismutase (SOD) is a key

antioxidant enzyme that breaks down O2- into

a less reactive species H2O2. Glutathione

peroxidase (GPx) and catalase (CAT) are other antioxidant enzymes that are involved in the process and help breakdown hydrogen peroxide into water but are not key regulators.

SOD

2O2- + 2H+ → H2O2 + O2

Mitochondrial MnSOD and cytoplasmic CuZnSOD are two main forms of SOD. This is important because damage may be related to superoxide leakage from the mitochondria (Hodgkinson et al. 2003).

In vitro, Ceriello et al. (1996) found that continuous high glucose induced overexpression of antioxidant enzyme CuZnSOD in cells. In contrast, Hodgkinson et al. (2003) found evidence that mRNA expression of CuZnSOD decreases in diabetic patients with complications suggesting that there is a defect in the antioxidant enzyme.

Recently, SOD activity was shown to be significantly lower in retinal pericytes and ARPE-19 cells subjected to oscillating glucose than in those exposed to continuous high glucose (Ihnat et al. 2007).

PCR60°C annealing temperature

30 cycles1μl cDNA

Primers from Padgaonkar et al. 2004.

Figure 1. Gel images of MnSOD and CuZnSOD cDNA. Isolated mRNA was quantified, 0.5μg of each sample was reverse transcribed. 30 cycles of PCR amplified 1μl cDNA for each sample. 2% agarose gel was loaded with 20µl of each cDNA sample and electrophoresis was run for 10min @200v.

Week 1

MnSOD CuZnSOD

Week 2

Week 3

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5, 3

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RESULTS

MnSOD Over the progression of 3 weeks across all

treatment groups, MnSOD expression increased.

30mM treatment showed over a 2-fold increase in MnSOD expression compared to 5mM week 1 treatment.

The oscillating treatment group shows similar trends as 30mM treatment.

Across all groups, alterations in MnSOD expression are not observed until week 3.

5 to 30mM and 30 to 5mM at week 1 both had extremely low levels of SOD expression.

CuZnSOD The alteration trends of CuZnSOD

expression were similar to the MnSOD expression over the 3 weeks, except the fold increase of CuZnSOD was not as dramatic.

FUTURE STUDIESFUTURE STUDIES

Look at greater sample size and use a cDNA specific loading control to do statistics and determine if alterations in SOD expression are truly significant. Determine if passage number of cells affects SOD expression by looking at treatment groups over a longer time period.

Gene Primer Target size

MnSOD Upstream: ggtagcaccagcactagcagcDownstream: gtacttctcctcggtgacgttc

228

CuZnSOD

Upstream: cactctcaggagaccattgc Downstream: ggcctcagactacatccaag

175

Week 1

Week 2

Week 3

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5 to

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Figure 2. A) Fold increase of MnSOD in human RPE cells in 5 different glucose treatments over 3 week period. B) Fold increase of CuZnSOD in human RPE cells in 5 different glucose treatments over 3 week period. RNA was isolated after each week of treatment followed by quantification, RT-PCR, and gel electrophoresis. Densities of bands on gel were averaged and divided by the average of Week 1 5mM (control) to obtain mean fold increase. Sample size was 2 except for Week 1 30mM and Week 1 5, 30mM oscillating; these had n=1 due to absence of band on gel. Each bar (and value above bar) represents the mean densitometry, and error bars represent SEM.

A) Fold Increase of MnSOD for Different Glucose Treatments Over 3 Weeks

0.0

0.5

1.0

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30mM5, 30mM 24hr oscillating5mM (2 weeks) to 30mM (1 week)30mM (2 weeks) to 5mM (1 week)

5mM (control)

1

0.92

5

1.88

5

1.09

0.91

3.65

5

1.09 1.

225

3.63

0.16

1.05

53.

3

0.16

5

1.67

5 2.51

Week

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Incr

ease

0.0

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1.0

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2.0

2.5

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5mM (control)30mM5 30mM 24hr oscillating5mM (2 weeks) to 30mM (1 week)

30mM (2 weeks) to 5mM (1 week)

B) Fold Increase of CuZnSOD for Different Glucose Treatments Over 3 Weeks

1

1.79

5

0.83

50.

55

0.11

0.74

1.80

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0.11

50.

745

2.05

0.64

5

0.8

1.74

0.87

0.58

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ease