Hyperoxia Affects the Expression of Mitochondrial Proteins UCP-3, SOD-1 and SOD-2
By Megan Schwarz
Many premature infants undergo aggressive oxygen and ventilator therapies to maintain oxygen saturation in under-developed lungs. Exposing rats to hyperoxia, an excess supply of oxygen, provides a well-accepted model for infants born prematurely who face these life-threatening circumstances . Here, I propose post-natal hyperoxia will decrease the expression of uncoupling protein-3 (UCP-3) — a protein that affects energy metabolism in the skeletal muscle of newborns — and will increase the expression of superoxide dismutase-1 (SOD-1) and superoxide dismutase-2 (SOD-2) — enzymes that prevent damage from oxygen radicals. Rats were exposed to either hyperoxia (HYP) or normoxia (NORM) for 14 days. Oxygen exposure had no significant effect on the expression of SOD-1. SOD-2 was found to be significantly lower in HYP rats compared to NORM rats. In addition, expression of UCP-3 among HYP rats was significantly lower compared to NORM rats. These findings suggest that high levels of post-natal oxygen lead to decreased expression of SOD-2 and UCP-3, which can lead to metabolic inefficiencies. If we are better able to understand the molecular impacts that premature birth has on infants, we can provide better care early on for these infants in order to decrease the risk of developing further pathologies in adulthood.
Conditions in the intrauterine environment and early postnatal life stages contribute to development of diseases later on in life, such as the development of hypertension, type 2 diabetes and heart disease . Reduced insulin sensitivity, which can lead to the pathogenesis of these diseases, has been recognized in premature infants . In a study conducted by Hovi et al. , adults who were born at term with normal birth weights (5 pounds, 8 ounces to 8 pounds,13 ounces) were tested for glucose tolerance and insulin sensitivity. Subjects who had a low birth weight had a 6.7% increase in fasting blood glucose, and an average increase of 40% in fasting insulin as compared to those who had normal birth weights, suggesting that adults who had a low birth weight had higher insulin resistance and lower glucose tolerance as compared to those who were born at term .
Premature infants face many challenges starting immediately after birth, including being born with immature lungs, weak breathing muscles and respiratory distress syndrome . These issues can lead to hypoxia, a shortage of oxygen, which can damage other parts of the body. Although hypoxia is often treated by administering supplemental oxygen , it is difficult to safely administer oxygen therapies to infants because it can lead to lung damage. For instance, bronchopulmonary dysplasia (BPD), a type of chronic lung disease, can result from long-term use of oxygen, which some infants may never recover from . In addition, supplemental oxygen therapies can lead to hyperoxia, an excess of oxygen in the lungs. Exposing neonates to hyperoxia can also overwhelm antioxidant defense mechanisms . This impacts the function and expression of superoxide dismutase-1 (SOD-1) and superoxide dismutase-2 (SOD-2 — enzymes that prevent damage from oxygen radicals. SOD-1 is responsible for destroying oxygen radicals in the body and is thought to function primarily in the cytoplasm. SOD-2 is responsible for forming diatomic oxygen from superoxide (O2–) in the electron transport chain and mainly affects superoxide in the mitochondria. 
Uncoupling protein-3 (UCP-3) is expressed in skeletal muscle and has been related to the risk of developing obesity and type 2 diabetes due to its role in the development of insulin resistance . UCP-3 is associated with protection from insulin resistance and is important in separating oxidative phosphorylation from the synthesis of ATP in mitochondria . In particular, UCP-3 is involved in lipid oxidation and has been shown to affect energy metabolism in the skeletal muscle of newborns . It has been theorized that UCP-3 is able to export fatty acids that cannot be oxidized out of the mitochondrial matrix . This function prevents these fatty acids from being built up in the mitochondria, reducing the risk of mitochondrial damage . However, this energy conversion mechanism may not be sufficiently developed among premature infants, which could greatly impact metabolic processes , such as the accumulation of fatty acids in non-adipose tissues, like skeletal muscle, which is characteristic of diabetes . Brauner et al.  took muscle samples from 40 low birth weight infants who had passed away and found a positive correlation between gestational age at birth and an increase in the expression of UCP-3. This data suggests that the length of intrauterine development has an impact on the expression of UCP-3, leading to insufficient development of key metabolic pathways and risk of disease if the time of intrauterine development is cut short via premature births .
The purpose of this study is to understand the presence of essential protective uncoupling and antioxidant proteins and the impact of oxygen therapy in premature infants. I decided to compare the difference in expression of UCP-3, SOD-1 and SOD-2 between rats that were either exposed to hyperoxia or normoxia for 14 days. I hypothesized that hyperoxia (HYP, 85% oxygen) would decrease the expression of UCP-3 expression and increase the expression of SOD-1 and SOD-2 compared to control rats exposed to normoxia (NORM, 21% oxygen). The rats were constantly exposed to the different oxygen conditions in order to simulate the same conditions premature infants face because all term-born rats are born with the same stage of organ development as premature infants . By using rats as our model organism, we can gain knowledge on the effects of premature birth in humans, and inform health care professionals providing care to premature infants. The results can help guide future studies regarding the risk factors associated with preterm birth to make progress toward changes in health care.
New-born Sprague Dawley rats were used for this study. Immediately after birth, 12 pups received constant exposure to hyperoxic gas (85% O2) and 12 pups received normal levels of oxygen (21% O2) to generate a control group for comparison. After 14 days of treatment, the rats were euthanized. Gastrocnemius muscle was harvested and flash frozen in liquid nitrogen.
Homogenized and protein isolated tissue was powderized in liquid nitrogen then lysed in a RIPA buffer with protease and phosphate inhibitors (ThermoFisher, 89900). The protein was isolated and quantified with Bradford protein assay (Bio-Rad, 5000001) . Protein samples were then aliquoted based on quantification and diluted with deionized water. Sample buffer and reducing agent were added to each sample of protein. Samples were heated at 95 degrees Celsius for five minutes. The Criterion XT gel (Bio-Rad, 3450120) was placed in the Criterion cell tank along with running buffer and each sample was loaded into the gel and run at 200 volts for 45 minutes. The gel was then transferred on to a nitrocellulose membrane (Bio-Rad, 1620112) and was run for 30 minutes at 100 volts. Proteins that were separated and then transferred on to the nitrocellulose membrane allow the protein expression to be analyzed. The membrane was then removed from the transfer tank and incubated in bovine serum albumin (BSA) (ThermoFisher, 15561020), and then in primary mouse anti-rabbit antibodies (SOD1, SOD2 and UCP3) (Cell Signaling, 3678S) at 4ºC  and then washed three times in TBST. Secondary infrared antibodies (infrared conjugated donkey anti-mouse (LI-COR, 68180) and donkey anti-rabbit (68073)) were added in solution with BSA in order to detect the protein, along with Vinculin (Abcam, 18058) to normalize the expression of the proteins. Vinculin is able to bind to the proteins so protein expression can be analyzed. By standardizing the expression of the proteins by Vinculin, protein expression is able to be quantified. The membrane was washed three times in TBST again. Then using the Licor Odyssey CLx (LI-COR, 9140) near-infrared fluorescence imaging system, the blot was imaged with Image Studio.
Analyzing band density, the expression of UCP-3, SOD-1, and SOD-2 were quantified and compared between HYP rats and NORM rats using a two-way ANOVA with post hoc analysis by Tukey’s for multiple comparisons tests. A two-way ANOVA test was used to measure the effects of the two independent variables. Because the rats were separated into two groups — each with equal numbers of males and females with each group receiving a different oxygen treatment — the test is able to determine the relationship between the independent variables (gender and treatment type) and the dependent variable (expression of proteins). Tukey’s for multiple comparisons test is able to determine which means are statistically significant, where statistical significance is indicated by a p-value less than 0.05.
In a two-way ANOVA multiple comparisons test on SOD-1 expression, there was no statistically significant effect of hyperoxia on the expression of SOD-1 (p-value= 0.8481). The mean value of SOD-1 expression shown in Figure 1, was similar in NORM males and HYP males. There was also no statistically significant difference between NORM females and HYP females (p-value= 0.9985), shown in Table 1.
However, by a two-way ANOVA analysis of SOD-2 expression, there was an overall statistically significant effect of hyperoxia on SOD-2 (p-value= 0.0016). Figure 2 shows that there was a significant difference in the mean SOD-2 value when comparing NORM males to HYP males (p-value= 0.0010). It also shows that there was a statistically significant difference in SOD-2 expression between NORM females and HYP females (0.0245).
A two-way ANOVA showed a statistically significant difference in gender (p-value= 0.02471) and treatment group (p-value= 0.0021) in the expression of UCP-3. Figure 3 highlights the difference in gender by showing the difference in average UCP-3 expression between HYP males (1.041) and NORM males (0.9280), as well as between HYP females (0.6988) and NORM females (0.7408). Table 1 shows that there was a statistically significant difference in UCP-3 expression between HYP males and HYP females (p-value= 0.0043), as well as NORM females and HYP females (p-value= 0.0240). There was also a statistically significant difference between NORM males and HYP females (p-value= 0.0015). Analysis showed that there was no significant difference between HYP males and NORM males (p-value= 0.9875).
Expression of essential protective proteins in rats can serve as a model for infants who are born prematurely and require life-sustaining therapies because all term-born rats are born with the same stage of organ development as premature infants . It was hypothesized that expression of UCP-3 would decrease with exposure to hyperoxia while expression of SOD-1 and SOD-2 would increase. Although SOD-1 has been known to be responsible for the metabolism of cellular oxygen radicals, my data showed the expression of this enzyme was not significantly different, with the expression of SOD-1 remaining relatively unchanged between HYP and NORM rats. Despite the fact there was no significant difference in SOD-1 expression, there was a significant difference in SOD-2 expression between HYP and NORM rats. However, in both males and females, the expression of SOD-2 was less among HYP rats compared to NORM rats. SOD-2 is responsible for forming diatomic oxygen from superoxide produced by the electron transport chain during normal metabolism, preventing oxidative damage . Thus, I would have expected SOD-2 expression to be higher in HYP rats. Further research needs to be conducted in order to confirm these results because lack in statistical significance could be due to procedural errors during Western Blotting. For example, during the transfer of the gel onto the membrane, not all of the air bubbles may have been rolled out, which may have caused the image of the blot to show these bubbles, making it difficult to quantify the expression of the enzymes and proteins. This lack of statistical significance could also be due to flaws in the theories of SOD-2 expression.
Gender played a significant role in expression of UCP-3. Analysis showed that UCP-3 expression was lower in females than in males. This could be due to the fact that females tend to have more slow-twitch muscle fibers than males, and studies have shown that fast-twitch muscle fibers contain higher levels of UCP-3 . Future studies can be conducted to show that females do have a decreased need for UCP-3. There was also a significant impact of hyperoxia on UCP-3 expression. This relationship suggests that there is a negative correlation between the amount of oxygen given to the rats and the expression of UCP-3. Post-natal hyperoxia exposure in rats is a model for premature infants because, similar to rats, they are born with underdeveloped organs and then treated with high levels of oxygen. Thus, it can be rationalized that premature infants may have lower levels of UCP-3. This correlation shows the energy mechanism may not be fully developed in premature infants, which can impact several metabolic processes, including insulin resistance . My results show that UCP-3 is significantly decreased among HYP rats, similar to premature infants. However, we need to further pursue the role of UCP-3 in the risk of development of metabolic disease in premature infants. Furthering our understanding of how oxygen therapy and other care neonates undergo may help reduce long-term complications. The incidence of type 2 diabetes of adults who were born prematurely has yet to be analyzed. Investigating this rate could lead to correlational research between UCP-3 and the development of insulin resistance .
In addition to more UCP-3 research, further research on the expression of SOD-1 and SOD-2 among rat models, as well as the expression of UCP-3 in fast-twitch compared to slow-twitch muscle, should be conducted in order to determine the relationship between gender and mitochondrial protein expression. Additional research on the effects of hyperoxia on organ development, also needs to be conducted. Those born prematurely face challenges not only immediately after birth, but later in life as well. Further research could lead to increased understanding of the care we provide to premature infants in order to reduce their risks for diseases, like type 2 diabetes and BPD, and increase their quality of life.
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