Effects of choline supplementation on expression of genes involved in choline synthesis and metabolism
Kali Blain
February 2, 2018
Abstract
An increase in choline intake has been shown to have preventative and mitigating effects on certain diseases. This study observed the effects of choline on the gene expression of PEMT, BHmT, and CTα, with possible implications of treating or preventing gestational diabetes. Pregnant mice were given either a control diet with no supplementations or a diet with supplemental choline. Through the analysis of RNA from liver samples, expression of the three genes of interest was analyzed twelve days into gestation. The results showed that gene expression was increased for all three genes.
Introduction
Gestational diabetes mellitus (GDM) is the onset of glucose intolerance during pregnancy in women who have no prior history of diabetes mellitus. There are many causes of GDM, such as genetics, diet, among other environmental factors. While GDM typically goes away after birth, the disease has various negative health effects on both the mother and child (Obeid et al., 2013). Additionally, women with GDM are at a higher risk of developing diabetes later in life (Obeid et al., 2013). Women with GDM also have a higher risk of preeclampsia, miscarriage, and preterm birth. GDM is also linked to offspring with higher body mass indexes (BMI) and an increase in future health risks such as type I diabetes (Jiang et al., 2014).
One factor that contributes to the onset of GDM is the alteration of glucose transporters, such as GLUT1 and GLUT4, which facilitate the transport of glucose across the cell membrane and help stabilize blood-glucose levels. Alteration of these glucose transporters can lead to levels of glucose entering the cell that are either too high or too low, which destabilizes blood-glucose levels and the metabolism of glucose (Jiang et al., 2016). Another factor for the onset of GDM is diet. Women on high-fat diets are at an increased risk of developing GDM (Jiang et al., 2014).
Choline is an essential vitamin that plays an important role in cellular functions and is found in most animal products, such as eggs, meat, and milk (Jiang et al., 2014). It is a methyl donor and, thus, serves a major role in the methylation of glucose transporter genes (Obeid et al., 2013). Choline donates a methyl group that binds to the base cytosine to produce a methylated cytosine. This methylation affects the interaction between DNA and transcription factors, which are proteins that are involved in converting DNA into RNA during DNA replication (Razin et al. 1991). It has been shown that without a substantial choline supply, DNA methylation is decreased, negatively impacting the function of glucose transporter genes (Karnieli et al., 1990).
Choline and its effects remain widely unknown to the general public, despite its importance in the body. Therefore, many people are unknowingly deficient, leading to an increased risk for diseases such as non-alcoholic fatty liver disease (NAFLD). The current adequate intake level for choline are shown in Figure 1 (Ziesel et al., 2009). Breast milk contains high concentrations of choline, thus increasing the adequate intake amount for lactating women. These values, shown in Figure 1, however, have been shown to be insufficient and can lead to choline deficiency.
An increase in choline intake has been shown to have preventative and mitigating effects on certain diseases. This study observed the effects of choline on the gene expression of PEMT, BHmT, and CTα, with possible implications of treating or preventing gestational diabetes. Pregnant mice were given either a control diet with no supplementations or a diet with supplemental choline. Through the analysis of RNA from liver samples, expression of the three genes of interest was analyzed twelve days into gestation. The results showed that gene expression was increased for all three genes.
Introduction
Gestational diabetes mellitus (GDM) is the onset of glucose intolerance during pregnancy in women who have no prior history of diabetes mellitus. There are many causes of GDM, such as genetics, diet, among other environmental factors. While GDM typically goes away after birth, the disease has various negative health effects on both the mother and child (Obeid et al., 2013). Additionally, women with GDM are at a higher risk of developing diabetes later in life (Obeid et al., 2013). Women with GDM also have a higher risk of preeclampsia, miscarriage, and preterm birth. GDM is also linked to offspring with higher body mass indexes (BMI) and an increase in future health risks such as type I diabetes (Jiang et al., 2014).
One factor that contributes to the onset of GDM is the alteration of glucose transporters, such as GLUT1 and GLUT4, which facilitate the transport of glucose across the cell membrane and help stabilize blood-glucose levels. Alteration of these glucose transporters can lead to levels of glucose entering the cell that are either too high or too low, which destabilizes blood-glucose levels and the metabolism of glucose (Jiang et al., 2016). Another factor for the onset of GDM is diet. Women on high-fat diets are at an increased risk of developing GDM (Jiang et al., 2014).
Choline is an essential vitamin that plays an important role in cellular functions and is found in most animal products, such as eggs, meat, and milk (Jiang et al., 2014). It is a methyl donor and, thus, serves a major role in the methylation of glucose transporter genes (Obeid et al., 2013). Choline donates a methyl group that binds to the base cytosine to produce a methylated cytosine. This methylation affects the interaction between DNA and transcription factors, which are proteins that are involved in converting DNA into RNA during DNA replication (Razin et al. 1991). It has been shown that without a substantial choline supply, DNA methylation is decreased, negatively impacting the function of glucose transporter genes (Karnieli et al., 1990).
Choline and its effects remain widely unknown to the general public, despite its importance in the body. Therefore, many people are unknowingly deficient, leading to an increased risk for diseases such as non-alcoholic fatty liver disease (NAFLD). The current adequate intake level for choline are shown in Figure 1 (Ziesel et al., 2009). Breast milk contains high concentrations of choline, thus increasing the adequate intake amount for lactating women. These values, shown in Figure 1, however, have been shown to be insufficient and can lead to choline deficiency.
Although the majority of choline comes from dietary intake, some choline is synthesized in the body. The three main genes involved in choline synthesis and metabolism are the phosphatidylethanolamine methyltransferase (PEMT), betaine homocysteine methyltransferase (BHmT), and phosphocholine cytidylyltransferase (CTα) genes. PEMT codes for phosphatidylcholine, the most abundant phospholipid in the body. Phosphatidylcholine is also important for transporting fats throughout the bloodstream as well as maintaining membrane structure (McEvoy et al. 2015). CTα is a regulatory gene for the rate of phosphocholine synthesis (Kast et al. 2017). BHmT codes for a shortcut of homocysteine to methionine (All Mutations 2017). An abundance of these three genes would increase the amount of choline synthesized by the body, which optimize global DNA methylation within the body.
Previous studies have shown a positive correlation between increased choline intake and the prevention—and in some cases reversal—of diseases such as NAFLD, neurological disorders, cardiovascular disease (Ziesel et al., 2009). The goal of the study was to observe whether choline supplementation could indirectly help prevent, treat, and possibly reverse GDM through increased DNA methylation of these aforementioned choline genes.
Methodology
Two groups of wild-type, female mice were started on diets of either a high-fat or a low-fat control. The fat content is The high-fat diet consisted of 60% fat, and the low-fat control diet contained 10% fat. All of the diets contained 2 millimolars (mM) of choline per 1000 kilocalories (kcal) of food. Six mice from both the high-fat and low-fat control diets were then selected to receive additional choline supplementation of 25mM.
Four weeks into the experimental diets, the mice were naturally impregnated by regular, wild-type, male mice. The diets were continued until embryonic day twelve (mid-gestation). On day twelve, the pregnant mice were euthanized, with tissue from the liver, fatty gonads, liver metabolites, fat, and serum samples were collected and stored in -80˚C until future use. The mass of the liver was recorded before collection. The fetuses were also removed from the womb, and a visual of each fetus was taken on a 1x1 centimeter grid for further analysis. Fetal tail and liver samples were collected for qPCR analysis and stored at -80˚C until future use.
RNA was extracted from the maternal liver and then either stored in -20˚C awaiting analysis, or immediately reverse transcribed to a more stable cDNA, following the regular reverse transcription protocol. The cDNA was then diluted in DNAse free water in a 1:10 solution. Reagents containing the forward and reverse primers for BHmT, CTα, PEMT, and the comparison gene Actβ, and the SYBR Green master mix were placed into four, one-milliliter centrifuge tubes, respectively. Two microliters of cDNA were poured into twelve consecutive wells on a 360-well plate. Eight microliters of reagent from each centrifuge tube were poured into sets of three consecutive wells, making sure each gene was tested in triplicates with each sample. Finally, the slide containing all of the samples and reagents was loaded into the Bio-Rad CFX96™ Real-time PCR Detection System, and run for forty cycles. After the PCR, samples were discarded appropriately, and the results analyzed.
Results
The gene expression for all samples tested was evaluated based on the quantification cycle (cq) at which the genes were first detected. The significant (less than 0.02 standard deviation) cq values of each gene for each sample were averaged, subtracted from the value of Actβ, and then plugged into the equation 2x, where x is the subtracted cq value, to produce a CT value which is comparable to the baseline cq value of the comparison gene. The CT values for all samples in each group—high fat choline control, high fat choline supplementation, low fat choline control, and low fat choline supplementation—were compared to each other and the of each calculated. The average CT values for the choline supplemented groups was then subtracted from the average CT values from the choline control groups to give summary of the relative difference in cq value for each gene. A lower CT value indicates a lower cq value, which means that it took fewer cycles to reach fluorescent detection levels, indicating a greater abundance of that gene transcription. The summative results for all samples collected are shown in Figure 2.
Previous studies have shown a positive correlation between increased choline intake and the prevention—and in some cases reversal—of diseases such as NAFLD, neurological disorders, cardiovascular disease (Ziesel et al., 2009). The goal of the study was to observe whether choline supplementation could indirectly help prevent, treat, and possibly reverse GDM through increased DNA methylation of these aforementioned choline genes.
Methodology
Two groups of wild-type, female mice were started on diets of either a high-fat or a low-fat control. The fat content is The high-fat diet consisted of 60% fat, and the low-fat control diet contained 10% fat. All of the diets contained 2 millimolars (mM) of choline per 1000 kilocalories (kcal) of food. Six mice from both the high-fat and low-fat control diets were then selected to receive additional choline supplementation of 25mM.
Four weeks into the experimental diets, the mice were naturally impregnated by regular, wild-type, male mice. The diets were continued until embryonic day twelve (mid-gestation). On day twelve, the pregnant mice were euthanized, with tissue from the liver, fatty gonads, liver metabolites, fat, and serum samples were collected and stored in -80˚C until future use. The mass of the liver was recorded before collection. The fetuses were also removed from the womb, and a visual of each fetus was taken on a 1x1 centimeter grid for further analysis. Fetal tail and liver samples were collected for qPCR analysis and stored at -80˚C until future use.
RNA was extracted from the maternal liver and then either stored in -20˚C awaiting analysis, or immediately reverse transcribed to a more stable cDNA, following the regular reverse transcription protocol. The cDNA was then diluted in DNAse free water in a 1:10 solution. Reagents containing the forward and reverse primers for BHmT, CTα, PEMT, and the comparison gene Actβ, and the SYBR Green master mix were placed into four, one-milliliter centrifuge tubes, respectively. Two microliters of cDNA were poured into twelve consecutive wells on a 360-well plate. Eight microliters of reagent from each centrifuge tube were poured into sets of three consecutive wells, making sure each gene was tested in triplicates with each sample. Finally, the slide containing all of the samples and reagents was loaded into the Bio-Rad CFX96™ Real-time PCR Detection System, and run for forty cycles. After the PCR, samples were discarded appropriately, and the results analyzed.
Results
The gene expression for all samples tested was evaluated based on the quantification cycle (cq) at which the genes were first detected. The significant (less than 0.02 standard deviation) cq values of each gene for each sample were averaged, subtracted from the value of Actβ, and then plugged into the equation 2x, where x is the subtracted cq value, to produce a CT value which is comparable to the baseline cq value of the comparison gene. The CT values for all samples in each group—high fat choline control, high fat choline supplementation, low fat choline control, and low fat choline supplementation—were compared to each other and the of each calculated. The average CT values for the choline supplemented groups was then subtracted from the average CT values from the choline control groups to give summary of the relative difference in cq value for each gene. A lower CT value indicates a lower cq value, which means that it took fewer cycles to reach fluorescent detection levels, indicating a greater abundance of that gene transcription. The summative results for all samples collected are shown in Figure 2.
Discussion
The goal of this study was to assess whether or not choline supplementation increased the gene expression of PEMT, BHmT, and CTα genes. Additionally, the goal was to determine if increased choline intake could prevent or treat GDM. This was done by analyzing the mean difference in CT value between choline control and choline supplementation for each diet. For mice on a high fat diet, the mean difference in CT values for all three genes was less than zero, indicating that, on average, for mice who had choline supplementation, there was a greater abundance of transcription of each gene. For the low fat diet, both PEMT and BHmT had a negative CT value, but CTα had a positive value, indicating that, on average, there was a higher cq value for the choline supplemented samples than the control and that there was less overall transcription of that gene compared to control samples (Figure 3). The negative mean difference in CT values for each gene from mice on either a high fat or low diet suggests that, when supplemented with choline on a high fat diet, choline synthesis within the body increases compared to non supplementation.
The hypothesis was that choline supplementation would increase the gene expression of PEMT, BHmT, and CTα, which may alleviate the symptoms and possibly reverse GDM in pregnant mice. The data suggests that choline supplementation has a positive effect on the expression of BHmT, and CTα, and PEMT. However, the significance of these results only suggest a possible trend. Further research with more samples is needed to strengthen the support for these findings. In addition, experimentation on the effects on expression of glucose transport genes would strengthen the relationship between choline supplementation and GDM. Research studying the effect of choline on human patients with GDM would be necessary before the adequate intake levels can be reexamined.
Acknowledgments
I want to thank Dr. Xinyin Jiang and Brooklyn College Lab facilities for providing me the resources to carry out this research. I would also like to thank Ms. Schmitz, Mr. Holzinger, and Ms. Machac, for running the Independent Science Research program, and allowing me to engage in this research.
References
The goal of this study was to assess whether or not choline supplementation increased the gene expression of PEMT, BHmT, and CTα genes. Additionally, the goal was to determine if increased choline intake could prevent or treat GDM. This was done by analyzing the mean difference in CT value between choline control and choline supplementation for each diet. For mice on a high fat diet, the mean difference in CT values for all three genes was less than zero, indicating that, on average, for mice who had choline supplementation, there was a greater abundance of transcription of each gene. For the low fat diet, both PEMT and BHmT had a negative CT value, but CTα had a positive value, indicating that, on average, there was a higher cq value for the choline supplemented samples than the control and that there was less overall transcription of that gene compared to control samples (Figure 3). The negative mean difference in CT values for each gene from mice on either a high fat or low diet suggests that, when supplemented with choline on a high fat diet, choline synthesis within the body increases compared to non supplementation.
The hypothesis was that choline supplementation would increase the gene expression of PEMT, BHmT, and CTα, which may alleviate the symptoms and possibly reverse GDM in pregnant mice. The data suggests that choline supplementation has a positive effect on the expression of BHmT, and CTα, and PEMT. However, the significance of these results only suggest a possible trend. Further research with more samples is needed to strengthen the support for these findings. In addition, experimentation on the effects on expression of glucose transport genes would strengthen the relationship between choline supplementation and GDM. Research studying the effect of choline on human patients with GDM would be necessary before the adequate intake levels can be reexamined.
Acknowledgments
I want to thank Dr. Xinyin Jiang and Brooklyn College Lab facilities for providing me the resources to carry out this research. I would also like to thank Ms. Schmitz, Mr. Holzinger, and Ms. Machac, for running the Independent Science Research program, and allowing me to engage in this research.
References
