Sex Differences in Cell Metabolism
Most of what we know about human physiology has been derived from clinical studies on men or experimental studies on male rats or mice. For a long time and for many different reasons, physiological research assumed that, except for obvious differences in reproductive organs and body shape, women are just like men. The clear behavioral differences (for example, women are more likely to be depressed than men) were thought to be explained by non-physiological causes better left to social scientists. Recent research shows that female physiology and male physiology are really different. During the years of menstruation, estrogen (and the androgens) effects many enzymes in cell metabolism. The differences are large and medically significant, showing that, in many cases, treatment and drug dosing should be different for men and women. This has opened up an exciting new field and, as always, mathematics is essential for the investigation and determination of underlying biological and medical mechanisms.
A few examples show major differences between men and women that are begging to be explained. American women live, on average, five years longer than men. Men are diagnosed with cancer more frequently than women and diagnosed with Parkinson’s disease twice as often as women. Women have less oxidative stress than men and are less vulnerable to drug overdoses and poisons in food. Women are more resilient to infectious diseases and topical estrogen improves wound healing in both sexes. This last suggests that a good place to start is the influence of the endocrine hormones on the differential regulation of human physiology in males and females.
The physiological differences between men and women are investigated in the context of human evolution in the recent New York Times bestseller: “Eve – How the Female body Drove 200 Million Years of Human Evolution,” by Cat Bohannon (Knopf, New York, 2024).
Our journey into the study of sex differences began in 2017 when Farrah Sadre-Marandi, a postdoc at the Mathematical Biosciences Institute at Ohio State, asked Mike Reed the question:
“Why do women have lower homocysteine than men?’’ This is an important question since homocysteine, which is produced in prodigious quantities by the liver, is the major biomarker for cardio-vascular disease. This is almost certainly the reason that women have fewer cardio-vascular events than men. If you have high homocysteine in your blood, your family physician will prescribe folate (vitamin B-9) to lower homocysteine. We thought maybe women had more folate, but no they don’t. So, we thought what’s different about women? Well, they have much more estrogen than men during the menstruating years. This led us to look at the literature to find out if the circulating form of estrogen, estradiol, affects any of the enzymes in one-carbon metabolism (OCM). We were stunned to find out that estradiol and testosterone affect (activate or inhibit) 5 major enzymes in OCM: SHMT, MTHFR, MS, BHMT, and PEMT (full names of the enzymes can be found below in our paper. The male-female differences in activation or inhibition were very large. We had been making mathematical models of different parts of OCM since 2002, so it was relatively straightforward to put in the effects of estradiol and testosterone into the models and find out the male-female differences in OCM, and this led to Project 1.
Project 1. Table 2 shows the ratios of enzyme activities and substrate concentrations in OCM.
The references in Table 2 can be found in our paper. As you can see, the changes in enzyme activities are large. Also, the sphingomyelin input (upper right corner in the diagram below) is 30% higher in females.
Figure 1. OCM for the male
In Figure 1, substrates are in boxes and enzyme abbreviations in blue and yellow ovals. Next to each substrate the concentration at steady state is in muM is in white. A few of the arrows have velocities, in muM/hr, in blue boxes.
Figure 2: OCM for the female
In Figure 2, the concentrations are in black letters for the male (as in Figure 1) and in red letters for the female. One can see that the Hcy concentration drops modestly for the female. In Project 2 we will see that estradiol directly activates CBS and in Project 4 we will see that glutathione activates CBS. These two effects make the gap between male and female Hcy concentrations even larger.
How does activation and inhibition of CBS affect its substrate Hcy? Mass comes in to the methionine cycle through methionine input from the blood. Mass leaves via the CBS reaction. Thus, in this model, the velocities of methionine input and CBS output must be equal at steady state. So what? This means that if you activate the enzyme (raising its Vmax), the Hcy concentration will drop so the flux through CBS remains the same at steady state. And, if you inhibit the enzyme (lowering its Vmax), the Hcy concentration will drop so the flux through CBS remains the same at steady state. Actually, this is not strictly true because there are two relatively small other drains of mass in the methionine cycle, the polyamine drain from SAM and the thiolactone drain from Hcy. So, the CBS flux is close to the methionine flux at steady state, and the argument above still works.
Why does raising folate lower Hcy? Everyone (including your family doctor) thinks that raising the folate in the system (in particular, 5mTHF) will lower Hcy because this will raise the MS flux thus lowering Hcy. This sounds plausible, but is false! All of the mass in the MS reaction goes around the methionine cycle and returns to Hcy, and thus, at steady state, the Hcy concentration wouldn’t change. We show this in the paper. But giving folate does lower folate. Why? The answer is in the long-range interactions indicated by the red arrows in Figure 1. If you raise the concentration of 5mTHF, that will increase the inhibition of GNMT, which will cause the concentration of SAM to go up. The increase in SAM provides more activation of CBS and therefore drives Hcy concentration down.
What is the (evolutionary) purpose of the female changes to the enzymes in OCM? In the case of the enzyme PEMT that is highly upregulated in females, the answer is clear. Because of the upregulation of PEMT, females have about twice as much choline and betaine as males (see Figure 2). Maternal choline is important for neural tube closure in the fetus and for neurodevelopment in the fetal hippocampus, which effects memory. In fact, pregnant subjects fed 4 times the dietary levels of choline had offspring with a 30% enhanced visuospatial and auditory memory, and these enhanced functions did not decrease as they aged. This and other important choline results were obtained in many experiments and clinical trials by Steve Zeisel at UNC.Thus, it seems extremely likely that the upregulation of PEMT by estrogen is an evolutionary mechanism for ensuring large choline supplies for the fetus and the mother. The evolutionary reasons for the other expression level differences caused by the sex hormones remain uncertain.
Project 1 was the joint work of Farrah Sadre-Marandi (consultant at qPharmetrica), Thabat Dahdoul (graduate student at UC Irvine), Michael Reed, and Fred Nijhout and appeared in paper.
Project 2. In 2021, Duke graduate student, Ruby Kim, enlarged and improved the mathematical model from Project 1. She included recent experimental evidence that estradiol activates the enzymes TS and DHFR in the folate cycle and directly activates CBS (see Figure 1, above). She also included the polyamine pathway which drains modest amounts of mass from SAM in the methionine cycle. In Project 1 we compared the steady state concentration and velocity variables for the male with those for the female. In Project 2 we wanted to study how these OCM variables change throughout the menstrual cycle, since the estradiol concentration changes dramatically during the cycle.
In Figure 2, we show our estradiol curve during the menstrual cycle created from clinical and experimental data in the literature (see the reference in paper2). The green curve shows the average serum concentration of estradiol in nM during the cycle. The blue curve shows the 95th percentile and the red curve shows the 5th percentile. Not surprisingly, there is a lot of variation between different women. In Project 1, we had two sets of parameters, one representing men and the other representing women. Here, we scaled the parameters between the male values and the females values proportionally to the scale of the green curve from lowest value (at day 1) to highest value (at day 13).
This technique allowed us to calculate, using the model, how the OCM variables change dramatically during the menstrual cycle (see Figure 3). The variables fluctuate up and down by 20%-30% during the cycle. Most notable, the Hcy concentration fluctuates in the opposite direction from the estradiol concentration. We gave the explanation for this phenomenon in Project 1. It is interesting to speculate how these changes could be related to how women feel during the cycle.
Figure 3. Fluctuations of OCM variables during the menstrual cycle. All the variables are scaled relative to their values at day 1. See Figure 1 for the variables.
Homocysteine and oral contraceptives. Most of the estrogen in women during the child-bearing years is produced by developing follicles in the ovaries. This naturally raises the question of homocysteine levels in women who take oral contraceptives (OCP), since those pills, containing mostly progesterone and some estrogen, prevent follicular development by inhibiting GnRH production in the hypothalamus and FSH and LH production in the pituitary. The serum levels of estradiol of women taking the pill is 20–80 pg/ml which corresponds approximately to estradiol levels early in the follicular phase. Using the model, we can compute the average estrogen over the menstrual cycle for normal women not taking the pill and women taking the pill and, therefore, can compute their average Hcy levels too (see Table 3).
Table 3. Homocysteine and the contraceptive pill in the Mathematical Model.
Average estradiol(nM) Average Hcy(muM)
Menstruating women 0.44 1.29
Women using OCPs 0.16 1.57
Men 0.09 1.80
As one can see in Table 3, women using oral contraceptives have higher homocysteine than menstruating women, but not as high as men. Just because these differences are large does not necessarily mean that they are important. Women, during the menstruating years are at quite low risk for cardio-vascular events, so increased homocysteine may just increase this risk to a higher, but still low, risk.
Is Homocysteine a good biomarker for deficiencies in vitamins B-6, B-9, and B-12? In another aspect of Project 2, we showed that the answer to this question is No! This does not have to do with sex differences, so we mention it only briefly here because our result is controversial. For many years clinicians and experimentalists who work on one-carbon metabolism have said that homocysteine is a good biomarker for vitamin deficiencies. Folate is vitamin B-9 and it is one of the substrates for the MS reaction. Vitamin B-12 is a co-factor for the MS reaction, and Vitamin B-6 is a cofactor for the CBS reaction. Since the MS and CBS reactions lead away from Hcy, it was natural to assume that the concentration of Hcy would be sensitive to the concentrations of these micro-nutrients. But NHANES data (that we give in the paper) shows otherwise. Over large ranges of these micronutrients, the Hcy concentration changes very little. It is only at extremely low concentrations that Hcy rises. Thus, Hcy is quite homeostatic with respect to variations in the micronutrients. In the paper, we explain the three different homeostatic mechanisms in one-carbon metabolism that keep the concentration of Hcy quite homeostatic. Since our ancestors’ diets were likely extremely variable and Hcy is bad for you, it is very reasonable that evolution should have produced these homeostatic mechanisms.
The results of Project 2 were published in paper2 .
Project 3. In the next project, we investigated glutathione metabolism with Duke graduate student Allison Cruikshank. Glutathione (GSH) is a tri-peptide, made up of the amino acids glutamate, cysteine and glycine. It is produced in all cells but is made in large quantities in the liver. It is the main anti-oxident in the body and plays a central role in cleansing the input from the gut to remove toxicants such as mercury and drugs. Starting at Hcy, GSH is synthesized by the transsulfuration pathway depicted in Figure 4. The enzyme CBS makes cystathionine from Hcy and serine, and then CTGL makes cysteine. The enzyme CTGL attaches a glutamate and then GS adds the glycine. GSH reduces oxidative species through the enzyme GPX.
There are several interesting aspects of the transsulfuation pathway. Estradiol (E2) activates the enzymes GCL and GPX, as well as activating CBS directly. In addition, GSH inhibits its own synthesis by inhibiting GS and GCL. It’s not so easy to guess the effects of E2 since the activation of GCL should increase GSH and the activation of GPX should lower GSH. The balance between these two effects determines the steady state concentration of GSH.
The full pathway including OCM for the male at steady state is shown in Figure 5, and
Figure 5. The transsulfuration pathway for the male.
Figure 6. The transsulfuration pathway for the female.
for the female in Figure 6. Full details of the model are in paper3.
Stability of GSH during the menstrual cycle. Figure 7 shows the steady state of GSH
for different levels of E2 in the blood, roughly the levels that E2 goes through during the menstrual cycle. This is alarming because it suggests that maybe GSH fluctuates wildly during the cycle since E2 varies so much (Figure 2). However, that is not the case.
Relative Estradiol
Figure 7. Steady state of GSH as a function of Estradiol concentration.
Figure 8. Stability of GSH during the menstrual cycle.
GSH does vary throughout the cycle but not as much as the steady states would suggest. This is because the dynamics of GSH is relatively slow, so essentially the GSH concentration is averaging over the E2 concentration over many days. Full discussion is in paper3.
Vulnerability to toxicants. GSH is a main player in liver metabolic pathways that detoxify toxicants and drugs coming from the gut that could harm the liver. It is known that women are less vulnerable to toxicants and drugs and the reason is almost certainly the higher concentration of GSH in women. In our paper, we illustrate this by examining responses to overdoses of acetaminophen in women and men by connecting a previous model of acetaminophen detoxification to the current model of GSH synthesis.
The results of Project 3 were published in paper3.
