Anecdotal and scientific evidence suggest that the sex hormone estrogen provides significant health benefits in women. Women have higher estrogen levels than men. Circulating estrogen reaches its highest level during the reproductive period and steadily declines with the onset of menopause. The role of estrogen and estrogen receptors in both cellular physiology and pathophysiology has been controversial. Estrogen has anti-inflammatory and anti-oxidant effects, which preserve cell viability during cardiovascular incidents, but it enhances disease progression in the context of breast cancer. Estrogen mediates these responses via activation of estrogen receptor subtypes located in the cell membrane, nucleus, and mitochondrion. Further, transcription of nuclear and mitochondrial genes by estrogen yields products that play an important role in regulating mitochondrial function. Mitochondria are part of a highly dynamic network and undergo fission and fusion, produce cellular energy, adenosine 5′ triphosphate (ATP), and regulate cell death. Herein, we review the cell and receptor specific effects of estrogen on mitochondrial structure, function, and cell death under normal physiological conditions and in the context of cardiovascular disease, inflammation, neurodegeneration, and cancer. Further research is needed to elucidate the specific role of estrogenic control of mitochondria in health and disease.
- estrogen receptors
The term estrogen refers to a family of chemically similar steroid hormones that include estrone, estradiol, and estriol. Estrogens are synthesized primarily by the ovarian follicles . An important rate limiting step in steroid hormone synthesis is the production of pregnenolone in follicular granulosa cells. Cholesterol is transported from the outer to the inner mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein, followed by conversion to pregnenolone by cytochrome P450 side chain cleavage (CYP11A1). Pregnenolone then diffuses to the theca cells where it is converted to androstenedione and then re-routed to the granulosa cells for the aromatase-mediated conversion to estrogen. Androstenedione can also be converted to testosterone. Thus, mitochondria play an important role in estrogen biosynthesis.
Estrogen serves a major role in determining female secondary sex characteristics during development and in regulating the estrous cycle. During puberty and throughout the female reproductive cycle, estrogen levels fluctuate, and as women age, sex steroid production decreases . Estrogen levels oscillate during the estrous cycle. They are lowest during menstruation, steadily rise during the follicular stage and reach a maximal level during ovulation. If a woman becomes pregnant, estrogen levels will remain high, but if fertilization does not occur, hormone levels decline during the luteal phase. Following the luteal phase, menstruation occurs, and the cycle resumes. As estrogen levels fluctuate during the estrous cycle, mitochondria alter the production of pregnenolone accordingly.
Estrogen is a pleiotropic hormone that exerts its effects
2. Estrogen receptor types
Estrogen modulates cellular function
ERα and ERβ are also found on the membranes of cellular organelles, including the endoplasmic reticulum and the mitochondrion, where they mediate various cellular functions. Localization to mitochondria was first confirmed using radioligand binding methods in mitochondria isolated from rat uterus and later by immunocytochemistry in rat pancreatic acinar cells . MALDI-TOF mass spectrometry studies have shown that mitochondrial ERs are identical to ERs located in the nucleus . ERβ is the predominant mitochondrial receptor in most tissues: for example ovary, uterus, spermatocytes, cerebral and hippocampal neurons, cardiomyocytes, and endothelial cells [17, 18]. In contrast, the identification of mitochondrial ERα has been limited to the uterus, ovary, and the MCF-7 breast cancer cell line . The presence of ERs on mitochondria and estrogen responsive elements on the mitochondrial DNA suggests a role for estrogen in regulating the structure and/or function of the organelle .
Estrogen also binds to a GPER on the plasma membrane. GPER specifically binds to estradiol and mediates numerous responses including cell proliferation, vasodilation, and regulation of glucose metabolism by non-genomic mechanisms . GPER has also been localized to intracellular sites. In the endoplasmic reticulum, GPER activation induces calcium release and activation of the phosphoinositide 3-kinase (PI3K)-
3. Mitochondrial function
The mitochondria are classically described as the powerhouse of the cell by virtue of its ability to generate ATP. Physiological processes underlying mitochondrial bioenergetics and respiration have been previously reviewed . Under aerobic conditions, mitochondria utilize electron transport and a protomotive force to produce ~36 ATP molecules for every glucose molecule. Reducing equivalents produced by the Krebs cycle (NADH and FADH2) are accepted by the respiratory chain at Complex I (NADH Dehydrogenase) and Complex II (Succinate Dehydrogenase), respectively. Electrons are shuttled through the complexes with oxygen acting as the final electron acceptor at Complex IV. Concurrently, hydrogen ions are pumped from the electronegative mitochondrial matrix into the more positively charged inner membrane space by Complexes I, III (cytochrome C reductase), and IV (cytochrome C oxidase). The protomotive force thus generated allows hydrogen ions to flow down their concentration gradient at Complex V (ATP Synthase), resulting in the formation of ATP. This chemiosmotic process is tightly regulated and highly efficient. Although ATP is the primary (and often most studied) product of cellular respiration, ROS and thermal energy/heat are also generated by mitochondria. ROS include chemical species produced by the incomplete reduction of O2. These molecules include superoxide anion (O2•−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH). Mitochondrial ROS are thought to perform a variety of cell signaling functions under normal physiological conditions.
Mitochondria are highly dynamic organelles that are components of a constantly changing, dynamic mitochondrial network. Damaged or old mitochondria can be cleared from the cell by autophagy/mitophagy or bulk clearance and degraded by lysosomal enzymes . Furthermore, mitochondria can also undergo fission and fusion. Fission is the process by which mitochondria bud off from the mitochondrial network. This is regulated by the proteins DRP-1 and FIZZ1. Fusion represents the incorporation of mitochondria in the mitochondrial network and is regulated by mitofusin1 (Mfn1), mitofusin2 (Mfn2), or optic atrophy 1 (OPA1) . These regulatory proteins help to maintain the balance between fission and fusion that is required to preserve cell viability. In different disease states, however, this balance can be disrupted causing mitochondrial dysfunction and cell death.
4. Mitochondria, cell injury, and apoptosis
Manganese superoxide dismutase (MnSOD) and glutathione represent endogenous molecules that minimize mitochondrial-derived ROS. Mitochondrial injury occurs, however, when ROS formation exceeds the capacity for their removal by these antioxidant mechanisms. ROS biochemically modify other molecules to produce cytotoxic species that induce cellular injury . For example, ROS induce the formation of 4-hydroxynonenal (HNE), a reactive lipid species that is associated with neuronal damage in brains of Parkinson’s disease patients [29, 30]. ROS production also leads to cell death
5. Mitochondrial responses to estrogen
Both nuclear and mitochondrial genes are subject to regulation by estrogen . Nuclear DNA encodes proteins that are incorporated in mitochondria and influence their function. For example, the binding of estrogen to nuclear ERα induces the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) . This protein plays an important role in mitochondrial biogenesis, a process by which new mitochondria are formed. The mitochondrion also contains its own maternally inherited DNA, which encodes 37 genes . A recent study in MCF-7 cells showed that estrogen regulates mitochondrial RNA production under serum starvation conditions . Subsequent to estrogen treatment, ERα translocated to the mitochondria and increased expression of mitochondrial tRNAs used to translate mitochondrial proteins. In GH4C1 pituitary cells, treatment with estrogen increases expression of mitochondrial-encoded RNA for subunit II of cytochrome C oxidase . In female rats, levels of mitochondrial-encoded 16S RNA, a housekeeping gene used as a marker for mitochondrial number, are four times higher than males of the same age [36, 37]. Ovariectomy (OVX) in rats is characterized by an increase in liver and brain peroxide production and formation of 8-oxo-2′deoxyguanosine, a marker of mitochondrial DNA damage. These changes were associated with a reduction in the antioxidant protein GSH and MnSOD . Estrogen treatment in OVX rats reversed these responses . Collectively, these data suggest that increased estrogen levels regulate mitochondrial and nuclear anti-oxidant protein expression.
Estrogen plays an important role in the regulation of apoptosis by stimulating Bcl-2 protein expression and translocation to the mitochondria. This is achieved
Estrogen effects on mitochondrial function vary in different cell types. For example, estrogen binds specifically to the oligomycin-sensitivity conferring protein of ATP-synthase (Complex V) in brain mitochondria and inhibits ATP production . In contrast, another study showed that the enzymatic activity of F0F1-ATPase, a Complex V subunit, is higher in mitochondria isolated from the heart than other tissues. Estrogen induced a further increase in cardiac ATPase activity implying a direct link between estrogen stimulation and ATP production [45, 46]. Estrogen can therefore exert different effects on mitochondria from different cell types. These differential responses may impact disease pathogenesis.
6. Estrogen, mitochondria, and cardiovascular disease
Cardiovascular disease (CVD) is the leading cause of death in men and women, and American Heart Association statistics reveal a significant increase in CVD mortality in women compared to men . Mitochondrial dysfunction has been implicated as a causative factor in CVD, with mitochondrial DNA damage being significantly increased in the heart and aorta of patients with CVD compared to healthy controls [25, 48, 49, 50]. Women do not generally present with CVD until the seventh decade, while the incidence of death due to CVD is high in men throughout life. This age-dependent increase in CVD in women has been linked to the onset of menopause and a reduction in circulating estrogen levels. The “Free Radical Theory of Aging” proposes that, with increased age, an increase in free radical formation initiates a vicious cycle of ROS formation that causes progressive cell injury . Data suggest that loss of the antioxidant and anti-inflammatory effects of estrogen after menopause contributes to the development of mitochondrial injury [52, 53]. Thus, maintaining high levels of estrogen may increase lifespan and/or health in postmenopausal women.
Studies using experimental animal models of CVD show that OVX increases vascular inflammation/injury in a manner that is prevented by estrogen treatment [54, 55]. Analysis of mitochondria isolated from hearts of OVX rats reveals increased levels of apoptotic markers compared to mitochondria of intact animals. Administration of estrogen to these animals significantly attenuated apoptosis . Since mitochondrial damage and apoptosis can be mediated by ROS, it has been hypothesized that the estrogen can decrease ROS by activating the antioxidant pathway. Treatment of human aortic endothelial cells (HAECs) with estradiol upregulates the mitochondrial antioxidant MnSOD by an ERα-dependent mechanism. The ability of estrogen to increase MnSOD levels is ablated in ERα KO mice but not in ERβ KO mice. Interestingly, while ERβ does not regulate MnSOD expression, it was shown to be essential for preventing atherosclerotic progression
Gender differences have been identified in mitochondrial genes isolated from rat hearts . Whole genome microarray analysis showed that expression of genes associated with mitochondrial apoptosis pathways is significantly elevated in male mice compared to females. In contrast, genes associated with fatty acid and glucose metabolism were upregulated in females. Female rats also displayed higher transcription levels for mitochondrial Complexes I and IV. These data suggest that genes related to cellular metabolism, including mitochondrial respiration, are upregulated in cardiac mitochondria from female rats while genes associated with mitochondrial apoptosis are increased in males. Whether this difference is directly related to the circulating levels of estrogen
Mitochondrial structure in the heart is influenced by estrogen. The hearts of OVX rats that underwent I/R injury had lower levels of mitochondrial respiratory function and increased myocardial cell death compared to intact animals. Transmission electron microscopy showed that the mitochondria in cardiomyocytes from OVX rats were more disordered within the cell and structurally damaged compared to mitochondria from intact animals . Interestingly, even male ERα KO mice that underwent cardiac I/R injury display lower coronary blood flow rates, increased calcium accumulation, and reduced nitrite production compared to non-ischemic hearts. Further, electron microscopic analysis revealed that the mitochondria from ERα KO mice were abnormally shaped . These studies suggest that estrogen signaling plays a role in regulating mitochondrial structure in both females and males. In another model of I/R injury, female wild-type mouse hearts were shown to have better functional recovery and an attenuated inflammatory response compared to female ERα KO mice and wild-type male mice . These data further suggest the importance of ER signaling as a cardioprotective mechanism in females.
Effects of estrogen on mitochondrial function have been tested in a genetic model of hypertrophic cardiomyopathy (cTnT-Q92). Estrogen treatment improved ATP production, the mitochondrial respiratory ratio, and diastolic function in OVX cTnT-Q92 mice compared to untreated OVX mice . OVX in cTnT-Q92 mice attenuated the expression of the mitochondrial biogenesis genes PGC1α, peroxisome proliferator-activated receptor alpha (PPARα), mitochondrial transcription factor A (tFAM), and the antioxidant protein nuclear respiratory factor 1 (NRF-1). Estrogen treatment improved cardiac mitochondrial organization and cristae structure and increased mitochondrial biogenesis. These data directly show that estrogen exerts cytoprotective effects at the level of the mitochondrion that translate into an improvement in cardiac function.
7. Estrogen, mitochondria, and inflammation
Macrophages contribute to the chronic inflammation associated with many diseases including CVD and neurodegeneration. Macrophages display plasticity in that they may adopt various phenotypes. The differentiation of these cells is highly dependent on the local microenvironment in which they are situated. M1 macrophages are pro-inflammatory and are induced by cytokines and lipopolysaccharide (LPS). M2 macrophages are anti-inflammatory, play a role in wound healing, and are induced by IL-4 and IL-13 [63, 64]. The metabolic characteristics of M1 and M2 macrophages are different. M1 macrophages rely on glycolysis for ATP formation while M2 macrophages are dependent on mitochondrial oxidative phosphorylation for energy [63, 64]. Damage to mitochondria induced by inflammatory stimuli can exacerbate cellular injury . It was shown that both ERα knockout and mitochondrial dysfunction inhibit the IL-4 mediated conversion to macrophages from an M1 to an M2 phenotype [64, 66].
Treatment of macrophages with LPS/interferon-γ (IFN-γ) favors an increase in the M1 phenotype. In macrophages isolated from premenopausal women, estrogen treatment was shown to reduce the M1/M2 ratio in cells exposed to LPS/IFN-γ to a greater extent than macrophages isolated from postmenopausal women . Recent studies from our group have shown that there is a significant decrease in ERα expression in macrophages from postmenopausal women compared to premenopausal women while estrogen therapy was able to preserve ERα expression . These data imply that estrogen and ER levels play a crucial role in macrophage polarization, but the role of estrogen on the mitochondrial function in these groups is still unknown. Determining the role of estrogen on the mitochondria and how it affects macrophage phenotype can help us to better understand the anti-inflammatory roles of estrogen.
8. Estrogen, mitochondria, and neurodegeneration
Neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by the progressive deterioration and death of specific neuron types in the brain . It is well known that mitochondrial dysfunction plays a pathogenic role in neurodegeneration . AD is characterized by atrophy of the cortex and hippocampus. Additionally, increases in glucose metabolism and a reduction in the activities of mitochondrial Complexes I, III, and IV in the brain have been reported [69, 70]. Plaques containing amyloid beta and tau protein can become incorporated in the remaining brain cells . These inclusions are known to increase oxidative stress and mitochondrial dysfunction.
In PD, the loss of dopaminergic neurons in the substantia nigra contributes to the development of motor disturbances. The protein alpha synuclein becomes entangled with these neurons, resulting in ROS production, mitochondrial dysfunction, and neuronal cell death . A decrease in mitochondrial Complex I activity has been observed in postmortem brains of PD patients that is linked to a reduction in oxidative phosphorylation .
Gender differences have been identified in the pathology of both AD and PD [75, 76]. These include alterations in brain weight, regional atrophy, distribution of white and gray matter, cerebral blood flow, expression of neurotransmitter transporters and receptors, and age of onset . In the case of PD, there is also an increased incidence of the disease in men compared to women [78, 79]. The gender difference in neurodegenerative phenotypes has led to the hypothesis that estrogen exerts neuroprotective effects and that these effects are mediated at the level of the mitochondrion . In mouse spinal cord neurons, estrogen treatment increased mRNA levels of nuclear-encoded mitochondrial electron transport chain genes ND1, CytB, Cox2, and ATP6 . ERα has been identified in the mitochondria of endothelial cells in the brain and forebrain. In these cells, estrogen increased expression of cytochrome C and reduced ROS formation . ERβ has been localized to hippocampal mitochondria [17, 81, 82]. Hippocampal cells isolated from ERβ KO mice had lower mitochondrial membrane potential and were more resistant to oxidative stress compared to control mice . Taken together, these data suggest opposing effects of ERα and ERβ in the brain. More cell-type specific studies are needed to better understand the role of these ERs in the brain and to help clarify these opposing views.
Estrogen also interacts with mitochondrial proteins in a non-genomic manner. Under
Estrogen preserves mitochondrial structure/function by upregulating the mitochondrial antioxidant enzyme MnSOD in the brain of female rodents [37, 86, 87]. In SK-N-SH neuroblastoma cells, estrogen inhibits the effects of the mitochondrial Complex II inhibitor, 3-nitroprionic acid (3-NPA), by preserving mitochondrial ATP production and inhibiting the 3-NPA induced hydrogen peroxide and peroxynitrite formation . These data suggest that estrogen also plays an anti-oxidant role in the brain. This has led many to hypothesize that the anti-oxidant effects of estrogen may play a role in slowing disease pathogenesis.
Estrogen also regulates mitochondrial dynamics in astrocytes in a gender-dependent manner. It reduces expression of the fusion protein Mfn1 in astrocytes isolated from male rodents but has no effect on astrocytes obtained from females . Treatment of cortical primary astrocytes with estrogen increases the expression of fission (Dyn 1 and Fis 1) and fusion (Mfn2) proteins to a greater extent in female mice than males. The upregulation of both fission and fusion proteins suggests that mitochondrial network is more dynamic in females than males. Although the exact mechanisms and reasons for the differences in fission and fusion regulation between male and female rodents are unknown, these responses may explain the sexual dimorphism seen in neurodegenerative diseases and other pathologies.
9. Estrogen, mitochondria, and cancer
Cancer is the second leading cause of death in the United States, and, among all cancers, breast cancer is the second most commonly diagnosed cancer in women. Breast tumor cells that express estrogen receptors are classified as ER-positive and account for 80% of all breast cancers . Further, the Women’s Health Initiative Study showed that menopausal hormone therapy (MHT) increases the incidence of breast cancer in women compared to controls . Modern day cancer treatment principally focuses on identifying estrogen signatures in breast cancers, and suppression of estrogen receptor function is a routine therapeutic strategy.
Estrogen is known to regulate mitochondrial function in the context of breast cancer by several mechanisms. First, it has been shown to alter mitochondrial morphology. Administration of physiologically relevant doses of estrogen to MCF-7 breast cancer cell lines results in enlargement of mitochondria . Mitochondrial cristae adopt an abnormal structure that is reminiscent of mitochondria that are oxygen-deprived and rely on glycolysis for ATP formation. This change in structure was associated with a 2.5-fold increase in the mitochondrial content of ERα and ERβ and an increase in the mitochondrial expression of cytochrome C oxidase subunits I and II. Alternatively, activation of cell membrane estrogen receptors is reported to induce changes in the cytoskeleton that indirectly influences mitochondrial structure . Alteration in mitochondrial structure not only affects the capacity of the energy production but also influences other crucial functions such as calcium signaling, ROS production, or biosynthetic processes.
Second, estrogen induces cellular ROS production in cancer cells by three main pathways: (1) direct inhibition of respiratory chain complexes; (2) accumulation of calcium within mitochondria; and (3) inhibition of the antioxidant response element (ARE) . The increase in ROS generation by the mitochondria and the decrease in antioxidant capacity cause a cellular shift to high ROS production that plays an important role in cancer cell proliferation and cell damage. Estrogen also promotes ROS formation in breast cancer cells by inducing cyclin D1 gene expression . NRF-1 regulates the expression of several nuclear-encoded mitochondrial genes  that encode respiratory protein subunits, mtDNA transcription/replication machinery, components of heme biosynthesis, and mitochondrial protein import . Cyclin D1 phosphorylates NRF-1, resulting in repression of its activity. This inactivation of NRF-1 reduces mitochondrial activity and shift glucose metabolism toward glycolysis . The reduction in mitochondrial ATP production, in part, increases mitochondrial ROS production.
Another characteristic of cancer cells is that they highjack apoptotic pathways in order to evade cell death. When normal cells are exposed to UV radiation, the generation of mitochondrial ROS activates c-jun N-terminal kinase (JNK) and protein kinase C (PKC)-δ. These signaling molecules trigger the translocation of
The role of estrogen receptors in breast cancer development has been known for almost over 30 years. Subsequent studies strongly suggested that ER status is the single most important predictive and prognostic biomarker in breast cancer. Clinicians use several strategies to battle estrogen-sensitive breast cancer, which affect not only estrogen levels but also mitochondrial function. One approach is to block ovarian function. Ovarian ablation can either be performed surgically to remove the ovaries (oophorectomy) or by radiation. An alternative approach is to temporarily suppress the ovarian function pharmacologically using gonadotropin-releasing hormone (GnRH) agonists. GnRH interferes with signals that are produced by the pituitary gland that stimulate the ovaries to produce estrogen. GnRH agonists also act by inducing mitochondrial depolarization, thereby decreasing mitochondrial oxidative capacity. Aromatase inhibitors represent another pharmacological approach to inhibit estrogen synthesis. Addition of aromatase inhibitors to MCF-7 cells was shown to induce caspase 9 expression . Thus, activation of the intrinsic cell death pathway may be an alternative mechanism by which aromatase inhibitors decrease cancer progression. Selective estrogen receptor modulators (SERMs) are another class of drugs that are used for treatment of breast cancers. The SERM tamoxifen blocks the ability of estrogen to stimulate the growth of breast cancer cells. Tamoxifen has also been shown to induce mitochondrial ROS and apoptosis by increasing mitochondrial nitric oxide synthase (mtNOS) . Tamoxifen decreases cellular respiration, increases mitochondrial cytochrome C release, and increases mitochondrial lipid peroxide formation. These data suggest that tamoxifen induces the mitochondrial cell death pathway. While current breast cancer therapeutics inhibit both estrogen signaling and mitochondrial function, the development of next generation drugs that can more efficiently inhibit these processes is required.
Estrogen is a multi-functional hormone that exerts its effects by both transcriptional and non-genomic mechanisms. Nuclear transcriptional responses are classically induced by binding of estrogen to ERα and ERβ. Estrogen-dependent gene transcription plays an important role in the development of female reproductive structures and regulation of the estrous cycle. The local production of estrogen is now also thought to regulate physiological responses in males. Non-genomic responses to estrogen are more rapid and include induction of signaling pathways that promote cell proliferation. The differential expression of ERs in different cell types and cellular loci dictates their specific function.
Studies in recent years have defined a role for estrogen in the regulation of mitochondrial structure and function. Estrogen increases expression of respiratory complexes, antioxidant molecules, and anti-apoptotic factors that directly impact mitochondrial structure and function. Aging in women is associated with a reduction in estrogen formation and the development of mitochondrial dysfunction. An increase in free radical damage in cells also occurs with aging. Damaged mitochondria are more likely to produce additional ROS, thus initiating a vicious cycle that progressively degrades cellular function. This includes estrogen biosynthesis. Transgenic mice with a mutation in the inner mitochondrial membrane peptidase-2 (IMMP-2) had hyperpolarized mitochondria, which produced increased levels of superoxide and ATP and resulted in impaired ovulation and reduced fertility . Other data show that defective mitochondrial DNA polymerase activity induces mitochondrial dysfunction and infertility in mice . It follows that cytoprotective responses to estrogen at the level of the mitochondrion are ablated. Thus, a reciprocal relationship exists between estrogen and mitochondrial function. Under normal physiological conditions, mitochondria are critical mediators of estrogen biosynthesis and are also targets for estrogen action.
Strong evidence suggests that estrogen plays a major role in promoting the proliferation of both normal and the neoplastic breast cancer cells. However, cancer represents a unique scenario in which estrogen exerts tumorigenic responses in susceptible cells. Prolonged exposure to high levels of estrogen is associated with an increased incidence of breast cancer, which supports models of estrogen-induced carcinogenesis. In breast cancer cells, estrogen is shown to not only stimulate the cell proliferation but also inhibit apoptotic pathways, which can therefore lead to uncontrolled tumor growth. Estrogen increases mitochondrial ROS production that can also promote cancer progression. Further research is needed to expand our understanding of how estrogen induces carcinogenesis.
Estrogen and ER signaling in the mitochondria play an important role in health and disease. We have shown that estrogen effects are cell type and receptor type specific, which explains the varied effects of estrogen treatment described in this review. We also understand that despite the protective role of estrogen in inflammation, cardiovascular disease, and neurodegeneration, it promotes breast cancer through both nuclear and mitochondrial regulation. Further research is needed to understand the specific mechanisms of estrogen-induced mitochondrial changes in health and disease.
This work was supported by grants from the National Institutes of Health (GM115367 and DK108836).