Gender bias in gastric emptying is well-documented in both health and gastroparesis; however, the role of endogenous sex hormones in regulating gastric motility remains unclear. Women during their reproductive ages, tend to be disproportionately affected by gastroparesis because their stomach motility is slower to begin with, likely due to elevated levels of sex steroid hormones and nitric oxide. The pathogenesis of diabetes-associated motility disorders are multifactorial, though much can be attributed to abnormalities in nitric oxide/nNOS expression, enzyme activity and oxidative stress . Our results show that in-vitro hyperglycemia reduces 1) gastric and duodenal Nrf2, nNOSa and ER alpha protein expression, 2) supplementation of estrogen and/or estrogen receptors restores Nrf2, nNOSa, total nitrite and nitrergic relaxation in hyperglycemic conditions. In addition, pre-incubation with ER antagonists inhibit Nrf2 and nNOSa protein expression and nitrergic relaxation. Collectively, the above studies suggest that estrogens regulate gastric motility via stimulating Nrf2/nNOSa signaling mechanism(s).
Estrogen receptor signaling is complex, but it is well understood to primarily mediate many of their biological effects via genomic regulation. Most of the actions of estrogens appear to be exerted via two estrogen receptor (ER) subtypes, denoted ERa and ERb, intracellular proteins that are members of a large superfamily of proteins that function as ligand-activated transcription factors . Although ER subtype surface membrane receptors exist, few studies have implicated these targets in the rapid vasodilator effects of E2 . Moreover, several lines of evidence suggest that both ERa and ERb proteins are expressed in enteric neurons within the nucleus and cytoplasm . Co-expression of ERa and ERb in enteric neurons indicates that estrogenic effects could also be mediated through neurogenic reflexes [16, 18]. Therefore, our study sought to understand the genomic changes associated with selective ER activation in gastric neuromuscular specimens.
To test whether sex hormone receptors contribute to gastric nitrergic function, we have first investigated the effects of non-selective antagonists: ICI 182, 780 (ER) and RU-486 (PR) in ex-vivo normoglycemic conditions. Our studies demonstrate that blockade of estrogen receptors, but not progesterone receptors, by antagonists showed a reduction in nitrergic relaxation in gastric neuromuscular strips; implicating ER signaling/effects. Estrogens have been shown to mediate both, rapid and genomic, effects. Genomic effects of steroid hormones have been shown to occur in as early as 2 hours . The results from our study revealed that mRNA and protein expression for Nrf2/nNOSa are altered by inhibition with ER or PR antagonists which clearly shows that total nitrite production is also altered in the presence of ICI and RU-486. The above data suggests that ERs could play a vital role in gastric motility function through regulating Nrf2/nNOSa expression and nitrergic relaxation.
Furthermore, several cofactors have been shown to be important for nNOSa activity, including BH4. The level of BH4 is tightly regulated by both de novo and salvage pathways. GCH-1 is a rate limiting enzyme and regulates BH4 levels via the de novo pathway, while DHFR reduces oxidized (inactive) BH2 and B to active BH4 via the salvage pathway. Previous studies from our laboratory suggest E2 deficiency reduced expression of GCH-1 and DHFR levels in female follitropin receptor knockout (FORKO) gastric neuromuscular tissue . In addition, reduced levels of E2 may augment impairment of BH4-nNOSa function and elevate oxidative stress, thus promoting gastroparesis in women. Our studies show that inhibition of ER, but not PR, reduces DHFR, but not GCH-1 suggesting that ERs perhaps synthesizing BH4 via the salvage pathway.
Our current studies demonstrate that in-vitro exposure to HG significantly reduced gastric protein expression of both ERa and ERb, Nrf2/nNOSa, total nitrite and nitrergic relaxation. Although the osmolarity of the culture medium was not assessed, the effects of elevated glucose levels on cellular osmolarity is well reported. Exposure to high glucose concentrations in vitro is often used to understand the cellular modifications occurring in diabetes. Furthermore, recent studies have documented that hyperosmolarity, as occurring in diabetic hyperglycemia, may represent important regulatory elements influencing cell fate and viability, both in physiological and pathological conditions. These studies further report using glucose concentrations between 24mM – 75 mM with incubation times up to 72 hr are sufficient to mimic the diabetic oxidative stress response in different cellular types (human gingival fibroblasts and erythrocytes) [39, 43]. Furthermore, exposure to a prolonged severe hyperglycemic (>30mM) load is correlated with increased susceptibility to cellular damage and severe inhibitory effects on nNOS/Nrf2. However, short term incubation (<48 hr) display little effect on cell viability while maintaining HG insult. In our study, we examined the gastric neuromuscular response to increasing concentrations of hyperglycemia (DMEM(5.5mM), 30 mM and 50 mM glucose) in the culture media for 48 hr. (Fig 6 A-C). Although we observed no significant differences between nNOS, ER, and nrf2 protein expression in 30 mM and 50 mM glucose media, the remaining HG experiments (incubation with hormones/ER agonists/antagonist and organ bath studies) employed a lower (30 mM) glucose concentration in the incubation media.
In addition, our studies show that nonselective (E2), and selective activation of ERa by PPT or ERb by DPN restored gastric Nrf2/nNOSa expression, total nitrite and nitrergic relaxation in vitro exposed to HG. After 90 min incubation, we observe differences in the efficacy of the selective ER agonists at various doses; though each ER agonists significantly enhanced nitrergic relaxation in gastric neuromuscular specimens exposed to in-vitro hyperglycemia. This is in line with Al-Shboul et al., reporting that estrogen-induced relaxation was greater in female gastric smooth muscle cells (GSMC) compared with that in male . Our current studies further asserted that ERα agonist, PPT and the ERβ agonist, DPN induced relaxation to a greater extent than PPT, although this result was not statistically significant. These differences may be due to variations in receptor subtype expression in GSMCs vs enteric neuronal cells. All of our data were generated from gastric full-thickness specimens that has several cellular subtypes including smooth muscle and enteric neuronal cells. Furthermore, as shown in Fig. 2., selective activation with PPT restored Nrf2, nNOSa, ERa, dihydrofolate reductase (DHFR) mRNA expression; selective ERβ activation (DPN) restored nNOS⍺, Nrf2, and ER⍺ mRNA expression. Collectively, the above studies suggest ER activators regulate nitrergic relaxation by restoring gastric Nrf2/BH4 synthesis in an experimental hyperglycemic condition.
Estrogen receptors (α/β) are encoded by separate genes and exhibit distinct tissue distributions[18, 23, 45–47]. ERα is well known to be majorly expressed in the uterus, liver, kidney and heart. Whereas, ERβ expression is primarily found in the ovary, lung, gastrointestinal tract, bladder, and central nervous systems. Much of the evidence describes spatially-specific actions in which ERs may possess converging or diverging pathways leading to cellular responses[27, 42]. It has been demonstrated in bone that ERβ can stimulate some of the same genes as does ERα, whereas ERβ almost always reduced the magnitude of gene stimulation by ERα when both receptors were present. It has also been reported through the use of subtype-selective ligands that ERβ modulation of ERα activity appears to be response specific. At experimental doses, PPT displays 410-fold selectivity for ERα over ERβ, whereas DPN displays a 70-fold selectivity for ERβ[34, 48]. However, Tamir et al. reported that oxidative stress, a well-known consequence of diabetes, differentially regulates the expression of ERα and ERβ in various cells . Furthermore, although not studied in this context, numerous mRNA splice variants exist for both ERs in diseased and healthy tissues. These splice variants are speculated to potentially alter full-length (active) protein expression and activity in rodent studies, potentially facilitating compensatory signaling mechanism[49–51]. Our findings demonstrate that ER beta is increased in hyperglycemia. We anticipate that PPT activation of ER alpha may restore other mechanisms (i.e inflammation) to prevent the upregulation of ER beta in HG. Future studies are needed to further understand the complexities of sex hormone receptor signaling. Furthermore, the expression of ERs, DHFR, Nrf2, and HO-1 could be from mucosal, muscle and neuronal layers. We did not conduct the cellular localization studies of the target proteins in the current study. Of note, several studies have demonstrated that nNOS and ERs are primarily localized within enteric neuronal cells of the gut [16, 52]. Furthermore, regionally specific co-localizations of nNOS and ERα have also been reported, suggesting potential interaction in this system within the neurons. Reports indicate that enteric neurons innervate throughout the layers of the stomach[12, 18, 19]. Therefore, we speculate that despite of localization of these specific markers in various cell types (mucosal -> neurons), may interact with via autocrine and/or paracrine fashion, to thus regulate nNOS-mediated motility of the stomach.
Moreover, our previous studies demonstrate that loss of Nrf2 reduced nitrergic relaxation and delayed gastric emptying in female mice [27, 28]. In-vivo activation of Nrf2 has been shown to regulate gastric nNOSa function and ERs in a high-fat diet fed obesity Type II DM model . Much of the work delineating the interactions between Nrf2 and ovarian hormone receptors is limited to breast cancer models [53, 54]. Estrogen (E2) increases Nrf2 activity in MCF7 breast cancer cells through activation of the PI3K/GSK3β non-genomic pathway . Similarly, E2 is known to regulate, to a lesser extent, antioxidant response element (ARE)-responsive genes through Nrf2 and co-activators within the promoter region of these genes [53, 55, 56]. Our study sought to provide evidence for genomic changes in Nrf2 and nNOS in response to selective ER activation.
In addition, Nrf2 is widely known to bind to a host of Phase II detoxifying enzymes to rid the cell from oxidative stressors. Most importantly, heme oxygenase 1 (HO-1) is a protective marker controlled by Nrf2-ARE. In mouse models of diabetes, increased expression of antioxidants such as HO-1 protected ICC from oxidative stress and reversed diabetic gastroparesis . Here, we provide evidence of a reduced expression of HO-1, that was restored by selective ER activators, suggesting ER alpha and beta can increase HO-1 expression. These findings suggest co-activation with nuclear ERs can facilitate HO-1 gene expression.
Furthermore, motor abnormalities associated with gastroparesis syndrome may not be limited to the smooth muscle function. Diabetic gastroparesis comprises a decrease in fundic and antral motor activity, a reduction or a lack of the inter-digestive migrating motor complex, gastric dysrhythmias, and pylorospasms [13, 57]. As reported earlier, NO donors were ineffective in relieving gastroparesis symptoms in humans suggesting that stomach motility is not solely regulated by smooth muscle . Numerous studies demonstrated that gut motility is regulated by enteric neuronal system including nNOS. Therefore, we suggest that although NO donors have an effect on relaxing smooth muscle in general, it may not relieve gastroparesis symptoms. In our study, hyperglycemia significantly reduced the expression of Nrf2 and nNOSa in gastric antrum and duodenum specimens. We further observed attenuation of Nrf2 and nNOSa protein expression with sex hormone (primarily E2) supplementation. Earlier studies in duodenum report the number of nNOS nerve cell bodies per ganglia was increased in type II DM rodent models of gastroparesis; however, the density index of nNOS varicosities was reduced [14, 59]. These studies further suggested that nitrergic neurons might be protected from hyperglycemia-related oxidative stress better in the duodenum. Similarly, the impairment of nNOS pathways in streptozotocin (STZ)-induced diabetic rats, the nitrergic myenteric neurons did not diminish in the duodenum, unlike the other gut segments. . In the diabetic duodenum, besides a decreasing number of nNOS neurons, the number of colocalized myenteric neurons did not alter significantly. Our studies so far align with a decreased expression of nNOS in the duodenum, however sex hormones may improve complications associated with this depletion of NO. It should be noted that Cellek, and others, suggest that nNOS expression is reversibly decreased (point of return) in the nitrergic axons while unaffected in the cell bodies in the early stages of diabetes in male rodents. The same studies further suggest that nNOS neurons are reduced in the long-term diabetes (point of no return). Since hormones and their receptor agonists were able to restore nNOS protein expression, we suggest that our experimental HG model is related to the early stages, but not long-term diabetic conditions in which we would expect a loss of nNOS neurons.