Carbohydrate intake and acid secretion decrease gastric estrogen secretion

Carbohydrate intake and acid secretion decrease gastric estrogen secretion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Carbohydrate intake and acid secretion decrease gastric estrogen secretion Yoshimitsu Kanai, Takao Ito, Yuichi Ozaki, Atsushi Tanaka This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4567800/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Gastric parietal cells (gastric acid secreting cells) secrete estrogen in response to blood lipid (triglycerides and fatty acids) levels. 1 Estrogen decreases blood lipids by suppressing appetite, lipogenesis and lipolysis and by increasing lipid storage and consumption, 2 – 5 so gastric estrogen plays an important role in maintaining blood lipids levels. 1 However, parietal cells use fatty acids to generate energy for estrogen production and acid secretion, so postprandial changes in blood lipid levels and acid secretion activities could affect production of gastric estrogen. Here we show that blood estrogen decreases after meals, especially after carbohydrate intake. Blood fatty acids also decrease, and intravenous injection of lipids partially restores blood estrogen levels. Gastric acid-secreting hormones decrease production of gastric estrogen, while antacid and gastric acid-suppressing hormones, including those secreted after lipid ingestion like glucagon-like peptide-1 (GLP-1), 6 increase gastric estrogen production and postprandially decreased blood estrogen levels. Secreted insulin and gastric estrogen directly enter the liver to enhance and suppress lipogenesis respectively, 7 before diluted in the systemic blood. We therefore conclude that diet and the subsequently secreted hormones regulate gastric estrogen production, as well as insulin secretion, for proper hepatic lipogenesis, taking into account ingested carbohydrate and lipid levels. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Maintaining energy homeostasis, especially keeping blood glucose and lipid (triglycerides and fatty acids) levels within appropriate ranges, is critical for life. Since the discovery of insulin in 1921, regulation of blood glucose levels has been studied in detail. 8 However, until recently it was unclear which organs monitor blood lipid levels and which hormones lower elevated blood lipid levels. In 2021, we reported that gastric parietal cells (gastric acid secreting cells) secrete estrogen in response to the blood lipid levels. 1 Estrogen is not just a sex hormone— it is a multi-target and multi-functional hormone. As well as from the ovaries in women, it is secreted from adipocytes and gastric parietal cells, 9 – 11 and in addition to follicular growth and ovulation, it regulates energy homeostasis, bone metabolism, brain function and skeletal growth. 12 Estrogen suppresses feeding behavior, lipogenesis, and lipolysis, while increasing lipid storage and consumption, 2 – 5 all of which have the effect of lowering blood lipid levels. We therefore previously proposed a model in which gastric parietal cells monitor and control the blood lipid levels through the secretion of gastric estrogen in response to the blood lipid levels. 1 However, we excluded hormonal and neural influences on gastric estrogen secretion, and they should be considered for proper understanding of the role of gastric estrogen in daily life. Parietal cells generate energy from fatty acids and use it not only for estrogen production, but also for acid secretion, 1 and diet could affect both blood lipid levels and gastric acid secretion activities. Here, we show that postprandially decreased blood fatty acids and activated gastric acid secretion decrease gastric estrogen production and subsequent blood estrogen levels. We found that direct elevation of blood fatty acid levels by intravenous injection partially restores postprandially decreased estrogen levels. We also found that gastric acid-secreting hormones decrease gastric estrogen production. On the other hand, gastric acid-suppressing hormones and an antacid increase gastric estrogen production in vitro and blood estrogen levels in postprandial (gastric acid active) rats, but this was not the case in fasted (gastric acid inactive) rats. Insulin and estrogen enhance and suppress hepatic lipogenesis, respectively, 3 , 7 so we suggest that insulin decreases gastric estrogen production by lowering blood fatty acids after meals. Postprandially-activated gastric acid secretion also decreases gastric estrogen production. However, if the meals are rich in lipids, the intestines secrete gastric acid-suppressing hormones, such as GLP-1, 13 which would reduce lipogenesis by increasing gastric estrogen production. We therefore conclude that diet and subsequent hormones regulate gastric estrogen production for adequate hepatic lipogenesis, taking into account ingested carbohydrate and lipid. Blood estrogen decreases after meals, especially after carbohydrate intake To understand how gastric estrogen works in daily life, rats fasted for 12 h were fed either a high-fat diet (HFD) or a normal diet (ND) ad libitum for 30 min, and we monitored the levels of blood triglycerides, fatty acids, glucose, and estradiol [E2], the most potent estrogen (Fig. 1 a). Blood triglycerides increased and peaked 2 h after feeding in the HFD-fed rats, but did not show apparent changes in the ND-fed rats. The stomach secretes estrogen in response to the blood triglyceride levels, 1 so it was thought that blood E2 would increase in HFD-fed rats but not ND-fed rats. However, blood E2 decreased from the end of feeding in both types of rats. After feeding, blood fatty acids decreased and glucose increased. HFD and ND are mainly composed of carbohydrates, triglyceride, and proteins, and their content ratios were 10:5:5 in HFD and 10:1:5 in ND in this study in accordance with the manufacturer’s instructions. To determine which nutrient is the key to lowering blood estrogen levels, rats were orally administered carbohydrate (4 g starch per kg body weight), triglyceride (2.5 mL olive oil per kg body weight), 1 or protein (2 g casein per kg body weight), and we monitored their blood triglyceride, fatty acid, glucose, and E2 levels (Fig. 1 b). The amounts of nutrients administered were determined according to their content in HFD. Olive oil-administered rats had increased blood triglyceride and E2 levels, as we previously observed. 1 Starch-administered rats had decreased blood E2 levels with increase in blood glucose and decrease in blood fatty acid levels. Casein-administered rats did not show apparent changes in blood E2 levels. Carbohydrate would therefore be the key nutrient for postprandial decrease in blood estrogen levels. We then examined the relationship between blood glucose, fatty acid, and E2 levels in starch-administered rats (before [0 h], and 1 and 2 h after administration) (Fig. 1 c). We found a negative correlation between blood glucose and fatty acid levels, and a positive correlation between blood fatty acid and E2 levels. Insulin decreases fatty acid release from adipose tissues 22 and isolated gastric gland epithelia produce estrogen in a fatty acid-dependent manner, 1 so insulin-induced decrease in blood fatty acid levels are thought to play an important role in the postprandial decrease in blood estrogen levels. Intravenous injection of lipids partially recovers postprandially-decreased blood estrogen levels To confirm that the postprandial decrease in blood estrogen levels was due to the decrease in blood fatty acid levels, we directly increased blood triglycerides or fatty acids by intravenous injection (Fig. 2 ). We previously used triglyceride emulsion in an injection study and lauric acid (C12-FFA) in an in vitro E2 production assay using isolated gastric gland epithelia (Fig. 2 a, left). 1 However, C12-FFA was unsuitable for the injection study because of its low water solubility. Estrogen-producing gastric parietal cells uptake and use fatty acids using heart-type fatty acid binding protein, 1 which binds to fatty acids of C10 to C18. 14 Capric acid (C10-FFA) is highly water soluble, so we incubated isolated gastric gland epithelia with C10-FFA in the presence of testosterone. We confirmed that epithelia produced E2 in a C10-FFA concentration-dependent manner (Fig. 2 a, right), so we used C10-FFA in the injection study. ND-fed (2 h after a 0.5 h feeding) and fasted control rats were intravenously injected with triglycerides (2 mL of 20% soy oil emulsion per kg body weight) or C10-FFA (2 mL of 150 mM C10-FFA [pH 7.8] per kg body weight), and we monitored their blood triglyceride, fatty acid, and E2 levels (Figs. 2 b and c). When rats were injected with triglycerides, blood triglyceride, fatty acid, and E2 levels peaked immediately after the injection and returned to the basal levels 1 h later, regardless of their feeding conditions (Fig. 2 b). Blood fatty acid and E2 levels of C10-FFA injected rats also peaked just after the injection but returned to the basal levels more slowly, while there were no apparent changes to their blood triglyceride levels (Fig. 2 c). It is therefore thought that blood E2 levels correlate with blood fatty acid levels, including those produced by lipolysis of blood triglycerides. To examine the effects of diet on the blood fatty acid-dependent increase of blood E2 levels, we compared changes in blood fatty acid and E2 levels after triglyceride or C10-FFA injection ([0.25 h] - [0 h]) between ND-fed and fasted rats (Fig. 2 d). Although not significant, ND-fed rats tended to have smaller increases in blood E2 levels than fasted rats after either lipid injection. It is therefore suggested that postprandially-decreased blood fatty acid levels play an important role in the decrease in blood estrogen levels, but the presence of other factors should also be considered. Activation of gastric acid secretion decreases gastric estrogen production Parietal cells use energy for acid secretion in addition to estrogen production. Therefore, if gastric acid secretion is activated, there would be a reduction in energy for production of gastric estrogen. Diet activates acid secretion through nerves and hormones, 15 so we examined the effects of 15 stomach-related and metabolism-related hormones including autonomic neurotransmitters on the production of gastric estrogen using isolated gastric gland epithelia (Fig. 3 ). We also examined the effect of an antacid, lansoprazole (LPZ). 16 Epithelia were cultured with a series of concentrations of hormones or LPZ in the presence of testosterone and C12-FFA, and we determined their relative E2 production levels. E2 production was decreased by ghrelin, gastrin, histamine, and acetylcholine (ACh), all of which activate gastric acid secretion (Fig. 3 a). 15 On the other hand, des-acyl ghrelin, somatostatin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK), cortisol, adrenaline, and LPZ increased E2 production (Fig. 3 b). All of these, with the exception of cortisol, suppress gastric acid secretion. 15 – 20 Insulin, leptin, and triiodothyronine (T3) did not affect E2 production (Fig. 3 c). Further, LPZ restored the decrease of E2 production by gastric acid-secreting hormones (Fig. 3 d). According to these data, activation of gastric acid secretion is thought to decrease the production of gastric estrogen. GLP-1 and lansoprazole increase blood estrogen levels in postprandial, but not fasted, rats Finally, we examined the effects of gastric acid inhibition on the postprandially-decreased blood estrogen levels. GLP-1 increased gastric E2 production of most of the hormones examined in vitro (Fig. 3 b), so we used GLP-1 as a representative of gastric acid-inhibiting hormone. We also used LPZ to inhibit gastric acid secretion. ND-fed (2 h after a 0.5 h feeding) and fasted control rats were intravenously injected with GLP-1 (2 mL of 12 ng/mL GLP-1 per kg body weight) or LPZ (2 mL of 4 µM LPZ per kg body weight), and we monitored their blood triglyceride, fatty acid, and E2 levels (Fig. 4 ). GLP-1 and LPZ increased and restored blood E2 levels in ND-fed rats 15 min after injection, while there were no apparent changes in fasted rats. There were no apparent changes to blood triglyceride and fatty acid levels 15 min after injection in either of the rat types. Inhibition of gastric acid secretion is suggested by our data to increase blood estrogen levels in postprandial rats, but not in fasted rats. Discussion Gastric parietal cells generate energy from fatty acids, including those lipolyzed from triglycerides, 1 , 21 and use it for gastric estrogen production and gastric acid secretion (Fig. 5 a). Diet affects blood nutrient levels and gastric acid secretion, so we examined the effects of diet and related hormones on gastric estrogen production and blood estrogen levels. After oral triglyceride administration, blood estrogen increased with an increase in blood triglycerides, as we have previously reported (Figs. 1 and 5 b, upper right). 1 However, blood estrogen decreased after meals, especially after carbohydrate intake. Blood fatty acids also decreased after meals, and intravenous injection of triglycerides or fatty acids partially restored postprandially-decreased blood estrogen levels (Fig. 2 ). Insulin suppresses fatty acid release from adipocytes and lowers blood fatty acid levels, 22 but we found it had no direct effect on gastric estrogen production in vitro (Fig. 3 c). Parietal cells produce energy from fatty acids, so it was thought that insulin-induced decrease in blood fatty acids levels play an important role in the postprandial decrease in gastric estrogen production and subsequent blood estrogen levels (Fig. 5 b, upper left). Hormones that activate gastric acid secretion (ghrelin, gastrin, histamine, and ACh) decreased gastric estrogen production (Fig. 3 a). 15 Conversely, hormones that suppress secretion of gastric acid (des-acyl ghrelin, somatostatin, glucagon, GIP, GLP-1, CCK, and adrenaline) 15 , 17 – 20 and an antacid (LPZ) 16 increased gastric estrogen production (Fig. 3 b), and LPZ restored gastric estrogen production decreased by gastric acid-secreting hormones (Fig. 3 d). Intravenous injection of a gastric acid-suppressing hormone (GLP-1) or LPZ increased blood estrogen in postprandial (high in acid secretion) rats, but not in fasted (low in acid secretion) rats (Fig. 4 ). Activation of gastric acid secretion would therefore reduce the energy for gastric estrogen production in parietal cells, resulting in a decrease in gastric estrogen production and subsequent blood estrogen levels (Fig. 5 b, lower). Gastric estrogen, like insulin, enters the liver directly before being diluted in the systemic blood, so it is thought that gastric estrogen plays an important role in the liver. After a carbohydrate-based diet, insulin lowers elevated blood glucose levels by, for example, stimulating glycogen synthesis and lipogenesis in the liver. 7 , 22 , 23 Insulin also decreases blood fatty acids by suppressing lipolysis in the adipose tissues. 22 Parietal cells use energy from fatty acids to produce estrogen, and estrogen inhibits hepatic lipogenesis. Insulin would thus suppress gastric estrogen by reducing blood fatty acid levels. Parietal cells use energy for estrogen production and acid secretion. Activation of gastric acid secretion after meals would therefore also decrease gastric estrogen production. When the diet is rich in lipids, however, the intestines secrete hormones such as GIP, GLP-1, and CCK, which suppress secretion of gastric acid. 13 Lipid intake therefore increases gastric estrogen and decreases hepatic lipogenesis. We collectively conclude that diet and subsequent hormones regulate gastric estrogen production to optimize hepatic lipogenesis taking into account ingested carbohydrates and lipids. Just as insulin dysfunction can cause hyperglycemia, gastric estrogen dysfunction could cause hyperlipidemia and fatty liver. Indeed, hyperlipidemia and fatty liver are common in elderly people, and gastric estrogen production decreases with decrease in gastric parietal cells (estrogen producing cells) and testosterone (precursor of estrogen) in old rats. 24 , 25 A new subcategory of “gastric estrogen dysfunction” can therefore be added to hyperlipidemia and fatty liver. Finally, dietary habits that continuously lower blood fatty acid levels and increase gastric acid secretion, such as “carbohydrate snacking,” are suggested to be risks of hyperlipidemia and fatty liver through the continuous decrease in gastric estrogen secretion and subsequent blood estrogen levels. This study therefore sheds new light on the fields of estrogen and lipid homeostasis, including hyperlipidemia and fatty liver. We showed that postprandially-decreased blood fatty acids and activated gastric acid secretion decrease gastric estrogen production and subsequently blood estrogen levels. However, our data do not exclude the presence of other effects of diet and hormones on the gastric estrogen production. Indeed, gastrin, histamine, and ACh activate gastric acid secretion via phosphorylation pathways, 15 and phosphorylation positively (Y361) 26 and negatively (S118) 27 regulates aromatase activity. Phosphorylation may also affect energy production in parietal cells via heart-type fatty acid binding protein. 28 Further investigation of the effects of hormones or kinases on aromatase and energy production activities in parietal cells are therefore required to properly understand the comprehensive intracellular signaling pathway for gastric estrogen production. Methods Animals . Wistar rats, purchased from Kiwa Laboratory Animals (Japan), were housed in an air-conditioned environment (24 ± 2 ºC and 50–60 % humidity) with 12 h light/dark cycle (lights on at 8:00) and allowed access to food (normal diet [ND, CE-2 diet, CLEA-Japan (Tokyo, Japan); carbohydrate : triglycerides : protein = 10 : 1 : 5] ) and water ad libitum unless otherwise stated. In high-fat diet studies, rats were fed a high-fat diet (HFD, D12451, Research Diets; carbohydrate : triglycerides : protein = 10 : 5 : 5) for one week before the experiment. All experiments were conducted according to the protocol approved by the Wakayama Medical University Animal Care and Use Committee (approval number: 1104). Hormones and antacid. Ghrelin (334-43731), des-acyl ghrelin (332-44371), insulin (093-06471), glucagon (339-40981), leptin (120-06591), cholecystokinin (CCK, 333-41001), gastric inhibitory polypeptide (GIP, 331-41781), glucagon-like peptide 1 (GLP-1, 333-43441), somatostatin (332-40231), gastrin (338-41431), histamine (088-00641) and acetylcholine (ACh, 011-00592) were purchased from FujiFilm (Tokyo, Japan). Triiodothyronine (T3, T2887), cortisol (H4001) and adrenaline (E4642) were purchased from Merck (Amsterdam, Netherland). Lansoprazole (LPZ, 123-05861), a proton pump inhibitor, was purchased from FujiFilm. Feeding of high-fat and normal diets. Eight-week-old male rats were deprived of food for 12 h. Rats were fed either a HFD or ND ad libitum for 30 min (from -0.5 to 0 h), and their blood samples were collected from the tail vein before (-0.5) and at 0, 1, 2, 3, 4, and 5 h after the feeding. Rats with initial tail-vein triglyceride levels > 200 mg/dL were excluded from this study. Oral administration of starch, olive oil, and casein. Oral administration studies were performed as previously described, with slight modifications. 1 Eight-week-old male rats were deprived of food for 12 h. Starch (4 g [10 mL of 40 % starch solution] per kg body weight), olive oil (2.5 mL per kg body weight), or casein (2 g [12.5 mL of 16 % casein solution] per kg body weight) were administered orally to rats using the intragastric gavage technique, and their blood samples were collected from the tail vein before (0) and then 1, 2, 3, 4, and 5 h after the administration. The amounts of starch (carbohydrate), olive oil (triglyceride), and casein (protein) administered were determined according to their content in HFD (carbohydrate : triglyceride : protein = 10 : 5 : 5). Starch (191-03985) and casein (030-01505) were purchased from FujiFilm. Olive oil (Yoshida) was purchased from Yoshida Pharmaceutical Company (Tokyo, Japan). Rats with initial tail vein triglyceride levels higher than 200 mg/dL were excluded from this study. Intravenous injection of triglycerides, fatty acids, and LPZ . Intravenous injection studies were performed as previously described, with modified conditions. 1 Eight-week-old male rats were deprived of food for 12 h. Triglyceride emulsion (2 mL of 20% soy oil emulsion per kg body weight; Intralipos Injection 20%, Otsuka, Tokyo, Japan), C10-FFA (2 mL of 150 mM capric acid [pH 7.8] per kg body weight; D0024, Tokyo Chemical Industry, Tokyo, Japan), or LPZ (2 mL of 4 µM LPZ per kg body weight) was injected intravenously into fasted rats or rats fed ND ad libitum for 30 min followed by 2 h rest. Blood samples were collected from the tail vein before (at 0 h [fasted] or at -2.5, -2, -1, 0 h [ND fed]) and at 0.25, 0.5, 1, 2, and 3 h after the injection. Rats with initial tail vein triglyceride levels > 200 mg/dL were excluded from this study. In vitro gastric estrogen production assay. Gastric gland epithelia were isolated as previously described with slight modifications. 1 Male rats aged 11-15 weeks were deprived of food for 4 h. Stomachs removed from rat anesthetized with isoflurane were opened along the greater curvature and washed with ice-cold phosphate-buffered saline (PBS). After removal of the serosal muscle, the fundic region was cut into < 5 mm pieces and shaken in the chelating buffer (5 mM EDTA in PBS) supplemented with Tosyl-L-lysyl-chloromethane hydrochloride (TLCK, 147 ng/mL, 200-20141, FujiFilm) for 2 h on ice. The supernatant was changed with the dissociation buffer (54.9 mM D-sorbitol and 43.4 mM sucrose in PBS), and the tube was shaken vigorously for 2 min to dissociate epithelia from the mucosa. After centrifugation at 160 g for 10 min at 4 ºC, the pellet was dissolved in DMEM (044-32955, FujiFilm). After filtration through a 100 µm cell strainer (VCS-100, As One), the isolated gastric gland epithelia were incubated in DMEM supplemented with testosterone (20 nM, T-1500, Sigma), lauric acid (C12-FFA, 500 µM, L0016, Tokyo Chemical Industry) or capric acid (C10-FFA, 250, 500, 1000 µM, D0024, Tokyo Chemical Industry), and TLCK (147 ng/mL) for 1 hour at 37 °C in the presence or absence of hormone or drug. As non-estrogen (E2) producing controls, the epithelia were incubated in DMEM supplemented with testosterone and TLCK only. E2 and phospholipid (PL) levels in the epithelia with culture medium were measured, and E2 levels were normalized by PL levels (relative total E2 levels). To obtain relative E2 production levels, relative total E2 levels were subtracted with that of the non-E2 producing control. Isolated gastric gland epithelia were observed under an Eclipse Ti microscope equipped with Plan Fluor 10/0.30 lens and DS-Fi1 camera (Nikon, Tokyo, Japan). Image processing was performed with ImageJ 2 2.3.0 (National Institutes of Health). Measurement of triglycerides, glucose, fatty acids, estrogen, and phospholipid concentrations . Measurement of triglyceride, estrogen, and phospholipid concentrations were performed as previously described. 1 Plasma glucose and fatty acids concentrations were measured using a LabAssay Glucose and a NEFA C (FujiFilm) according to the manufacturer’s instructions. Statistics and reproducibility. Data are mean ± s.d. P values of different two groups were determined by two-sided Student’s t -test. R and P values determined by Pearson’s product-moment correlation with 95% density ellipse. Statistical analyses were performed using JMP Pro ver. 16 (SAS Institute Japan, Tokyo, Japan). P values 0.5 and < -0.5 were considered to be positively and negatively correlated, respectively. Every experiment was performed multiple times with essentially the same results. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Declarations Data availability The source data for the Figures are available in Supplementary Data 1-4. All other data that support the findings of this study are available from the corresponding author on reasonable request. Acknowledgements . We thank Yuta Yamamoto, Naoko Yamagishi and Keiko Ikemoto from the Kanai Laboratory for their assistance. This study was supported by Medical Research Support from Wakayama Medical University (Y.K.). We also acknowledge proofreading and editing by Benjamin Phillis at the Clinical Study Support Center at Wakayama Medical University. Author contributions. Y.K. conceived the project, designed the experiments, interpreted the results, wrote the manuscript, and prepared the figures. Y.O. and A.T. found the postprandial decrease in blood E2 levels. T.I. performed in vivo experiments using rats. Y.K. and T.I. performed in vitro experiments using isolated gastric gland epithelia. T.I. performed statistical analysis. Competing interests. The authors declare no competing interests. Additional Information Supplementary information The online version contains supplementary material available at Correspondence and requests for materials should be addressed to Yoshimitsu Kanai. Online content Any methods, Nature Portfolio reporting summaries, source data, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data are available at https:// References Ito, T., Yamamoto, Y., Yamagishi, N. & Kanai, Y. Stomach secretes estrogen in response to the blood triglyceride levels. Commun Biology 4 , 1364 (2021). Dhillon, S. S. & Belsham, D. D. Estrogen inhibits NPY secretion through membrane-associated estrogen receptor (ER)-α in clonal, immortalized hypothalamic neurons. Int J Obes 1–10 (2019) doi:10.1038/ijo.2010.124. Qiu, S. et al. Hepatic estrogen receptor α is critical for regulation of gluconeogenesis and lipid metabolism in males. 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Lipid Res. 34 , 1355–1366 (1993). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryData1.xlsx Supplementary Data 1 SupplementaryData2.xlsx Supplementary Data 2 SupplementaryData3.xlsx Supplementary Data 3 SupplementaryData4.xlsx Supplementary Data 4 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4567800","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":319338122,"identity":"148e846c-2f74-4d05-998f-271976f2ebb4","order_by":0,"name":"Yoshimitsu Kanai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDACHhBhYANlQIABiGAmoCWNZC0MhzG14AQGZw4/e/Cj4Hxif8/hZxI/c+wYdNubNzD8qGFgN8el5WybuWGPwe3EGWfbzCR7tyUzmJ05VsDYc4yB2bIBh5bzDGYSPAa3cxtADN5tzPXbbuQYMPA2MDAbHMClhf2b5B+Dc7nzQYy/2+oZzO6/MWD8i0/L2R4zaR6DA7kbQAzebYcZzG7wGDDjs0XyzJkyaRmD5PqNZ84UW8tuOw70S1rBYZljEjj9wncmfZvkmz92xnJn0jfefLutmsHs+OGND9/U2CTjCjEFJNtZJGAsoKBEMq7YkUeynfkDsowd/ggdBaNgFIyCEQQAPxldrxab9SEAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0375-6853","institution":"Wakayama Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yoshimitsu","middleName":"","lastName":"Kanai","suffix":""},{"id":319338123,"identity":"b28a54d7-62ff-4084-ac2a-adf2d410cab9","order_by":1,"name":"Takao Ito","email":"","orcid":"https://orcid.org/0000-0002-6408-9572","institution":"Wakayama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Takao","middleName":"","lastName":"Ito","suffix":""},{"id":319338124,"identity":"7dc3f115-a1cb-492b-8187-adccaa24e6f2","order_by":2,"name":"Yuichi Ozaki","email":"","orcid":"","institution":"Wakayama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuichi","middleName":"","lastName":"Ozaki","suffix":""},{"id":319338125,"identity":"e69d9601-316e-49d6-a6ae-5b3dffcc055f","order_by":3,"name":"Atsushi Tanaka","email":"","orcid":"","institution":"Wakayama Medical University","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Tanaka","suffix":""}],"badges":[],"createdAt":"2024-06-12 06:00:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4567800/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4567800/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61607309,"identity":"06557bba-a019-4f1e-9085-f4cd2fc400f1","added_by":"auto","created_at":"2024-08-01 22:29:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":448259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlood estrogen decreases after meals, especially after carbohydrate intake. \u003c/strong\u003eEight-week-old male rats were deprived of food for 12 h. \u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003e b \u003c/strong\u003eRats were fed either a high-fat diet (HFD, red) or a normal diet (ND, blue) \u003cem\u003ead libitum\u003c/em\u003efor 30 min (from -0.5 to 0 h, bars). Triglyceride (TG), fatty acids (FFA), glucose, and estrogen (E2) in the tail venous blood were measured before (-0.5) and at 0, 1, 2, 3, 4, and 5 h after the feeding. \u003cstrong\u003ec \u003c/strong\u003eRats were orally administrated either starch (4 g per kg body weight, blue), olive oil (2.5 mL per kg body weight, red), or casein (2 g per kg body weight, green) using intragastric gavage technique. TG, FFA, glucose, and E2 in the tail venous blood were measured before (0) and at 1, 2, 3, 4, and 5 h after the administration. \u003cstrong\u003ec\u003c/strong\u003e The correlation diagram between blood glucose, FFA, and E2 of glucose-administered rats using data before (0 h) and 1 and 2 h after the administration. Data are represented as mean ± s.d. \u003cem\u003en\u003c/em\u003e = 8. \u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eP\u003c/em\u003e values were determined by two-sided Student’s t-test between before and 2 h after the feeding or administration.\u003cstrong\u003e c\u003c/strong\u003e, \u003cem\u003eR\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003evalues determined by Pearson’s product-moment correlation with 95% density ellipse. Raw data are provided in Supplementary Data 1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/c34a8fa2c2c1d710d781147e.png"},{"id":61607353,"identity":"b6b0b97b-b5bc-433b-9cf1-2eb158d4c043","added_by":"auto","created_at":"2024-08-01 22:37:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":468269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntravenous injection of lipids partially recovers postprandially-decreased blood estrogen levels. a\u003c/strong\u003e Isolated gastric gland epithelia from male rat (left) were incubated with testosterone (20 nM) and capric acid (C10-FFA: 0, 250, 500 and 1000 µM) at 37 \u003csup\u003e◦\u003c/sup\u003eC for 1 h, and their relative E2 levels, normalized by phospholipid (PL), were determined. Bar: 50 µm.\u003cstrong\u003e b \u003c/strong\u003eand\u003cstrong\u003e c \u003c/strong\u003eEight-week-old male rats were deprived of food for 12 h before the experiment. TG (2 mL of 20% soy oil emulsion per kg body weight, \u003cstrong\u003eb\u003c/strong\u003e) or C10-FFA (2 mL of 150 mM capric acid [pH 7.8] per kg body weight, \u003cstrong\u003ec\u003c/strong\u003e) was injected intravenously into rats fed ND \u003cem\u003ead libitum\u003c/em\u003e for 30 min (-2.5 to 2 h, bars) followed by 2 h rest (ND + TG or C10-FFA iv, blue) or control fasted rats (fasted + TG or C10-FFA iv, brown) (arrows, at 0 h). TG, FFA, and E2 in the tail venous blood were measured before (ND-fed: at -2.5, -2, -1, 0 h; fasted: at 0 h) and at 0.25, 0.5. 1, 2, 3 h after the injection. \u003cstrong\u003ed \u003c/strong\u003eChanges in blood FFA and E2 levels in ND-fed and fasted rats after TG or C10-FFA injection (from [0 h] to [0.25 h]).\u003cem\u003e \u003c/em\u003eData are represented as mean ± s.d. \u003cem\u003en\u003c/em\u003e = 8. \u003cem\u003eP\u003c/em\u003e values were determined by two-sided Student’s t-test between the indicated data sets. Raw data are provided in Supplementary Data 2.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/7ba6e004094aa955e641d304.png"},{"id":61607352,"identity":"4ee38262-e6ba-467c-927f-d237c1e395aa","added_by":"auto","created_at":"2024-08-01 22:37:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":361727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of gastric acid secretion decreases gastric estrogen production. a-c \u003c/strong\u003eIsolated gastric gland epithelia from male rats were cultured with testosterone (20 nM), lauric acid (C12-FFA: 500 µM), and stomach- or metabolism-related hormones and an antacid, lansoprazole (LPZ) at 37 \u003csup\u003e◦\u003c/sup\u003eC for 1 h, and their relative E2 production levels were determined. \u003cstrong\u003ea\u003c/strong\u003e Ghrelin, gastrin, histamine, and acetylcholine (ACh) decreased gastric E2 production. \u003cstrong\u003eb\u003c/strong\u003e Des-acyl-ghrelin, somatostatin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK), cortisol, adrenaline, and LPZ increased gastric E2 production. \u003cstrong\u003ec\u003c/strong\u003e Insulin, leptin, triiodothyronine (T3) did not affect gastric E2 production. \u003cstrong\u003ed\u003c/strong\u003e Epithelia were cultured with gastrin, histamine and Ach in the presence or absence of LPZ, and their relative E2 production levels were determined. Data are represented as mean ± s.d. \u003cem\u003en\u003c/em\u003e = 8. \u003cem\u003eP\u003c/em\u003e values between hormone-added and control hormone-free samples (\u003cstrong\u003eb \u003c/strong\u003eand\u003cstrong\u003e c, \u003c/strong\u003e*: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) or between the indicated data sets (\u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003e d\u003c/strong\u003e) were determined by two-sided Student’s t-test. Raw data and actual \u003cem\u003eP\u003c/em\u003e values are provided in Supplementary Data 3.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/39f938ec888fda5f3225315b.png"},{"id":61607622,"identity":"2d2f1410-904a-41d9-9bd2-c7fa13d367e4","added_by":"auto","created_at":"2024-08-01 22:45:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":227673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1 and LPZ increase blood estrogen levels in postprandial, but not fasted, rats. \u003c/strong\u003eEight-week-old male rats were deprived of food for 12 h before the experiment. GLP-1 (2 mL of 12 ng/mL GLP-1 per kg body weight, \u003cstrong\u003ea\u003c/strong\u003e) or LPZ (2 mL of 4 µM LPZ per kg body weight, \u003cstrong\u003eb\u003c/strong\u003e) was injected intravenously into rats fed ND \u003cem\u003ead libitum\u003c/em\u003e for 30 min (-2.5 to 2 h, bars) followed by 2 h rest (ND + GLP-1 or LPZ iv, blue) or control fasted rats (fasted + GLP-1 or LPZ iv, brown) (arrows, at 0 h). TG, FFA, and E2 in the tail venous blood were measured before (ND-fed: at -2.5, -2, -1, 0 h; fasted: at 0 h) and at 0.25, 0.5. 1, 2, 3 h after the injection. Data are represented as mean ± s.d. \u003cem\u003en\u003c/em\u003e= 8. \u003cem\u003eP\u003c/em\u003e values between before (at 0 h) and after (at 0.25 h) the injection were determined by two-sided Student’s t-test. Raw data are provided in Supplementary Data 4.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/f17b53fd962029aee59973a4.png"},{"id":61607310,"identity":"edfb7578-c599-4bcc-84fb-384ef51127b3","added_by":"auto","created_at":"2024-08-01 22:29:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":448334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of diet and hormones on gastric estrogen production. a\u003c/strong\u003e Gastric parietal cells produce estrogen and secrete acids using energy from lipids (fatty acids, including those lipolyzed from triglycerides). Decrease in blood lipid levels and activation of gastric acid secretion therefore decrease gastric estrogen production. \u003cstrong\u003eb\u003c/strong\u003e Carbohydrate intake (insulin) decreases blood fatty acid (upper left). Lipid intake, adrenaline and cortisol increase blood lipids (upper right). Gastric acid-secreting hormones, including those induced by food intake (gastrin, histamine, and ACh), decrease gastric estrogen production (lower left). Gastric acid-suppressing hormones, including those induced by lipid intake (GIP, GLP-1, and CCK) or fasting (des-acyl ghrelin, glucagon, and adrenaline), increase gastric estrogen production (lower right). Gastric estrogen directly enters the liver and suppresses hepatic lipogenesis before being diluted in the systemic blood.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/7d20efcd965d0efb35b2fb86.png"},{"id":61607735,"identity":"c1cd6209-00b3-4094-9f78-c1b022472203","added_by":"auto","created_at":"2024-08-01 22:53:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2215746,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/a5136525-6e56-4f0d-9236-3cdc1d7301ae.pdf"},{"id":61607302,"identity":"c45fbef5-0dbe-4baf-b139-f714d5bc4003","added_by":"auto","created_at":"2024-08-01 22:29:07","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26242,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 1\u003c/p\u003e","description":"","filename":"SupplementaryData1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/b848472f76620c9b84d179a7.xlsx"},{"id":61607308,"identity":"86f2b3ef-97cb-4cdb-920b-8cd6a75dcbb8","added_by":"auto","created_at":"2024-08-01 22:29:07","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":32549,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 2\u003c/p\u003e","description":"","filename":"SupplementaryData2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/a20342233ff6d07d591fccd3.xlsx"},{"id":61607305,"identity":"fec85be1-0811-406e-a833-3abcfb6b5566","added_by":"auto","created_at":"2024-08-01 22:29:07","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":96656,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 3\u003c/p\u003e","description":"","filename":"SupplementaryData3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/9f68d3f6b521d3c794c68336.xlsx"},{"id":61607303,"identity":"dea751da-62d4-46e3-929f-9afadceac083","added_by":"auto","created_at":"2024-08-01 22:29:07","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21384,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 4\u003c/p\u003e","description":"","filename":"SupplementaryData4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4567800/v1/1a8ccff2c5903154a8986b35.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Carbohydrate intake and acid secretion decrease gastric estrogen secretion","fulltext":[{"header":"Main","content":"\u003cp\u003eMaintaining energy homeostasis, especially keeping blood glucose and lipid (triglycerides and fatty acids) levels within appropriate ranges, is critical for life. Since the discovery of insulin in 1921, regulation of blood glucose levels has been studied in detail.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e However, until recently it was unclear which organs monitor blood lipid levels and which hormones lower elevated blood lipid levels. In 2021, we reported that gastric parietal cells (gastric acid secreting cells) secrete estrogen in response to the blood lipid levels.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Estrogen is not just a sex hormone\u0026mdash; it is a multi-target and multi-functional hormone. As well as from the ovaries in women, it is secreted from adipocytes and gastric parietal cells,\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and in addition to follicular growth and ovulation, it regulates energy homeostasis, bone metabolism, brain function and skeletal growth.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Estrogen suppresses feeding behavior, lipogenesis, and lipolysis, while increasing lipid storage and consumption,\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e all of which have the effect of lowering blood lipid levels. We therefore previously proposed a model in which gastric parietal cells monitor and control the blood lipid levels through the secretion of gastric estrogen in response to the blood lipid levels.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e However, we excluded hormonal and neural influences on gastric estrogen secretion, and they should be considered for proper understanding of the role of gastric estrogen in daily life. Parietal cells generate energy from fatty acids and use it not only for estrogen production, but also for acid secretion,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and diet could affect both blood lipid levels and gastric acid secretion activities.\u003c/p\u003e \u003cp\u003eHere, we show that postprandially decreased blood fatty acids and activated gastric acid secretion decrease gastric estrogen production and subsequent blood estrogen levels. We found that direct elevation of blood fatty acid levels by intravenous injection partially restores postprandially decreased estrogen levels. We also found that gastric acid-secreting hormones decrease gastric estrogen production. On the other hand, gastric acid-suppressing hormones and an antacid increase gastric estrogen production \u003cem\u003ein vitro\u003c/em\u003e and blood estrogen levels in postprandial (gastric acid active) rats, but this was not the case in fasted (gastric acid inactive) rats.\u003c/p\u003e \u003cp\u003eInsulin and estrogen enhance and suppress hepatic lipogenesis, respectively,\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e so we suggest that insulin decreases gastric estrogen production by lowering blood fatty acids after meals. Postprandially-activated gastric acid secretion also decreases gastric estrogen production. However, if the meals are rich in lipids, the intestines secrete gastric acid-suppressing hormones, such as GLP-1,\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e which would reduce lipogenesis by increasing gastric estrogen production. We therefore conclude that diet and subsequent hormones regulate gastric estrogen production for adequate hepatic lipogenesis, taking into account ingested carbohydrate and lipid.\u003c/p\u003e"},{"header":"Blood estrogen decreases after meals, especially after carbohydrate intake","content":"\u003cp\u003eTo understand how gastric estrogen works in daily life, rats fasted for 12 h were fed either a high-fat diet (HFD) or a normal diet (ND) \u003cem\u003ead libitum\u003c/em\u003e for 30 min, and we monitored the levels of blood triglycerides, fatty acids, glucose, and estradiol [E2], the most potent estrogen (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Blood triglycerides increased and peaked 2 h after feeding in the HFD-fed rats, but did not show apparent changes in the ND-fed rats. The stomach secretes estrogen in response to the blood triglyceride levels,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e so it was thought that blood E2 would increase in HFD-fed rats but not ND-fed rats. However, blood E2 decreased from the end of feeding in both types of rats. After feeding, blood fatty acids decreased and glucose increased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e HFD and ND are mainly composed of carbohydrates, triglyceride, and proteins, and their content ratios were 10:5:5 in HFD and 10:1:5 in ND in this study in accordance with the manufacturer’s instructions. To determine which nutrient is the key to lowering blood estrogen levels, rats were orally administered carbohydrate (4 g starch per kg body weight), triglyceride (2.5 mL olive oil per kg body weight),\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e or protein (2 g casein per kg body weight), and we monitored their blood triglyceride, fatty acid, glucose, and E2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The amounts of nutrients administered were determined according to their content in HFD. Olive oil-administered rats had increased blood triglyceride and E2 levels, as we previously observed.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Starch-administered rats had decreased blood E2 levels with increase in blood glucose and decrease in blood fatty acid levels. Casein-administered rats did not show apparent changes in blood E2 levels. Carbohydrate would therefore be the key nutrient for postprandial decrease in blood estrogen levels.\u003c/p\u003e \u003cp\u003eWe then examined the relationship between blood glucose, fatty acid, and E2 levels in starch-administered rats (before [0 h], and 1 and 2 h after administration) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We found a negative correlation between blood glucose and fatty acid levels, and a positive correlation between blood fatty acid and E2 levels. Insulin decreases fatty acid release from adipose tissues\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and isolated gastric gland epithelia produce estrogen in a fatty acid-dependent manner,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e so insulin-induced decrease in blood fatty acid levels are thought to play an important role in the postprandial decrease in blood estrogen levels.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\n \u003cp\u003e \u003c/p\u003e \n\n \u003cp\u003e \u003c/p\u003e"},{"header":"Intravenous injection of lipids partially recovers postprandially-decreased blood estrogen levels","content":"\u003cp\u003eTo confirm that the postprandial decrease in blood estrogen levels was due to the decrease in blood fatty acid levels, we directly increased blood triglycerides or fatty acids by intravenous injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We previously used triglyceride emulsion in an injection study and lauric acid (C12-FFA) in an \u003cem\u003ein vitro\u003c/em\u003e E2 production assay using isolated gastric gland epithelia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, left).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e However, C12-FFA was unsuitable for the injection study because of its low water solubility. Estrogen-producing gastric parietal cells uptake and use fatty acids using heart-type fatty acid binding protein,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e which binds to fatty acids of C10 to C18.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Capric acid (C10-FFA) is highly water soluble, so we incubated isolated gastric gland epithelia with C10-FFA in the presence of testosterone. We confirmed that epithelia produced E2 in a C10-FFA concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, right), so we used C10-FFA in the injection study.\u003c/p\u003e\u003cp\u003eND-fed (2 h after a 0.5 h feeding) and fasted control rats were intravenously injected with triglycerides (2 mL of 20% soy oil emulsion per kg body weight) or C10-FFA (2 mL of 150 mM C10-FFA [pH 7.8] per kg body weight), and we monitored their blood triglyceride, fatty acid, and E2 levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and c). When rats were injected with triglycerides, blood triglyceride, fatty acid, and E2 levels peaked immediately after the injection and returned to the basal levels 1 h later, regardless of their feeding conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Blood fatty acid and E2 levels of C10-FFA injected rats also peaked just after the injection but returned to the basal levels more slowly, while there were no apparent changes to their blood triglyceride levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). It is therefore thought that blood E2 levels correlate with blood fatty acid levels, including those produced by lipolysis of blood triglycerides.\u003c/p\u003e\u003cp\u003eTo examine the effects of diet on the blood fatty acid-dependent increase of blood E2 levels, we compared changes in blood fatty acid and E2 levels after triglyceride or C10-FFA injection ([0.25 h] - [0 h]) between ND-fed and fasted rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Although not significant, ND-fed rats tended to have smaller increases in blood E2 levels than fasted rats after either lipid injection. It is therefore suggested that postprandially-decreased blood fatty acid levels play an important role in the decrease in blood estrogen levels, but the presence of other factors should also be considered.\u003c/p\u003e"},{"header":"Activation of gastric acid secretion decreases gastric estrogen production","content":"\u003cp\u003eParietal cells use energy for acid secretion in addition to estrogen production. Therefore, if gastric acid secretion is activated, there would be a reduction in energy for production of gastric estrogen. Diet activates acid secretion through nerves and hormones,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e so we examined the effects of 15 stomach-related and metabolism-related hormones including autonomic neurotransmitters on the production of gastric estrogen using isolated gastric gland epithelia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We also examined the effect of an antacid, lansoprazole (LPZ).\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eEpithelia were cultured with a series of concentrations of hormones or LPZ in the presence of testosterone and C12-FFA, and we determined their relative E2 production levels. E2 production was decreased by ghrelin, gastrin, histamine, and acetylcholine (ACh), all of which activate gastric acid secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e On the other hand, des-acyl ghrelin, somatostatin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK), cortisol, adrenaline, and LPZ increased E2 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). All of these, with the exception of cortisol, suppress gastric acid secretion.\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e–\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Insulin, leptin, and triiodothyronine (T3) did not affect E2 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Further, LPZ restored the decrease of E2 production by gastric acid-secreting hormones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). According to these data, activation of gastric acid secretion is thought to decrease the production of gastric estrogen.\u003c/p\u003e"},{"header":"GLP-1 and lansoprazole increase blood estrogen levels in postprandial, but not fasted, rats","content":"\u003cp\u003eFinally, we examined the effects of gastric acid inhibition on the postprandially-decreased blood estrogen levels. GLP-1 increased gastric E2 production of most of the hormones examined \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), so we used GLP-1 as a representative of gastric acid-inhibiting hormone. We also used LPZ to inhibit gastric acid secretion.\u003c/p\u003e\u003cp\u003eND-fed (2 h after a 0.5 h feeding) and fasted control rats were intravenously injected with GLP-1 (2 mL of 12 ng/mL GLP-1 per kg body weight) or LPZ (2 mL of 4 µM LPZ per kg body weight), and we monitored their blood triglyceride, fatty acid, and E2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). GLP-1 and LPZ increased and restored blood E2 levels in ND-fed rats 15 min after injection, while there were no apparent changes in fasted rats. There were no apparent changes to blood triglyceride and fatty acid levels 15 min after injection in either of the rat types. Inhibition of gastric acid secretion is suggested by our data to increase blood estrogen levels in postprandial rats, but not in fasted rats.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGastric parietal cells generate energy from fatty acids, including those lipolyzed from triglycerides,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and use it for gastric estrogen production and gastric acid secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Diet affects blood nutrient levels and gastric acid secretion, so we examined the effects of diet and related hormones on gastric estrogen production and blood estrogen levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter oral triglyceride administration, blood estrogen increased with an increase in blood triglycerides, as we have previously reported (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, upper right).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e However, blood estrogen decreased after meals, especially after carbohydrate intake. Blood fatty acids also decreased after meals, and intravenous injection of triglycerides or fatty acids partially restored postprandially-decreased blood estrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Insulin suppresses fatty acid release from adipocytes and lowers blood fatty acid levels,\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e but we found it had no direct effect on gastric estrogen production \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Parietal cells produce energy from fatty acids, so it was thought that insulin-induced decrease in blood fatty acids levels play an important role in the postprandial decrease in gastric estrogen production and subsequent blood estrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, upper left).\u003c/p\u003e \u003cp\u003eHormones that activate gastric acid secretion (ghrelin, gastrin, histamine, and ACh) decreased gastric estrogen production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Conversely, hormones that suppress secretion of gastric acid (des-acyl ghrelin, somatostatin, glucagon, GIP, GLP-1, CCK, and adrenaline)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and an antacid (LPZ)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e increased gastric estrogen production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), and LPZ restored gastric estrogen production decreased by gastric acid-secreting hormones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Intravenous injection of a gastric acid-suppressing hormone (GLP-1) or LPZ increased blood estrogen in postprandial (high in acid secretion) rats, but not in fasted (low in acid secretion) rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Activation of gastric acid secretion would therefore reduce the energy for gastric estrogen production in parietal cells, resulting in a decrease in gastric estrogen production and subsequent blood estrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, lower).\u003c/p\u003e \u003cp\u003eGastric estrogen, like insulin, enters the liver directly before being diluted in the systemic blood, so it is thought that gastric estrogen plays an important role in the liver. After a carbohydrate-based diet, insulin lowers elevated blood glucose levels by, for example, stimulating glycogen synthesis and lipogenesis in the liver.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Insulin also decreases blood fatty acids by suppressing lipolysis in the adipose tissues.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Parietal cells use energy from fatty acids to produce estrogen, and estrogen inhibits hepatic lipogenesis. Insulin would thus suppress gastric estrogen by reducing blood fatty acid levels. Parietal cells use energy for estrogen production and acid secretion. Activation of gastric acid secretion after meals would therefore also decrease gastric estrogen production. When the diet is rich in lipids, however, the intestines secrete hormones such as GIP, GLP-1, and CCK, which suppress secretion of gastric acid.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Lipid intake therefore increases gastric estrogen and decreases hepatic lipogenesis. We collectively conclude that diet and subsequent hormones regulate gastric estrogen production to optimize hepatic lipogenesis taking into account ingested carbohydrates and lipids.\u003c/p\u003e \u003cp\u003eJust as insulin dysfunction can cause hyperglycemia, gastric estrogen dysfunction could cause hyperlipidemia and fatty liver. Indeed, hyperlipidemia and fatty liver are common in elderly people, and gastric estrogen production decreases with decrease in gastric parietal cells (estrogen producing cells) and testosterone (precursor of estrogen) in old rats.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e A new subcategory of \u0026ldquo;gastric estrogen dysfunction\u0026rdquo; can therefore be added to hyperlipidemia and fatty liver. Finally, dietary habits that continuously lower blood fatty acid levels and increase gastric acid secretion, such as \u0026ldquo;carbohydrate snacking,\u0026rdquo; are suggested to be risks of hyperlipidemia and fatty liver through the continuous decrease in gastric estrogen secretion and subsequent blood estrogen levels. This study therefore sheds new light on the fields of estrogen and lipid homeostasis, including hyperlipidemia and fatty liver.\u003c/p\u003e \u003cp\u003eWe showed that postprandially-decreased blood fatty acids and activated gastric acid secretion decrease gastric estrogen production and subsequently blood estrogen levels. However, our data do not exclude the presence of other effects of diet and hormones on the gastric estrogen production. Indeed, gastrin, histamine, and ACh activate gastric acid secretion via phosphorylation pathways,\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and phosphorylation positively (Y361)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and negatively (S118)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e regulates aromatase activity. Phosphorylation may also affect energy production in parietal cells via heart-type fatty acid binding protein.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Further investigation of the effects of hormones or kinases on aromatase and energy production activities in parietal cells are therefore required to properly understand the comprehensive intracellular signaling pathway for gastric estrogen production.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e. Wistar rats, purchased from Kiwa Laboratory Animals (Japan), were housed in an air-conditioned environment (24 \u0026plusmn; 2 \u0026ordm;C and 50\u0026ndash;60 % humidity) with 12 h light/dark cycle (lights on at 8:00) and allowed access to food (normal diet [ND, CE-2 diet, CLEA-Japan (Tokyo, Japan); \u0026nbsp;carbohydrate : triglycerides : protein = 10 : 1 : 5] ) and water \u003cem\u003ead libitum\u003c/em\u003e unless otherwise stated. In high-fat diet studies, rats were fed a high-fat diet (HFD, D12451, Research Diets; carbohydrate : triglycerides : protein = 10 : 5 : 5) for one week before the experiment. All experiments were conducted according to the protocol approved by the Wakayama Medical University Animal Care and Use Committee (approval number: 1104).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHormones\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and antacid.\u003c/strong\u003e Ghrelin (334-43731), des-acyl ghrelin (332-44371), insulin (093-06471), glucagon (339-40981), leptin (120-06591), cholecystokinin (CCK, 333-41001), gastric inhibitory polypeptide (GIP, 331-41781), glucagon-like peptide 1 (GLP-1, 333-43441), somatostatin (332-40231), gastrin (338-41431), histamine (088-00641) and acetylcholine (ACh, 011-00592) were purchased from FujiFilm (Tokyo, Japan). Triiodothyronine (T3, T2887), cortisol (H4001) and adrenaline (E4642) were purchased from Merck (Amsterdam, Netherland). Lansoprazole (LPZ, 123-05861), a proton pump inhibitor, was purchased from FujiFilm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeeding\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of high-fat and normal diets.\u003c/strong\u003e Eight-week-old male rats were deprived of food for 12 h. Rats were fed either a HFD or ND \u003cem\u003ead libitum\u003c/em\u003e for 30 min (from -0.5 to 0 h), and their blood samples were collected from the tail vein before (-0.5) and at 0, 1, 2, 3, 4, and 5 h after the feeding. Rats with initial tail-vein triglyceride levels \u0026gt; 200 mg/dL were excluded from this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOral\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;administration of starch, olive oil, and casein.\u003c/strong\u003e Oral administration studies were performed as previously described, with slight modifications.\u003csup\u003e1\u003c/sup\u003e Eight-week-old male rats were deprived of food for 12 h. Starch (4 g [10 mL of 40 % starch solution] per kg body weight), olive oil (2.5 mL per kg body weight), or casein (2 g [12.5 mL of 16 % casein solution] per kg body weight) were administered orally to rats using the intragastric gavage technique, and their blood samples were collected from the tail vein before (0) and then 1, 2, 3, 4, and 5 h after the administration. The amounts of starch (carbohydrate), olive oil (triglyceride), and casein (protein) administered were determined according to their content in HFD (carbohydrate : triglyceride : protein = 10 : 5 : 5). Starch (191-03985) and casein (030-01505) were purchased from FujiFilm. Olive oil (Yoshida) was purchased from Yoshida Pharmaceutical Company (Tokyo, Japan). Rats with initial tail vein triglyceride levels higher than 200 mg/dL were excluded from this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntravenous\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;injection of triglycerides, fatty acids, and LPZ\u003c/strong\u003e. Intravenous injection studies were performed as previously described, with modified conditions.\u003csup\u003e1\u003c/sup\u003e Eight-week-old male rats were deprived of food for 12 h. Triglyceride emulsion (2 mL of 20% soy oil emulsion per kg body weight; Intralipos Injection 20%, Otsuka, Tokyo, Japan), C10-FFA (2 mL of 150 mM capric acid [pH 7.8] per kg body weight; D0024, Tokyo Chemical Industry, Tokyo, Japan), or LPZ (2 mL of 4 \u0026micro;M LPZ per kg body weight) was injected intravenously into fasted rats or rats fed ND \u003cem\u003ead libitum\u003c/em\u003e for 30 min followed by 2 h rest. Blood samples were collected from the tail vein before (at 0 h [fasted] or at -2.5, -2, -1, 0 h [ND fed]) and at 0.25, 0.5, 1, 2, and 3 h after the injection. Rats with initial tail vein triglyceride levels \u0026gt; 200 mg/dL were excluded from this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gastric estrogen production assay.\u0026nbsp;\u003c/strong\u003eGastric gland epithelia were isolated as previously described with slight modifications.\u003csup\u003e1\u003c/sup\u003e Male rats aged 11-15 weeks were deprived of food for 4 h. Stomachs removed from rat anesthetized with isoflurane were opened along the greater curvature and washed with ice-cold phosphate-buffered saline (PBS). After removal of the serosal muscle, the fundic region was cut into \u0026lt; 5 mm pieces and shaken in the chelating buffer (5 mM EDTA in PBS) supplemented with Tosyl-L-lysyl-chloromethane hydrochloride (TLCK, 147 ng/mL, 200-20141, FujiFilm) for 2 h on ice. The supernatant was changed with the dissociation buffer (54.9 mM D-sorbitol and 43.4 mM sucrose in PBS), and the tube was shaken vigorously for 2 min to dissociate epithelia from the mucosa. After centrifugation at 160 g for 10 min at 4 \u0026ordm;C, the pellet was dissolved in DMEM (044-32955, FujiFilm). After filtration through a 100 \u0026micro;m cell strainer (VCS-100, As One), the isolated gastric gland epithelia were incubated in DMEM supplemented with testosterone (20 nM, T-1500, Sigma), lauric acid (C12-FFA, 500 \u0026micro;M, L0016, Tokyo Chemical Industry) or capric acid (C10-FFA, 250, 500, 1000 \u0026micro;M, D0024, Tokyo Chemical Industry), and TLCK (147 ng/mL) for 1 hour at 37 \u0026deg;C in the presence or absence of hormone or drug. As non-estrogen (E2) producing controls, the epithelia were incubated in DMEM supplemented with testosterone and TLCK only. E2 and phospholipid (PL) levels in the epithelia with culture medium were measured, and E2 levels were normalized by PL levels (relative total E2 levels). To obtain relative E2 production levels, relative total E2 levels were subtracted with that of the non-E2 producing control. Isolated gastric gland epithelia were observed under an Eclipse Ti microscope equipped with Plan Fluor 10/0.30 lens and DS-Fi1 camera (Nikon, Tokyo, Japan). Image processing was performed with ImageJ 2 2.3.0 (National Institutes of Health).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of triglycerides, glucose, fatty acids, estrogen, and phospholipid concentrations\u003c/strong\u003e. Measurement of triglyceride, estrogen, and phospholipid concentrations were performed as previously described.\u003csup\u003e1\u003c/sup\u003e Plasma glucose and fatty acids concentrations were measured using a LabAssay Glucose and a NEFA C (FujiFilm) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and reproducibility.\u0026nbsp;\u003c/strong\u003eData are mean \u0026plusmn; s.d. \u003cem\u003eP\u003c/em\u003e values of different two groups were determined by two-sided Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. \u003cem\u003eR\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e values determined by Pearson\u0026rsquo;s product-moment correlation with 95% density ellipse. Statistical analyses were performed using JMP Pro ver. 16 (SAS Institute Japan, Tokyo, Japan). \u003cem\u003eP\u003c/em\u003e values \u0026lt; 0.05 were considered to be significant. \u003cem\u003eR\u003c/em\u003e values \u0026gt; 0.5 and \u0026lt; -0.5 were considered to be positively and negatively correlated, respectively. Every experiment was performed multiple times with essentially the same results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReporting summary.\u003c/strong\u003e Further information on research design is available in the Nature Research Reporting Summary linked to this article.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe source data for the Figures are available in Supplementary Data 1-4. All other data that support the findings of this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e We thank Yuta Yamamoto, Naoko Yamagishi and Keiko Ikemoto from the Kanai Laboratory for their assistance. This study was supported by Medical Research Support from Wakayama Medical University (Y.K.). We also acknowledge proofreading and editing by Benjamin Phillis at the Clinical Study Support Center at Wakayama Medical University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions.\u003c/strong\u003e Y.K. conceived the project, designed the experiments, interpreted the results, wrote the manuscript, and prepared the figures. Y.O. and A.T. found the postprandial decrease in blood E2 levels. T.I. performed \u003cem\u003ein vivo\u003c/em\u003e experiments using rats. Y.K. and T.I. performed \u003cem\u003ein vitro\u003c/em\u003e experiments using isolated gastric gland epithelia. T.I. performed statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;interests.\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material available at\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u003c/strong\u003e should be addressed to Yoshimitsu Kanai.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eOnline content\u003c/h3\u003e\n\u003cp\u003eAny methods, Nature Portfolio reporting summaries, source data, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data are available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://\u003c/span\u003e\u003cspan address=\"https://\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIto, T., Yamamoto, Y., Yamagishi, N. \u0026amp; Kanai, Y. 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Lipid Res.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 1355\u0026ndash;1366 (1993).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4567800/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4567800/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGastric parietal cells (gastric acid secreting cells) secrete estrogen in response to blood lipid (triglycerides and fatty acids) levels.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Estrogen decreases blood lipids by suppressing appetite, lipogenesis and lipolysis and by increasing lipid storage and consumption,\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e so gastric estrogen plays an important role in maintaining blood lipids levels.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e However, parietal cells use fatty acids to generate energy for estrogen production and acid secretion, so postprandial changes in blood lipid levels and acid secretion activities could affect production of gastric estrogen. Here we show that blood estrogen decreases after meals, especially after carbohydrate intake. Blood fatty acids also decrease, and intravenous injection of lipids partially restores blood estrogen levels. Gastric acid-secreting hormones decrease production of gastric estrogen, while antacid and gastric acid-suppressing hormones, including those secreted after lipid ingestion like glucagon-like peptide-1 (GLP-1),\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e increase gastric estrogen production and postprandially decreased blood estrogen levels. Secreted insulin and gastric estrogen directly enter the liver to enhance and suppress lipogenesis respectively,\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e before diluted in the systemic blood. We therefore conclude that diet and the subsequently secreted hormones regulate gastric estrogen production, as well as insulin secretion, for proper hepatic lipogenesis, taking into account ingested carbohydrate and lipid levels.\u003c/p\u003e","manuscriptTitle":"Carbohydrate intake and acid secretion decrease gastric estrogen secretion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-01 22:29:02","doi":"10.21203/rs.3.rs-4567800/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"10e09fb4-7594-49a3-ad7a-e7847ad73af0","owner":[],"postedDate":"August 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T22:29:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-01 22:29:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4567800","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4567800","identity":"rs-4567800","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}