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| ORIGINAL ARTICLE |
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| Year : 2020 | Volume
: 63
| Issue : 3 | Page : 128-136 |
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Glucose reduces the osmopressor response in connection with the tyrosine phosphorylation of focal adhesion kinase in red blood cells
You-Hsiang Chu1, Ying-Hsuan Tai2, Chun-Chang Yeh3, Mei-Yung Tsou4, Herng-Sheng Lee5, Shung-Tai Ho6, Min-Hui Li7, Tso-Chou Lin3, Chih-Cherng Lu8
1 Department of Anesthesiology, Taipei Veterans General Hospital; Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
2 Department of Anesthesiology, Taipei Veterans General Hospital; School of Medicine, National Yang-Ming University, Taipei; Department of Anesthesiology, Shuang Ho Hospital, Taipei Medical University, New Taipei City; Department of Anesthesiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
3 Department of Anesthesiology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan
4 Department of Anesthesiology, Taipei Veterans General Hospital; School of Medicine, National Yang-Ming University, Taipei, Taiwan
5 Department of Pathology and Laboratory Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
6 Department of Anesthesiology, Taipei Veterans General Hospital, Taipei; Department of Anesthesiology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
7 Department of Physical Medicine and Rehabilitation, Kaohsiung Veterans General Hospital, Kaohsiung; Institute of Aerospace Medicine, National Defense Medical Center, Taipei, Taiwan
8 Department of Anesthesiology, Taipei Veterans General Hospital; Graduate Institute of Life Sciences, National Defense Medical Center; Department of Anesthesiology, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan
| Date of Submission | 15-Apr-2020 |
| Date of Decision | 23-May-2020 |
| Date of Acceptance | 28-May-2020 |
| Date of Web Publication | 23-Jun-2020 |
Correspondence Address:
Prof. Chih-Cherng Lu
Department of Anesthesiology, Taipei Veterans General Hospital, No. 201, Sec. 2, Shipai Road, Beitou District, Taipei
Taiwan
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/CJP.CJP_32_20
Glucose ingestion attenuates the water ingestion-induced increase in the total peripheral vascular resistance and orthostatic tolerance. We investigated the gastrointestinal physiology of glucose by examining the effect of glucose ingestion on the functional expression of focal adhesion kinase (FAK) in red blood cell (RBC) membrane. This study was performed in 24 young, healthy subjects. Blood samples were collected at 5 min before and 25 min and 50 min after an ingestion of 10% glucose water 500 mL, water 500 mL, or normal saline 500 mL. We determined glucose and osmolality in plasma, and phosphorylation of aquaporin 1 (AQP1), glucose transporter 1 (Glut1), and FAK in RBC membrane. Our results showed that glucose ingestion reduced the rise of peripheral vascular resistance after water ingestion and upregulated the serine phosphorylation of Glut1. It also lowered both the serine phosphorylation of FAK and tyrosine phosphorylation of AQP1, compared with the ingestion of either water or saline. In an ex vivo experiment, glucose activated the Glut1 receptor and subsequently reduced the expression of FAK compared with 0.8% saline alone. We concluded that glucose activates Glut1 and subsequently lowers the functional expression of FAK, a cytoskeleton protein of RBCs. The functional change in the RBC membrane proteins in connection with the attenuation of osmopressor response may elucidate the pathophysiology of glucose in postprandial hypotension.
Keywords: Focal adhesion kinase, glucose, osmopressor, red blood cells
How to cite this article:
Chu YH, Tai YH, Yeh CC, Tsou MY, Lee HS, Ho ST, Li MH, Lin TC, Lu CC. Glucose reduces the osmopressor response in connection with the tyrosine phosphorylation of focal adhesion kinase in red blood cells. Chin J Physiol 2020;63:128-36 |
How to cite this URL:
Chu YH, Tai YH, Yeh CC, Tsou MY, Lee HS, Ho ST, Li MH, Lin TC, Lu CC. Glucose reduces the osmopressor response in connection with the tyrosine phosphorylation of focal adhesion kinase in red blood cells. Chin J Physiol [serial online] 2020 [cited 2023 May 29];63:128-36. Available from: https://www.cjphysiology.org/text.asp?2020/63/3/128/287453 |
| Introduction | |  |
Water ingestion-induced osmopressor response is typically characterized by increased peripheral vascular resistance in young, healthy subjects, which is effective in decreasing the symptomatic burden of vasovagal reactions.[1],[2] In contrast, glucose ingestion exacerbates the presyncope symptoms in young, healthy subjects undergoing head-up tilt table testing. Glucose ingestion significantly reduces blood pressure in patients with baroreflex failure, slightly reduces blood pressure in elderly subjects, and induces tachycardia in young healthy subjects.[3],[4]
Postprandial hypotension, defined as a decrease in systolic blood pressure (SBP) ≥20 mmHg, occurring within 2 h of the end of a meal,[5],[6] commonly affects those with an autonomic dysfunction, especially the elderly and diabetic patients. Postprandial hypotension may lead to syncope, angina, and even stroke.[7],[8] It has been proposed that stomach modulates the hypotensive response to a high-carbohydrate meal through controlling the absorption rate of glucose in the small intestine.[9],[10] However, the pathophysiological mechanism of postprandial hypotension is still poorly understood.
There is a great concern about the long-term effect of sugar-sweetened beverages on health, but their immediate effect on cardiovascular system is relatively unexplored.[11],[12] Our previous study demonstrated that glucose reduced the water ingestion-induced orthostatic tolerance and abolished water-induced osmopressor response.[4] Recent report showed that aquaporin 1 (AQP1) in the red blood cell (RBC) membrane was phosphorylated at tyrosine 253 after water ingestion and abolished by glucose ingestion.[13] A study of human-induced pluripotent stem cells demonstrated that hyperglycemia-related hyperosmolarity upregulated the expression of AQP1.[14]
Glucose transporter 1 (Glut1) accounts for 10% of the total protein mass in the RBC membrane.[15] Glut1 is the main functional transporter of glucose.[16] Moreover, it also transports L-dehydroascorbic acid, the two-electron oxidized intermediate of ascorbic acid, and transmits extracellular signals of glucose to the cell interior, where multiple signaling pathways are activated with a myriad of cellular effects.[17],[18] The activation of Glut1 is associated with its phosphorylation on serine 226.[19] The activation of Glut1 may play an important role in regulating the physiology of RBCs following glucose intake. In addition, the functional expression of focal adhesion kinase (FAK), a cytoskeletal protein of RBCs, is highly involved in the regulation of RBC deformability.[20] However, there is no strong evidence implicating such a function of Glut1 in the RBC membrane and signaling pathways is associated with RBC deformation. Thus, we supposed that glucose activates Glut1 and regulates FAK in the RBCs, which may clarify the cardiovascular physiology of glucose ingestion.
This study was intended to examine the expression state of proteins in the RBCs under hyperglycemic environment and the cardiovascular physiology of glucose intake, which counteracts the osmopressor response of water ingestion. The primary aim was to examine how glucose acts through RBC membrane receptors to transmit extracellular signals to the cell interior, which may elucidate how glucose attenuates orthostatic tolerance. The second aim was to elucidate the molecular mechanism of FAK in the signal transduction of RBC membrane after glucose ingestion. Finally, we also analyzed the functional changes in RBC membrane protein under different osmotic and glycemic environments in vitro. Accordingly, we hypothesized that an ingestion of glucose water abolishes osmopressor response to water ingestion through modulating tyrosine phosphorylation of AQP1 and functional expression of FAK in the RBC membrane.
| Materials and Methods | | |
This study protocol was approved by the Institutional Review Board of Tri-Service General Hospital (IRB-TSGH No. 1-101-05-117) and Taipei Veterans General Hospital in Taiwan (IRB-TPEVGH No. 2014-05-003 ACF and 2015-02-002 AC). The experiment was performed strictly in accordance with the relevant guidelines and ethical regulations.
Study subjects and protocol
We enrolled 24 healthy, young adults with no history of syncope or any current use of over-the-counter medications. All adults gave written informed consent in advance. The volunteers did not ingest any food or water after midnight until the test session in the morning. We utilized a randomized crossover design, including the ingestion of 10% glucose water (D10W) 500 mL, water 500 mL, or normal saline (NS) 500 mL in a quiet and comfortable room during 3 consecutive days.
Instrumentation
We measured the hemodynamic parameters using Finometer® (Finapres Medical Systems, FMS, Arnhem, Netherlands), including heart rate (HR), SBP, diastolic blood pressure (DBP), stroke volume (SV), cardiac index (CI), and total peripheral vascular resistance (TPR).[21] One-minute average of the hemodynamic parameters was collected from the continuous recording of Finometer®. Skin blood flow was measured in the other hand by a laser Doppler flowmeter (DRT4 Instrument, Moor Instruments, Devon, UK).
Blood collection and glucose detection
Blood was collected and stored in K2 ethylenediaminetetraacetic acid (EDTA)-containing tubes (Becton Dickinson, NJ, USA) for protein extraction and in red blood collection tubes for serum osmolality measurement by Advanced® Instruments Model 3900 (Advanced Instruments, Norwood, MA, USA).[5],[9] RBCs were isolated by Ficoll Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). To measure the level of blood glucose, a sample of whole blood 2 mL from each subject was placed on a check strip and was measured with an EASY TEST Blood Glucose Meter®. All samples were tested in triplicate to calculate the mean value.
Phosphorylation assay of glucose transporter 1 receptor, aquaporin 1, and focal adhesion kinase
The level of AQP1 phosphorylation was detected by an immunoprecipitation assay. The method of AQP1 immunoprecipitation has been described in our previous article.[13] The level of Glut1 phosphorylation was also detected by immunoprecipitation assay. We used specific antibodies, anti-phospho-FAK (Tyr397)/anti-FAK (#8556 and #3285, Cell Signaling Technology, MA, USA), to detect total and phosphorylated FAK proteins (Tyr397) and antibodies, Abcam and ThermoPierce (Abcam, Cambridge, UK; Thermo Fisher Scientific, MA, USA), to detect total and phosphorylated Glut1. Specific bands from an immunoblotting reaction were visualized using the Enhanced Chemiluminescence System (Millipore, MA, USA). The intensity of each band was analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Ex vivo treatment under different osmotic and glycemic environments
We used an ex vivo model under hyperglycemic environment to determine the effect of glucose on RBC membrane proteins, including Glut1, AQP1, and FAK. A blood sample was collected from participants in EDTA-containing tubes before water ingestion and incubated under different conditions at 37°C. We also added mannitol to evaluate the independent effect of glucose on the expression of RBC membrane proteins. To mimic hypo-osmotic, iso-osmotic, and hyperosmotic and hyperglycemic environments, 0.8% saline, NS 400 mL, D10W 400 mL, and mannitol water 400 mL were added to equal volumes of blood, respectively.
Statistical analysis
Given a type I error rate of 0.05 and power of 0.8, a sample size of 24 was estimated to detect an effect size of 0.8 by a paired t-test with a two-sided significance level of 0.05. The hemodynamic data were further classified into nine subgroups based on the intake solutions (water, D10W, and NS) and time frames (baseline, 25 min, and 50 min). ANOVA tests were used to determine the significant difference in the outcome variables across subgroups. Post hoc pairwise comparisons were performed using the Bonferroni correction. A repeated-measure ANOVA was used to assess the changes in Glut1 receptor, AQP1, and FAK phosphorylation between the baseline and 25 min or 50 min after solution ingestion among the three groups. We considered P < 0.05 as statistically significant. All the statistical analysis was conducted using SPSS Version 18.0 (SPSS Inc., Chicago, IL, USA). Scientific graphing was performed using Prism 5.0 (GraphPad Software, CA, USA).
| Results | | |
Hemodynamic variables
[Table 1] shows the demographic data and baseline hemodynamic variables of the subjects. Compared with baseline values, HR decreased significantly from 70.5 ± 10.8 bpm to 66.1 ± 8.8 and 67.1 ± 9.5 bpm at 25 and 50 min after water ingestion, respectively (P = 0.001). However, no difference was observed after solution ingestion in the D10W and NS groups. Post hoc pairwise comparisons with Bonferroni's correction revealed no significant difference in HR at 25 or 50 min, compared with baseline, regardless of type of ingested solution. CI significantly decreased from 3.12 ± 0.37 L·min−1·m−2 to 2.87 ± 0.41 and 2.89 ± 0.50 L·min−1·m−2 at 25 and 50 min after water ingestion, respectively. In contrast, CI significantly increased from 3.05 ± 0.60 L·min−1·m−2 to 3.37 ± 0.56 and 3.14 ± 0.57 L·min−1·m−2 at 25 and 50 min after D10W ingestion (P = 0.001). TPR significantly increased from 1443 ± 186.6 dyne·s·cm−5 to 1858 ± 333.9 and 1804 ± 264.6 dyne·s·cm−5 at 25 and 50 min after water ingestion, respectively. There was no significant change in hemodynamics in D10W and NS groups, neither at 25 nor 50 min after solution ingestion [Table 2]. | Table 1: Characteristics of the 24 subjects examined 5 min before water ingestion (baseline)
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 | Table 2: Hemodynamic variables at -5 min (baseline), 25 min, and 50 min after ingestion of water, glucose, or normal saline in the same subjects (n=24)
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Serum osmolality and glucose concentration
Ingestion of water 500 mL reduced serum osmolality from 283.5 ± 3.6 mOsm·kg−1 to 279.3 ± 3.6 mOsm·kg−1 at 25 min and 280.3 ± 4.7 mOsm·kg−1 at 50 min, respectively. In either D10W or NS group, there was no significant difference in serum osmolality across the baseline, 25 min, and 50 min values. However, the water group was significantly different between D10W and NS groups in serum osmolality [Figure 1]a. The level of serum glucose increased significantly at 25 and 50 min after solution ingestion in the D10W group and reached a maximum at 25 min [Figure 1]b. | Figure 1: Changes of serum osmolality and glucose level. (a) Serum osmolality was measured in the same subject following ingestion of water 500 mL, normal saline 500 mL, or 10% glucose water 500 mL at baseline, 25, and 50 min after ingestion. Serum osmolality significantly decreased at 25 min after water ingestion. (b) Blood glucose significantly increased at 25 and 50 min after ingestion of 10% glucose water. In the other two groups, serum glucose was significantly decreased at 25 and 50 min after ingestion of water and normal saline. The data are presented as the mean ± standard deviation. Significant differences among the three groups were evaluated using ANOVA tests (N = 24, *P < 0.05, ***P < 0.001).
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Western blotting for aquaporin 1, glucose transporter 1, and focal adhesion kinase phosphorylation
AQP1 tyrosine phosphorylation significantly increased at 25 and 50 min after an ingestion of water but not NS or D10W [Figure 2]. Glut1 serine phosphorylation significantly increased at 25 min after D10W ingestion (1.35 ± 0.15-fold, P = 0.002) but not water or NS [Figure 3]. FAK tyrosine phosphorylation (Tyr397) significantly decreased (0.76 ± 0.09-fold, P = 0.001) at 25 min after D10W ingestion but not NS. There was an inverse correlation between the levels of FAK tyrosine phosphorylation and Glut1 serine phosphorylation at 25 min after D10W ingestion (R2 = 0.7389) [Figure 3]. | Figure 2: Phosphotyrosine of aquaporin 1 in red blood cell membrane. (a) A representative gel is shown. (b) Aquaporin 1 tyrosine phosphorylation in red blood cell membrane was recognized. The level of aquaporin 1 tyrosine phosphorylation was significantly increased at 25 and 50 min after ingestion of water but not normal saline or 10% glucose water. The data are presented as the mean ± standard deviation. Significant differences among the three groups were evaluated using repeated measurements by ANOVA tests and compared with the baseline using paired t-test (N = 6, *P < 0.05, **P < 0.01).
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 | Figure 3: Phosphorylation of glucose transporter 1 and focal adhesion kinase in red blood cell membrane. (a) A representative gel is shown. (b) Glucose transporter 1 serine phosphorylation in the red blood cells under three different conditions and time points were recognized. The levels of glucose transporter 1 serine phosphorylation were significantly increased at 25 and 50 min after ingestion of 10% glucose water but not water or normal saline. (c) The level of focal adhesion kinase tyrosine phosphorylation was significantly decreased at 25 min after ingestion of glucose water. The level of focal adhesion kinase tyrosine phosphorylation was slightly increased after water ingestion and not significantly changed by ingestion of normal saline. (d) Delta focal adhesion kinase phosphorylation levels in three groups. p-FAK/focal adhesion kinase ratios were internally normalized to each group within a blot. (e) The correlation between the change of focal adhesion kinase and glucose transporter 1 at 25 min after ingestion of glucose water. The data are presented as the mean ± standard deviation. Significant differences across the three groups were evaluated using ANOVA tests and post hoc comparisons between groups were adjusted by Bonferroni correction. The comparisons between baseline and measurements at other time points were conducted using paired t-test (N = 6, **P < 0.01).
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Aquaporin 1 phosphorylation in ex vivo hyperglycemic environment
A significant decrease in osmolality was observed from 284.75 ± 0.95 mOsm·kg−1 in the control to 280.5 ± 1.73 mOsm·kg−1 (P = 0.006) at 15 min and 281.0 ± 2.30 mOsm·kg−1 at 25 min after the addition of 0.8% saline in ex vivo [Figure 4]a. No significant difference in osmolality was observed at 15 and 25 min after the addition of 10 mM glucose water. The glucose level in the blood significantly increased after the addition of glucose water, from baseline (70.1 ± 2.7 mg·dL−1) to 231.0 ± 12.0 at 15 min and 220.0 ± 21.9 mg·dL−1 at 25 min [Figure 4]b. The level of AQP1 tyrosine phosphorylation slightly increased at 5 min and reached a significant plateau at 15 min after the addition of 0.8% saline. There was no significant change in AQP1 activation following the addition of NS or glucose water at the time points of 5, 15, and 25 min (data not shown). | Figure 4: Serum osmolality and glucose changes in an ex vivo model. (a) Osmolality in the blood was analyzed in the three groups of 0.8% saline, 0.9% normal saline, and glucose water. The osmolality significantly decreased at 15 and 25 min after the addition of 0.8% saline but not normal saline or glucose water. (b) Glucose level in the blood significantly increased at 15 min and 25 min after addition of glucose water but not 0.8% saline or normal saline. The data are presented as the mean ± standard deviation. Significant differences among the three groups were evaluated using repeated measurements ANOVA tests and post hoc comparisons between groups were adjusted by Bonferroni correction. The comparisons between baseline and measurements at other time points were conducted using paired t-test (N = 6, *P < 0.05).
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Glucose transporter 1 and focal adhesion kinase phosphorylation in ex vivo hyperglycemic environment
The ex vivo level of glucose reached 231.0 ± 12.0 mg·dL−1 at 15 min and maintained at 25 min after addition of glucose water [Figure 4]b. Glut1 serine phosphorylation was significantly increased at 15 min after addition of 10 mM glucose water [Figure 5]. However, there was no significant difference in Glut1 serine phosphorylation at 15 min after treatments with 0.8% saline, NS, or hyperosmotic mannitol solution. Glut1 serine phosphorylation was significantly increased at 25 min after addition of glucose water (P = 0.01). FAK tyrosine phosphorylation (Tyr 397) significantly decreased at 15 and 25 min after addition of 10 mM glucose water (0.58 ± 0.14-fold [P = 0.0027] and 0.51 ± 0.19-fold [P = 0.0045], respectively) and significantly increased at 15 and 25 min after addition of 0.8% saline group (1.43 ± 0.23-fold [P = 0.0134] and 1.43 ± 0.26-fold [P = 0.0209], respectively) [Figure 6]. However, there was no significant change in FAK tyrosine phosphorylation after addition of NS. | Figure 5: Glucose transporter 1 serine phosphorylation level was detected in four groups of normal saline, 0.8% saline, glucose water, and mannitol water. (a) A representative gel is shown. (b) Glucose transporter 1 serine phosphorylation level was significantly increased at 15 min after addition of 10% glucose water. The level of glucose transporter 1 serine phosphorylation was not significantly changed by the addition of normal saline, 0.8% saline, or mannitol water. The data are presented as the mean ± standard deviation. The comparisons between baseline and measurements at other time points were conducted using paired t-test (N = 5, *P < 0.05).
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 | Figure 6: The phosphorylation of focal adhesion kinase in red blood cells was detected under different environments. (a) Focal adhesion kinase tyrosine phosphorylation level was significantly increased at 15 and 25 min after addition of 0.8% saline. The level of focal adhesion kinase tyrosine phosphorylation was not significantly changed by addition of normal saline. Focal adhesion kinase tyrosine phosphorylation was significantly reduced by addition of glucose. (b) A significant correlation between changes of focal adhesion kinase phosphorylation and total peripheral vascular resistance. (c) The delta value of focal adhesion kinase phosphorylation level and total peripheral vascular resistance were calculated by measuring the gap between baseline and 25 min after ingestion. The data are presented as the mean ± standard deviation. The comparisons between baseline and measurements at other time points were conducted using paired t-test (N = 5, *P < 0.05, **P < 0.01). Focal adhesion kinase phosphorylation and total peripheral vascular resistance were highly and positively correlated (N = 24, R2 = 0.3208).
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Correlation between focal adhesion kinase phosphorylation and peripheral vascular resistance
A considerable skewed distribution was observed in the raw data of hemodynamic variables. TPR was significantly increased at 25 min after ingestion of water but not D10W or NS. The level of FAK tyrosine phosphorylation was significantly decreased after D10W ingestion and increased after water ingestion. The level of FAK tyrosine phosphorylation was highly and positively correlated with TPR at 25 min after solution ingestion among three groups (R2 = 0.3208) [Figure 6]b.
| Discussion | | |
There are three major findings in this study. First, glucose attenuates the osmopressor response to water ingestion. Second, the upregulation of tyrosine phosphorylation of AQP1 after water ingestion is abolished, especially after ingestion of D10W. Third, ingestion of D10W activates serine phosphorylation of Glut1 and consequently lowers the tyrosine phosphorylation of FAK in the RBC membrane compared to water ingestion alone.
Our analysis showed that the level of FAK tyrosine phosphorylation was highly positively correlated with TPR after solution ingestion. The study has showed shear stress applied on aortic endothelial cells triggered the tyrosine phosphorylation and the kinase activity of FAK.[22] In our study, ingestion of glucose water downregulated FAK phosphorylation of RBC membrane in association with the lower TPR compared to water alone, which suggests that RBCs in the peripheral blood may have a role in the regulation of vascular tone.[23] Previous studies demonstrated that water ingestion activates both the sympathetic and vagal branches in the cardiovascular autonomic system.[24],[25] Peripheral vascular resistance is in accordance with the observed increase in sympathetic vasomotor discharge after water ingestion, although the slow time course of the response could implicate a humoral mechanism.[24],[25] In young, healthy subjects, water ingestion elicited a series of cardiovascular changes, including an increase in TPR and a decrease in HR and skin blood flow, but had a little effect on blood pressure.[13],[26] Water ingestion raises blood pressure in patients with perturbed autonomic control, thereby improving orthostatic tolerance and reducing the risk of presyncope or vasovagal reactions in young, healthy subjects.[27] Thus, water ingestion is widespread used as a prophylaxis against syncope under certain circumstances, such as postblood donation syncope.[25]
Our study showed water ingestion produced a maximal rise in TPR and an apparent decrease in the skin blood flow without a prominent change in blood pressure. However, NS ingestion did not induce such an osmopressor response. NS ingestion did not change HR or TPR, suggesting that the relative hypo-osmotic solution triggers this cardiovascular response. Hypo-osmolality itself is considered as a major afferent stimulus to elicit the osmopressor response. The present study demonstrated that NS ingestion attenuates the activation of AQP1 tyrosine phosphorylation and the osmopressor response induced by water alone. Moreover, the ex vivo model showed that the upregulation of AQP1 tyrosine phosphorylation in the RBC membrane could be induced by the co-administration of 0.8% saline but not NS. Bothin vivo and ex vivo models clarified the effect of water ingestion on AQP1 tyrosine phosphorylation in RBC membrane, elicited by the hypo-osmotic stimulus of ingested water. Recent study reported that the role of transient receptor potential channel 4 was also highly correlated with water-induced pressor response.[28]
Ingestion of glucose water exacerbates orthostatic tachycardia in patients with orthostatic intolerance and precipitates syncope in orthostatic hypotensive patients.[4],[25],[29] Glucose reduces the water-promoted orthostatic tolerance and exacerbates orthostatic tachycardia in head-up tilt table testing for young, healthy subjects. The thermogenesis effect of glucose is one of the possible mechanisms accounting for the slightly increased HR after glucose water ingestion.[30] Glucose somehow abrogates the vasoconstrictor response to water ingestion, which provides a study model for the cardiovascular pathophysiology of postprandial hypotension or dumpling syndrome.
In the elderly, glucose ingestion causes a fall in blood pressure, though an intravenous infusion of glucose has a little effect on blood pressure, indicating that the response is mediated primarily by the action of glucose in the gastrointestinal tract.[9] The magnitude of the postprandial fall in blood pressure appears dependent on the absorption rate of nutrients (especially glucose) in the small intestine, which may explain the action of glucose in changing splanchnic vascular flow and sympathetic nerve activity. The absorption rate of glucose in the small intestine determines the postprandial fall in blood pressure and increase in HR in healthy, elderly subjects. Previous molecular studies also showed hyperglycemia facilitated the glycosylation of hemoglobin, RBC membrane, and cytoskeleton proteins.[31] It could cause oxidative damage of RBC membrane with oxidation of spectrin in the RBC membrane or reduced enzyme activities, due to defective Na-K-ATPase pump.[31],[32] A study of cancer cells showed Glut1 expression could affect the phosphorylation of FAK via intergrin β1/Src pathway.[33] In glycosylation, oxidative stress could alter RBCs' morphology and leads to the formation of echinocytes or swollen and impaired deformability.[34] The present study demonstrated ingestion of glucose water activated Glut1 serine phosphorylation and then subsequently reduced FAK serine phosphorylation in the RBCs. The phosphorylation of Glut1 at serine 226, the primary glucose transport protein in the human RBCs, regulates glucose transport.[19]
Our experiments showed ingestion of glucose water activated serine phosphorylation of Glut1 and consequently lowered the tyrosine phosphorylation of FAK in RBC membrane. Similarly, under an ex vivo hyperglycemic environment, glucose increased the serine phosphorylation of Glut1 and consequently lowered the tyrosine phosphorylation of FAK in RBCs. These findings suggested that the effect of glucose activates the Glut1 and subsequently reduces FAK phosphorylation in RBC membrane, which proposes a possible mechanism for the autonomic cardiovascular physiology of glucose acting through the gastrointestinal tract.
The localization of FAK to the sites of integrin clustering initiates downstream signaling that could play a role in focal adhesion (FA) targeting.[35],[36],[37] FAK is a part of a hierarchical cascade that involves FA compositional changes during FA maturation. In this process, FAs grow in size and facilitate the spatiotemporal transduction of distinct biological signals.[38] FAs can control the actin cytoskeletal organization and thus drive cell migration. One of the mechanisms regulating the dynamic organization of FAs involves the activity of tyrosine-specific kinases and phosphatases within the FAs.[36],[38] Our previous study demonstrated that FAK tyrosine phosphorylation was associated with improved RBC deformability in cardiac surgery.[39] The upregulation of FAK tyrosine phosphorylation may also serve as an indicator for RBC deformability. Overall, we found that the tyrosine phosphorylation of FAK in RBCs was attenuated by glucose ingestion and hyperglycemic environment, implicating that glucose downregulates the cytoskeletal proteins in the RBCs.
RBC membrane and cytoskeletal proteins are extensively developed and mainly responsible for the resistance of RBCs against the mechanical deformation during the passage through small capillaries.[40] Recent study demonstrated that high glucose levels have deleterious effects on RBC deformability in the circulating blood and perhaps change the capability of RBCs to deliver oxygen to peripheral tissues. The effect of glucose on the functional expression of FAK in RBCs was consistent with the findings from peritoneal mesothelial cells in rats.[41] The interaction between Glut1 and FAK activity after glucose ingestion may indicate the important role of glucose in transducing FAK-mediated signals and underlies the attenuation of circulating RBC deformability induced by hyperglycemia. It also reflects changes in the RBC deformability in response to the glucose of circulating blood.[42] The insight gained from these studies has improved our understanding in the molecular mechanism underlying glucose in the human RBCs and formed the basis for future studies on RBCs. It may also provide an implication for the treatment of diabetes and cardiovascular diseases.
In conclusion, Glut1 serine phosphorylation in the RBCs was increased and FAK tyrosine phosphorylation was subsequently attenuated after glucose ingestion. Downregulation of FAK tyrosine phosphorylation in the RBCs by glucose implicates its potential effects on RBC deformability. This study demonstrated that glucose ingestion lowers the phosphorylation of FAK, which is well correlated with the reduced peripheral vascular resistance. The functional change of RBC membrane protein in connection with the attenuation of osmopressor response may elucidate the pathophysiology of glucose in postprandial hypotension. The dynamic change of FAK tyrosine phosphorylation also offers a new avenue for pharmacological therapy to protect both diabetic and nondiabetic patients against the hazards of acute glycemic surges.
Acknowledgments
We thank the Clinical Research Core Laboratory of Taipei Veterans General Hospital and Department of Medical Research of Tri-Service General Hospital, National Defense Medical Center, for the experimental space and facilities.
Financial support and sponsorship
This work was supported by the grants from the Taiwan Ministry of Science and Technology (NSC-102-2314-B-016-012-MY3), Taipei Veterans General Hospital (V103C-159 and V104C-147), and Taipei Veterans General Hospital-National Defense Medical Center Joint Research Program (DV103-7).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]
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