© 1995 American Heart Association, Inc.
Articles |
Lymphocytic Na+-H+ Exchange Increases After an Oral Glucose Challenge
From Medizinische Universitäts-Poliklinik (M.T., R.S., M.B., K.K., T.K., C.S., W.Z.) and Institut für Klinische Chemie und Laboratoriumsmedizin (M.W.), University of Münster (Germany), and Medizinische Klinik und Poliklinik (C.M., R.G.B.), University of Giessen (Germany).
Correspondence to Prof W. Zidek, Med. Univ.-Poliklinik, Albert-Schweitzer-Str. 33, D-48129 Münster, Germany.
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Abstract |
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Abstract The effects of oral glucose challenge on plasma glucose concentration, plasma insulin concentration, arterial blood pressure, cytosolic pH (pHi), cytosolic free Na+ concentration ([Na+]i), and cellular Na+-H+ exchange activity were investigated in 16 healthy subjects. The pHi, [Na+]i, and Na+-H+ exchange activity were measured in intact lymphocytes by using the fluorescent dye technique. The oral glucose challenge significantly increased plasma glucose, plasma insulin, and the lymphocytic Na+-H+ exchange activity, measured as change of pHi per second (control [0 hours], 5.20±0.53x10-3 dpHi/s; 1 hour after glucose administration, 8.28±1.07x10-3 dpHi/s; 2 hours after glucose administration, 8.15±1.18x10-3 dpHi/s; P=.002). The lymphocytic Na+-H+ exchange was significantly correlated with plasma glucose concentration (r=.357, P=.041). During steady state euglycemic hyperinsulinemic clamp, the Na+-H+ exchange activity was not significantly changed compared with baseline values. The study shows that changes of blood glucose levels can induce an acute increase in Na+-H+ exchange activity. Systolic blood pressure and Na+-H+ exchange activity were significantly (P<.001) but weakly correlated during an oral glucose challenge.
Key Words: Na+-H+ exchange • glucose • insulin
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Introduction |
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The concurrence of hypertension with hyperinsulinemia and insulin resistance is noted in a significant portion of patients with essential hypertension,1 2 3 but the relation between the metabolic and hemodynamic abnormalities still remains obscure. Several actions of insulin, such as Na+ retention,4 5 6 stimulation of Ca2+ transport,7 8 9 and increased sympathetic activity10 have been suggested to link hypertension with hyperinsulinemia and/or insulin resistance.
The Na+-H+ exchange is known to be stimulated by insulin in red blood cells and skeletal muscle11 12 as well as in the condition of essential hypertension.13 14 Accordingly, in diabetic rats a decreased Na+-H+ exchange activity was described.15 Although the stimulatory effect of insulin on Na+-H+ exchange has been demonstrated in vitro, there is no experimental evidence of this relation in humans. Therefore, in the present study the effects of a physiological increase of insulin concentration after oral glucose challenge on cytosolic pH (pHi), cytosolic free Na+ concentration ([Na+]i), and activity of the Na+-H+ exchange were investigated in healthy subjects during the oral glucose tolerance test (OGTT). The results indicate that the oral glucose challenge is followed by increased levels of plasma glucose and insulin and increased cellular Na+-H+ exchange activity. Furthermore, the Na+-H+ exchange activity is weakly, but significantly, correlated with the systolic blood pressure.
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Subjects and Methods |
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Subjects
Sixteen healthy men (age, 30.4±1.2 years; body mass index, 24.5±0.7 kg/m2) volunteered for the study. None of the subjects had hypertension, diabetes mellitus, or gastrointestinal, hepatic, or renal disease. No subject had taken any medication for at least 8 weeks before the study. All subjects were nonsmokers and had a diastolic blood pressure of <90 mm Hg and a systolic blood pressure <160 mm Hg, obtained by repeated conventional sphygmomanometric measurements with patients in the sitting position. None had a familial history of non–insulin-dependent diabetes mellitus. All participants gave their informed consent. The study was approved by the local ethical committee.
OGTT and Blood Pressure Measurements
In the 16 subjects, an OGTT was performed with
simultaneous determination of plasma glucose and plasma
insulin concentrations. Simultaneously, blood pressure,
pHi, activity of the Na+-H+
exchange, and [Na+]i in lymphocytes were
measured immediately before (0 hours) and 1 and 2 hours after oral
glucose intake. Control studies were performed in these subjects on a
separate day under otherwise identical circumstances; eg, plasma
glucose and plasma insulin levels, blood pressure,
pHi, activity of the Na+-H+
exchange, and [Na+]i in lymphocytes were
measured after an overnight fast at 0, 1, and 2 hours by using the same
protocol without the administration of oral glucose.
The OGTT was performed according to the recommendations of the National
Diabetes Data Group.16 The OGTT started at 8:00
AM after at least 3 days of unrestricted diet (
150 g
carbohydrate intake) and physical activity. The subjects had fasted for
at least 10 but no more than 16 hours; water was permitted during this
period. The OGTT took place in a quiet room with constant temperature.
A polytetrafluoroethylene (Teflon) cannula
was inserted into an antecubital vein. The subjects were in the
recumbent position throughout the test. After a fasting blood sample
was collected, 100 g glucose in 400 mL flavored water
(Boehringer) was drunk in 
3 minutes, and blood samples were
collected at 60-minute intervals for 2 hours after the glucose intake.
Under control conditions, 400 mL water was ingested without glucose.
Glucose concentrations were determined by using a glucose
analyzer (Boehringer). None of the subjects met or
exceeded the plasma glucose concentrations after a 100 g glucose
challenge recommended for the diagnosis of diabetes mellitus in adults.
Plasma insulin concentrations were analyzed by radioimmunoassay
(Incstar Corp).
The blood pressure was measured in the recumbent subject over a
10-minute period at 0, 1, and 2 hours after oral glucose intake with an
automatic device (Finapres, Ohmeda). By using the
finger-plethysmographic technique, 600 readings of the
systolic and diastolic blood pressure were
collected during the 10-minute period and averaged.
Euglycemic Hyperinsulinemic Clamp
Technique
Euglycemic hyperinsulinemic clamp
studies were performed after an overnight fast of 10 to 16 hours, and
the fast was maintained until the end of the studies according to
previously described methodology.1 17 18
The subjects (n=4) lay supine in a quiet room with constant
temperature. Between 7:00 and 7:30 AM, an
intravenous catheter was placed in an antecubital vein for
infusion of glucose and insulin. Another catheter was placed
retrogradely in a dorsal wrist vein of the contralateral arm, which was
surrounded by a thermoregulated Plexiglas box (65°C) for blood
sampling. Use of the box ensures arterialization of venous
blood within 20 to 40 minutes.19 After a 60-minute
equilibration period, blood samples were obtained every 30 minutes
until the end of the study.
A primed continuous infusion of insulin (Hoechst) was administered at a
rate of 1.2 mU/kg body wt per minute for 2 hours. An infusion of 20%
glucose was begun to maintain the plasma glucose concentration at 90
mg/dL throughout.
Blood glucose was assayed every 5 minutes by a glucose analyzer. Plasma insulin was measured by radioimmunoassay as described above. The lymphocytic Na+-H+ exchange was measured during the baseline period and at steady state euglycemic hyperinsulinemia, ie, 2 hours after the start of the insulin infusion.
Preparation of Lymphocytes
Lymphocytes were obtained according to established
techniques.20 21 Briefly, 20 mL heparinized blood was
drawn by venipuncture from the antecubital vein and
centrifuged at 240g for 15 minutes. After removing
the supernatant, lymphocytes were isolated by layering 5 mL of diluted
blood (1:1 [vol/vol] with isotonic NaCl) on 3 mL of
Lymphoprep (5.6% [wt/vol] Ficoll, Boehringer; density, 1.077
g/mL) and centrifugation at 240g for 20
minutes. The lymphocyte interphase was carefully aspirated, washed
three times in isotonic NaCl by centrifugation at
400g for 5 minutes, and resuspended in HBSS containing
(mmol/L) NaCl 136, KCl 5.4, KH2PO4 0.44,
Na2HPO4 0.34, CaCl2 1,
D-glucose 5.6, and HEPES 10, pH 7.4. The cell preparation
consisted of 22±1% monocytes and 78±1% lymphocytes (83±2% T
lymphocytes and 17±6% B lymphocytes) as revealed by FACScan (Becton
Dickinson) using fluorescein isothiocyanate–labeled or
phycoerythrine-labeled monoclonal antibodies (Dianova).
Measurement of pHi in Lymphocytes
Measurements of pHi were performed according to
established methodology using a pH-sensitive fluorescent
dye.22 23 24 A stock solution of the pH-sensitive
fluorescent dye BCECF-AM (Sigma Chemical Co) at a final
concentration of 1 mmol/L was prepared in dimethyl sulfoxide. The
lymphocyte counts were adjusted to 106 cells/mL. The
lymphocyte suspension (106 cells/mL) was incubated
with 10 µmol/L of cell-permeant BCECF-AM for 15 minutes at
37°C. After centrifugation at 240g for 15
minutes to remove extraneous dye, the lymphocyte pellet was again
resuspended in HBSS. This procedure efficiently removes extraneous dye.
The leakage of dye during the experiment was <5%, as confirmed by
fluorescence measurements of the supernatant after
centrifugation of the lymphocyte suspension. The
fluorescence intensity of a 1000-µL suspension of
BCECF-loaded lymphocytes was measured in a thermostated quartz cuvette
with constant stirring in a fluorescence spectrophotometer
(model F-2000, Hitachi Ltd) by use of a ROM board (model 251-0250,
Hitachi Ltd). The light source used was a 150-W xenon lamp with ozone
self-dissociation function. Monochromators were large stigmatic
concave gratings (having 900 lines per millimeter) used on both
excitation and emission sides. The wavelength drive motors and slit
control motors were operated by the computer. The wavelength accuracy
was >±5 nm. Output signals from the monitor detector and
fluorescence detector (photomultiplier) were processed via the
A/D converter. Data sampling interval was 0.5 second, with excitation
wavelengths of 440 nm (representing the isosbestic
pH-insensitive fluorescence excitation wavelength of BCECF) and
495 nm (representing the pH-sensitive fluorescence
excitation wavelength of BCECF; bandwidth, 10 nm), and the emission
wavelength was 530 nm (bandwidth, 10 nm). The
autofluorescence of lymphocytes was measured before the
addition of the fluorescent dye and represented
<1% of the fluorescence of BCECF-loaded lymphocytes.
At each lymphocyte preparation, the fluorescence intensities were converted to estimates of pHi by using the high-K+/nigericin method described by Thomas et al.25 Briefly, lymphocytes were resuspended in a buffer solution in which NaCl had been replaced by KCl (final concentration, 141.4 mmol/L) containing 5 µmol/L nigericin. The calibration was performed by sequential titrations with 0.1 mol/L potassium hydroxide while recording fluorescence intensity and pHo. The calibration curve was linear at pHi 6.3 to 7.9, with correlation coefficient values >.95.
The Na+-H+ exchange was activated by the addition of sodium propionate solution (final concentration, 100 mmol/L) from a 1 mol/L stock solution, pH 7.4. The undissociated propionic acid permeates the cell membrane and acidifies the cytosol, thereby activating Na+-H+ exchange. No changes of the fluorescence intensity after the addition of propionic acid occurred at the pH-insensitive fluorescence excitation wavelength of 440 nm. In control experiments, 100 mmol/L NaCl was added; this addition did not change the pHi. The recovery of pHi was inhibited in the absence of extracellular Na+, where Na+ had been replaced by choline, indicating that the recovery of pHi after intracellular acidification is mediated by the Na+-H+ exchange. Rate constants of the pHi recovery after intracellular acidification were obtained through iterative curve fitting of the experimental data to the following model: pHi,t=pHi,x-(pHi,x-pHi,0)xe-kt, where pHi,t is pHi at a given time t, pHi,x is pHi at the new steady state, pHi,0 is the initial pHi at the moment of activation of the Na+-H+ exchange, and k is the rate constant. The rate of pHi recovery after intracellular acidification was computed by using the software GRAPHPAD-INPLOT 4.03 (GraphPad Software Inc) and expressed as change of pHi (dpHi) per second. Control experiments showed that a concentration of 100 mmol/L propionic acid produces maximum activation of the Na+-H+ exchange in lymphocytes. Therefore, the initial rates of pHi recovery after maximum stimulation of lymphocytes by 100 mmol/L propionic acid were used for the comparisons.
Measurements of [Na+]i in
Lymphocytes
Fluorescence measurements of
[Na+]i in intact lymphocytes were performed
by using Na+-binding
benzofuran-isophthalate-acetoxymethyl ester (SBFI-AM,
Calbiochem) according to recently described
methodology.26 27 28 29 30 Briefly, a stock solution of SBFI-AM
(final concentration, 1 mmol/L) was prepared in dimethyl sulfoxide. To
5 mL of lymphocyte suspension (106 cells per
milliliter), 30 µL of the membrane-permeant SBFI-AM at a final
concentration of 6 µmol/L and 5 µL of the nonionic detergent
Pluronic F-127 (Molecular Probes) at a final concentration of 0.1%
[wt/vol] were added. Lymphocytes were incubated with the dye for 60
minutes at 37°C. After centrifugation at
400g for 5 minutes to remove extraneous dye, the lymphocytes
were resuspended in fresh HBSS, pH 7.4. After loading, a cell viability
of >95% was observed by trypan blue exclusion. The
fluorescence intensity of a 1000-µL suspension of SBFI-loaded
lymphocytes (106 cells per milliliter) in a
thermostated quartz cuvette with constant stirring was measured in a
fluorescence spectrophotometer (model F-2000, Hitachi Ltd). The
data sampling interval was 0.5 seconds, with alternate excitation
wavelengths of 340 and 385 nm (bandwidth, 10 nm), and emission was
collected at 500 nm (bandwidth, 10 nm). The fluorescence
intensity at 340- and 385-nm excitation decreased by 10% within
1000 seconds. The decrease in fluorescence had been attributed
to bleaching and leakage of the fluorescent dye.31
In contrast, the fluorescence excitation ratio at 340 and 385
nm (F340 nm/F385 nm) was calculated and remained constant during the
measurements. The autofluorescence of lymphocytes was >110
times lower than the fluorescence of lymphocytes loaded with
SBFI. The autofluorescence was subtracted from the observed
fluorescence values before the fluorescence excitation
ratio was calculated. For assessment of leakage of SBFI from the cells,
the lymphocyte suspension was centrifuged at
11 000g for 3 minutes, and the fluorescence
intensity of the supernatant was measured and subtracted from the
observed fluorescence values of the lymphocyte suspension. The
fluorescence intensity of the supernatant was only <6% of the
fluorescence intensity of the whole lymphocyte suspension.
Since intracellular microviscosity may contribute to
fluorescence intensity of intracellular SBFI, the calibration
of the F340 nm/F385 nm ratio in terms of
[Na+]i was performed in situ on each
lymphocyte preparation.27 28 29 30 32 SBFI-loaded lymphocytes
were added to solutions of known [Na+]o that
were made by appropriate mixtures of high-Na+ and
high-K+ solutions in the presence of 5 µmol/L monensin
and 5 µmol/L nigericin (Sigma). When equilibrations of
[Na+]i and [Na+]o
were performed by 1 µmol/L gramicidin (Sigma), similar results were
obtained. The high-Na+ solution contained (mmol/L) sodium
gluconate 110, NaCl 30, CaCl2 1.2, MgCl2 0.6,
and sodium HEPES 10. The high-K+ solution was identical
except for complete replacement of Na+ by K+.
With increasing [Na+]i, an increase of
the F340 nm/F385 nm ratio could be observed. Therefore, the
fluorescent dye SBFI in lymphocytes responds sufficiently to
changes in [Na+]i, as was confirmed by
recent literature.27 31 The dye trapped in organelles
should be nearly constant, so that the perturbing effect of
compartmentation of the intracellular trapped SBFI is eliminated by the
in situ calibration.27 Treatment of the lymphocytes with
the detergent digitonin released 95% of the fluorescence,
indicating that no significant dye compartmentation had taken place.
Day-to-day intrasubject variation of
[Na+]i in resting lymphocytes was 4%, as
tested in 5 subjects.
Statistics
All data are presented as mean±SEM. Where error bars do
not appear on figures, errors are within the symbol size. The original
tracings shown in the figures were superimposed by using the graphic
software GRAPHPAD-INPLOT 4.03. Statistical analysis
was performed by using the SPSS FOR WINDOWS 5.01 software
package (SPSS Inc). For statistical evaluation of the data, Friedman’s
two-way ANOVA was used, and two-tailed values of
P<.05 were considered to be significant. The relation
between selected variables was tested by performing linear
regression.
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Results |
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Effects of Oral Glucose Challenge on Plasma Glucose and Plasma Insulin
After the administration of 100 g glucose, the plasma glucose concentration in 16 healthy subjects was elevated from 72±5 to 128±11 mg/dL (1 hour after administration, P<.01) and then dropped to 115±9 mg/dL (2 hours after administration, P<.01; Fig 1
). The changes of plasma glucose concentrations after
the administration of glucose were significant, as determined by
Friedman’s two-way ANOVA (
2 test, 21.4;
P=.0001). In parallel with the increase of plasma glucose
concentration, a significant increase of plasma insulin concentration
could be observed (2 test, 24.8;
P=.0001; Fig 1). Under control conditions, there were no
significant changes of plasma glucose concentrations or plasma insulin
concentrations within 2 hours.
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). Values represent the mean±SEM from 16
subjects. After the administration of glucose, the plasma glucose
concentrations (P=.0001) and plasma insulin concentrations
(P=.0001) increased significantly, as determined by
Friedman’s two-way ANOVA. Under control conditions, no significant
changes of plasma glucose concentration of plasma insulin concentration
could be observed. **P<.001 compared with baseline
values.
Effects of Oral Glucose Challenge on pHi,
Na+-H+ Exchange, and
[Na+]i
The changes of lymphocytic pHi,
Na+-H+ exchange activity, and lymphocytic
[Na+]i were measured before (0 hours) and 1
and 2 hours after the administration of 100 g glucose. The
Na+-H+ exchange activity was determined by
using the pHi recovery after intracellular acidification by
sodium propionate. The initial rates of pHi recovery after
maximum stimulation of lymphocytes by 100 mmol/L propionic acid were
used for the comparison of the Na+-H+ exchange
activity of the lymphocytes during the OGTT (Fig 2). The
data on resting pHi and pHi immediately after
the addition of propionic acid and after 100 seconds of recovery are
summarized in Table 1.
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After the administration of 100 g glucose, there was a significant
increase of the activity of the Na+-H+ exchange
in lymphocytes. The Na+-H+ exchange activity
significantly increased from 5.20±0.53x10-3 to
8.28±1.07x10-3 dpHi/s (1 hour) and to
8.15±1.18x10-3 dpHi/s (2 hours;
2 test, 12.1; P=.0024). It is
noteworthy that neither resting pHi nor resting
[Na+]i was significantly changed during the
OGTT. The data are summarized in Fig 3. Under control
conditions, there were no significant changes of
Na+-H+ exchange activity, resting
pHi, or resting [Na+]i
within 2 hours (Fig 3). In 5 subjects, the changes of
Na+-H+ exchange activity were pursued over 10
hours. As shown in Table 2, after the maximum at 1 hour,
Na+-H+ exchange activity slowly returned to
baseline within 10 hours.
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Effects of Oral Glucose Challenge on Blood Pressure
There were no significant changes of systolic or
diastolic blood pressure in the overall group (0 hours,
129±3/72±2 mm Hg; 1 hour, 130±3/71±3 mm Hg; and 2 hours,
130±3/72±3 mm Hg; 2 test, 3.5
[P=.173 for systolic pressure];
2 test, 1.15 [P=.56 for
diastolic pressure]). However, during OGTT the
systolic blood pressure increased in 14 of 16 subjects
studied.
Correlation of Na+-H+ Exchange Activity and
Blood Pressure
As shown in Fig 4, there was a significant
correlation between the activity of the Na+-H+
exchange and systolic blood pressure (r=.457,
P=.001) after oral glucose challenge, whereas under control
conditions there was no significant correlation (r=.114,
data not shown). No correlation between resting
[Na+]i and blood pressure could be observed
(r=.06, data not shown).
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Correlation of Plasma Glucose, Plasma Insulin, and
Na+-H+ Exchange
Since plasma glucose and plasma insulin concentrations increased
during the oral glucose challenge together with the lymphocytic
Na+-H+ exchange activity, it was determined
whether these parameters were correlated (Fig 4
). The
lymphocytic Na+-H+ exchange activity was
significantly correlated with the plasma glucose concentration
(r=.357, P=.041), whereas no significant
correlation could be obtained with the plasma insulin concentration
(r=.275, P=.084).
Blood Pressure and Lymphocytic Na+-H+
Exchange During Euglycemic
Hyperinsulinemic Clamp
To clarify whether the increase in glucose or insulin
concentrations caused the stimulation of lymphocytic
Na+-H+ exchange, euglycemic
hyperinsulinemic clamp studies were performed in 4
subjects. During steady state euglycemic
hyperinsulinemia, the plasma glucose concentrations
were similar to those under baseline conditions (95±0.8 mg/dL),
whereas plasma insulin concentrations were markedly increased compared
with baseline levels (63±5 versus 7±5 µU/mL). During steady state
euglycemic hyperinsulinemia, the
systolic and diastolic blood pressures were not
significantly different compared with baseline values (129±2/84±1
versus 126±2/83±1 mm Hg).
The lymphocytic Na+-H+ exchange was not significantly different at steady state euglycemic hyperinsulinemia compared with baseline values (5.71±0.34x10-3 versus 6.52±0.43x10-3 dpHi/s), indicating that hyperinsulinemia did not change the maximum Na+-H+ exchange activity.
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Discussion |
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In primary hypertension, an increased Na+-H+ exchange activity has been described in vascular smooth muscle cells,33 34 lymphocytes,22 35 red blood cells,36 37 and platelets.38
It was speculated that an increased Na+-H+ exchange may be related to the growth of vascular smooth muscle cells.39 If such a relation exists, it could only explain long-term changes in blood pressure subsequent to vascular hypertrophy. In the present study, a close relation between Na+-H+ exchange and systolic blood pressure in an acute experiment suggests that other interrelations between blood pressure and Na+-H+ exchange also exist.
Plasma glucose concentrations, but not insulin concentrations, are correlated with the changes in Na+-H+ exchange. Therefore, glucose seems to be the stimulator of Na+-H+ exchange under the conditions of oral glucose challenge. Indeed, glucose was shown to stimulate Na+-H+ exchange in vitro.40 Furthermore, the absence of insulin receptors on T lymphocytes,41 42 which represent the majority of circulating lymphocytes, points to a causal role of the changes in glucose concentration rather than of the insulin changes. Therefore, the predominant role of glucose compared with the role of insulin to stimulate Na+-H+ exchange may be unique to lymphocytes. Admittedly, in tissues possessing insulin receptors, an additional direct effect of insulin may be more evident. Furthermore, the experiments with the euglycemic hyperinsulinemic clamp technique support the role of glucose for the observed changes of Na+-H+ exchange. With markedly elevated insulin levels, but normal glucose levels, Na+-H+ exchange failed to increase. Taken together, changes of glucose concentrations rather than changes of insulin concentrations may be responsible for the increased activity of Na+-H+ exchange during the oral glucose challenge. Similar findings concerning the lack of blood pressure changes during steady state euglycemic hyperinsulinemia were obtained by Fliser et al43 and Widgren et al.44
How can the observed relation between blood pressure and Na+-H+ exchange be explained? Either changes in Na+-H+ exchange can influence vascular tone, or changes in Na+-H+ exchange are a consequence of blood pressure changes, but the latter possibility seems rather unlikely. Last, changes in plasma insulin concentration could directly influence blood pressure. However, there is ample evidence that the acute administration of insulin lowers blood pressure in humans. Observations on the vasopressor effect of insulin have been made mostly in rodents.45
In summary, the present study demonstrates that in humans Na+-H+ exchange is activated by an oral glucose challenge. The changes in Na+-H+ exchange activity appear to be related to plasma glucose concentration.
Received July 15, 1994; accepted August 14, 1995.
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