The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 7 2405-2410
Copyright © 1998 by The Endocrine Society
Modifications Induced by Plasma from Insulin-Dependent Diabetic Patients and by Lysophosphatidylcholine on Human Na+,K+-Adenosine Triphosphatase1
R. A. Rabini,
P. Fumelli,
G. Zolese,
E. Amler,
E. Salvolini,
R. Staffolani,
N. Cester and
L. Mazzanti
Department of Diabetology (R.A.R., P.F.), INRCA Ancona, and the
Institutes of Biochemistry (G.Z., E.S., R.S., L.M.) and Obstetrics and
Gynecology (N.C.), University of Ancona, Ancona, Italy; and the
Institute of Physiology, Czech Academy of Sciences (E.A.), Prague,
Czech Republic
Address all correspondence and requests for reprints to: Prof. L. Mazzanti, Istituto di Biochimica, Universitià di Ancona, Via P. Ranieri 65, 60131 Ancona, Italy.
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Abstract
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To investigate the molecular mechanisms of the inhibition of
Na+,K+-adenosine triphosphatase
(Na+,K+-ATPase) in diabetes mellitus, we
incubated Na+,K+-ATPase purified from human
placenta of six healthy nondiabetic women with plasma from six
insulin-dependent diabetic (IDDM) men and six healthy controls and with
different concentrations of lysophosphatidylcholine (LPC). We
determined the enzyme activity, anthroyl ouabain-binding capacity,
dissociation constant (Kd), and average lifetime values
(
) by the static and dynamic fluorescence of anthroyl ouabain. The
lipid annulus of the enzyme was studied by static and dynamic
fluorescence of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene
(TMA-DPH). Moreover, we studied the lipid microenvironment surrounding
the Na+,K+-ATPase purified from the
placentas of six healthy women and six insulin-dependent diabetic
women, determining the percent composition of phospholipids of the
lipid annulus. The addition of total and protein-free IDDM plasma to
normal Na+,K+-ATPase significantly inhibited
the enzymatic activity even at the lowest concentration studied
(1:100), whereas the ouabain-binding capacity, Kd, and
were not affected by IDDM plasma. The fluorescence polarization and
lifetime values of TMA-DPH were significantly decreased by diabetic
plasma. The incubation of Na+,K+-ATPase with
LPC caused an inhibition of the enzymatic activity without
modifications of the anthroyl ouabain-binding capacity and dissociation
constant. The fluorescence polarization and lifetime values of TMA-DPH
were significantly decreased by 5 µmol/L LPC. The study of the
phospholipids surrounding Na+,K+-ATPase
demonstrated a significant increase in the percent LPC content in IDDM
patients compared with controls together with a concomitant decrease in
phosphatidylcholine. These observations indicate that the inhibition
caused by diabetic plasma on Na+,K+-ATPase is
not dependent on a modification of the ouabain-binding site and that it
seems to mimic the effect of LPC addition. A link between modification
of the lipid moiety of the enzyme and
Na+,K+-ATPase inhibition might be hypothesized.
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Introduction
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ALTERATIONS in
Na+,K+-ATPase activity have
been widely described in human diabetes, and a link between this
modification and the chronic complications of the disease has been
hypothesized (1, 2). Lipid substances, such as nonesterified fatty
acids and lysophosphatidylcholine (LPC), are able to inhibit the
Na+,K+-adenosine triphosphatase
(Na+,K+-ATPase) activity
(3). More recently, we observed a relation between the plasma LPC
concentrations and
Na+,K+-ATPase activity in
both erythrocyte and platelet membranes from diabetic patients (4).
To investigate the molecular mechanisms of the enzyme inhibition
in diabetes, we incubated
Na+,K+-ATPase obtained from
human placenta (5) of healthy nondiabetic women with plasma from
insulin-dependent diabetic (IDDM) patients and determined the enzyme
activity and the anthroyl ouabain-binding capacity and dissociation
constant. The determination of the anthroyl ouabain binding capacity
allows an estimate of the number of active
Na+,K+-ATPase molecules
present in the preparations, whereas the study of its dissociation
constant (Kd) measures the affinity for ouabain of the
binding site. We also analyzed the average lifetime values () of
anthroyl ouabain fluorescence, which might indicate modifications of
the Na+,K+-ATPase molecule
occurring at the level of the ouabain-binding site.
It has been suggested that modifications in transmembrane cation fluxes
can be due to an altered composition of the membrane lipid fraction in
both hypertensive (6) and diabetic subjects (7). To elucidate the role
of the membrane lipid moiety in the modulation of enzymatic activity,
we also studied the effect of diabetic plasma on macro- and
microheterogeneity of the lipid annulus of the purified
Na+,K+-ATPase by means of
the static and dynamic fluorescence of
1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene
(TMA-DPH), which is a fluorescent probe providing information on the
lipid matrix at the interface between hydrophobic and hydrophilic
environments.
The effects of LPC on
Na+,K+-ATPase purified from
healthy subjects were also investigated to evaluate whether this
substance might mimic the action of IDDM plasma. Finally, we studied
the phospholipid composition of the lipid annulus of the enzyme
purified from healthy and diabetic women.
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Materials and Methods
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Plasma was obtained after overnight fasting from six men
affected by IDDM (age, 27 ± 4 yr; duration of disease, 6 ±
4 yr). All of the subjects with IDDM were in good metabolic control
(hemoglobin A1c, 7.6 ± 0.5%; normal range,
4.5–6.5%). No patient was affected by hypertension or by the
microvascular complications of diabetes, except for one man presenting
background retinopathy. Plasma from six age-matched healthy men
(hemoglobin A1c, 5.1 ± 0.4%) was used as a control.
All of the subjects studied were normotensive, had no family history of
hypertension, and gave their informed consent to the study.
In these experiments, plasma from each subject was obtained by blood
centrifugation at 600 x g for 15 min at room
temperature. Plasma protein denaturation was performed at 0 C. Plasma
(0.5 mL) was added to 0.66 N perchloric acid (0.5 mL). The
samples were centrifuged at 10,000 x g for 10 min, and
the supernatants were neutralized with 2 mol/L KHCO3. The
insoluble material was removed by centrifugation (10,000 x
g for 10 min), and the clear supernatant was tested at
scalar dilutions (8).
Enzyme purification
Placentas were obtained within 15 min after delivery from six
healthy pregnant women (age, 30 ± 4 yr) and from six age-matched
IDDM women (age, 28 ± 5 yr). Gestational ages at delivery were
similar in the two groups (controls, 40 ± 2 weeks; IDDM, 38
± 2 weeks). Na+,K+-ATPase
was purified from villous tissue of placentas at term according to a
modification of Jorgensen’s method (9), as previously described
(10).
The preparation consisted of complete membrane fragments, including
phospholipids and cholesterol, and showed two major bands (96,000 and
57,000 daltons) on SDS-PAGE corresponding to the two subunits of
Na+,K+-ATPase. The protein
concentration was determined by Lowry’s method using BSA as the
standard. Na+,K+-ATPase
activity was measured by means of the colorimetric determination of
released inorganic phosphate after Na2-ATP hydrolysis at 37
C according to the method of Esmann (11). The protein purity of the
enzyme preparation estimated from the contents of 
- and ß-peptides
on Coomassie blue-stained SDS gel was about 90%.
Incubation experiments
Na+,K+-ATPase
preparations purified from healthy subjects were incubated for 2 h
at 37 C with scalar dilutions of total and deproteinated plasma from
healthy and IDDM subjects, and the following determinations were made:
enzyme activity, anthroyl ouabain-binding capacity, dissociation
constant (Kd) and average lifetime values (),
and TMA-DPH fluorescence polarization and lifetime values.
To test the effect of LPC on
Na+,K+-ATPase, the enzymes
from healthy women were incubated for 2 h at 37 C with LPC
(1-palmitoyl-glycerol-3-phosphorylcholine; Sigma Chemical Co., St.
Louis, MO) at increasing concentrations (0, 1, 2.5, 5, and 10 µmol/L)
for determination of enzymatic activity and with 5 µmol/L LPC for the
fluorescence studies.
Anthroyl ouabain binding
Binding studies with anthroyl ouabain purchased from Molecular
Probes (Eugene, OR) were performed in purified enzymes incubated with
the agent at 37 C as described in our previous work (12). Steady state
fluorescence measurements were made either on a Perkin-Elmer LS-5 or a
Perkin-Elmer 7300 fluorometer (Norwalk, CT). The samples were excited
at 362 nm; the emission wavelength was 469 nm. The increase in
fluorescence intensity after anthroyl ouabain binding to the enzyme was
used to determine both the number of binding sites and their affinity
for ouabain, as described by Fortes (13). To obtain further information
on the microenvironment where the probe is embedded, dynamic
fluorescence measurements were performed on the labeled enzyme using an
ISS K2 Multifrequency Cross-Correlation Phase and Modulation
Fluorometer, as previously described (12). The average lifetime value
() was calculated as described by Ferretti et al.
(14).
Fluidity studies
The fluorescent probe TMA-DPH was incubated with the purified
enzyme at 37 C for 5 min as described in our previous work (12)
according to the method of Shinitzky and Barenholz (15). After
labeling, the steady state fluorescence measurements were made using a
Perkin-Elmer MPF 66 spectrofluorometer equipped with fluorescence
polarization accessory and a controlled temperature cell holder. The
excitation and emission wavelengths were 360 and 430 nm, respectively.
The fluorescence polarization value (P) was obtained from the
fluorescence intensities parallel (I||) and perpendicular
(I
) to the polarization direction of excitation light
using the equation of Schachter and Shinitsky (16). P indicates the
freedom of rotation of the fluorescent probe; a decrease in the P value
indicates a higher mobility of the probe TMA-DPH in the lipid
microenvironment where the probe is localized (i.e.
increased fluidity). Dynamic fluorescence measurements were also
performed on the enzyme labeled with TMA-DPH using the procedures above
described for the study of anthroyl ouabain binding.
Phospholipid composition
Lipids were extracted from
Na+,K+-ATPase obtained from
healthy and diabetic women by the method of Folch et al.
(17) and separated by thin layer chromatography on silica gel 60 plates
with chloroform-methanol-7 N ammonia (76:30:5,
vol/vol/vol). The lipids were visualized by iodine vapor, scraped, and
analyzed for phosphate by the method of Ames (18). Phospholipid classes
were identified by comigration with standards and expressed as a
percentage of the total phospholipids.
Statistical analysis
The significance of the results was assessed by Student’s
t test for paired data.
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Results
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The addition of IDDM plasma to normal
Na+,K+-ATPase significantly
inhibited the enzymatic activity even at the lowest concentration
(1:100; Table 1
). Protein-free plasma
showed the same behavior, although it determined a lower inhibition
compared with total plasma at the 1:50 and 1:10 dilutions (Table 1).
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Table 1. Effects of total and protein-free IDDM and healthy
subjects (C) plasma on Na+/K+-ATPase activity
(expressed as micromoles of Piper mg protein/h) of enzymes purified
from healthy subjects
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Anthroyl ouabain binding is known to occur at the active enzyme only
and was therefore used to determine both the number of binding sites
(binding capacity) and their affinity for ouabain by means of the
dissociation constant (13). As shown in Table 2, the binding capacity, dissociation
constant (Kd), and average lifetime values ()
of anthroyl ouabain were not significantly affected by the addition of
total or protein-free plasma from IDDM patients and healthy
subjects.
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Table 2. Effects of total (T) and protein-free (PF) IDDM and
control (C) plasma on binding capacity (B; nanomoles per mg protein),
dissociation constant (Kd; nanomoles per L), and average
lifetime () of anthroyl-ouabain bound to placental
Na+/K+-ATPase purified from healthy subjects
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The fluorescence polarization of TMA-DPH was significantly decreased by
a dilution of 1:100 of diabetic plasma, showing no further decrease at
the highest concentrations (Table 3).
This behavior indicates a higher fluidity of the lipid annulus
surrounding the
Na+,K+-ATPase, as also
suggested by the decreased lifetime values, consistent with an
increased polarity of the microenvironment (Table 3). Figure 1 shows the distribution analysis of
TMA-DPH fluorescence decay in lipids surrounding
Na+,K+-ATPase. The center
of the curve, representing the average lifetime value, was shifted
toward lower levels by the addition of IDDM plasma. Plasma from healthy
subjects did not cause any alteration in TMA-DPH fluorescence
polarization or lifetime values (Table 3).
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Table 3. Effects of IDDM and healthy subjects (C) plasma on
polarization (p) values and lifetimes () of TMA-DPH in
Na+/K+-ATPase isolated from normal placenta
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Figure 1. Lorentzian distribution analysis of TMA-DPH
fluorescence decay in lipids surrounding placental purified
Na+,K+-ATPase. Upper panel,
Control sample. Lower panel, Sample incubated with IDDM
plasma (dilution, 1:300).
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In vitro addition of LPC to
Na+,K+-ATPase purified from
healthy subjects significantly inhibited enzymatic activity in a
concentration-dependent manner (Fig. 2).
As shown in Table 4, 5 µmol/L LPC,
which caused a 20% decrease in enzymatic activity, did not modify the
anthroyl ouabain-binding capacity, dissociation constant, or lifetime.
On the contrary, the same LPC concentration significantly decreased the
polarization and lifetime values of TMA-DPH (Table 5).
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Figure 2. Effect of increasing concentrations of LPC
on Na+,K+-ATPase isolated from human
placenta. The mean ± SD are shown.
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Table 4. Effect of 5 µmol/L lysophosphatidylcholine (LPC)
on binding capacity (B; nanomoles per mg protein), dissociation
constant (Kd: nanomoles per liter), and average lifetime
() of anthroyl-ouabain bound to placental
Na+/K+-ATPase purified from healthy subjects
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Table 5. Effect of 5 µmol/L lysophosphatidylcholine (LPC)
on polarization (p) and lifetime () values of TMA-DPH bound to
placental Na+/K+-ATPase purified from healthy
subjects
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Study of the phospholipid composition of the lipid annulus of
Na+,K+-ATPase showed a
significant increase in LPC content in IDDM patients compared with that
in control subjects together with a reduction in phosphatidylcholine
(Table 6).
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Discussion
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Na+,K+-ATPase activity
is modulated by a wide number of factors, such as hormones and growth
factors, that act at the level of enzymatic gene expression or at the
posttranslational stage (19, 20, 21). The activity can also be modified by
ligands such as monovalent cations, ATP and related molecules, and
cardiac glycosides or ouabain-like substances (22, 23, 24). An ouabain-like
compound with sodium pump inhibitory properties can be released from
the mammalian adrenal cortex (25). The secretion of this ouabain-like
substance appears to be stimulated by Na+ retention and/or
extracellular fluid volume expansion (26), conditions described in IDDM
(27).
Diabetes mellitus shows a reduction in
Na+,K+-ATPase activity in
the plasma membranes of different cell types (28, 29, 30). The cause of
this reduction has not yet been clarified. Recently, we demonstrated
that the number of active enzymatic molecules is not modified in
placental tissue from IDDM subjects (12) and that LPC significantly
inhibits the Na+,K+-ATPase
activity of erythrocyte membranes from normal subjects (4).
On the basis of the hypothesis of the presence of a circulating
inhibitory substance in diabetes mellitus, we performed incubation
experiments with IDDM plasma using human
Na+,K+-ATPase purified from
normal placenta. Both total and protein-free plasma from diabetic
subjects inhibited
Na+,K+-ATPase even at low
concentrations. Neither total nor protein-free plasma influenced the
ouabain-binding capacity, its dissociation constant, or the anthroyl
ouabain lifetime values. This finding indicates that the inhibition
caused by diabetic plasma is not dependent on a reduction of the number
of active molecules or on a factor acting on the ouabain-binding site.
Therefore, it can be argued that the hypothetical inhibitory substance
is not a digitalis-like factor or a peptide. This inhibitory substance
does not seem to totally inactivate part of the
Na+,K+-ATPase molecule,
but, rather, might decrease the phosphorylation rate of all molecules,
producing a lower efficiency of the enzyme.
Diabetic plasma affected also the lipid annulus of the enzyme, as
demonstrated by the data for TMA-DPH fluorescence polarization and
lifetime. It might, therefore, be hypothesized that a lipid-soluble
inhibitory substance could act at the level of the head groups of the
phospholipids where TMA-DPH is anchored. The modification of the lipid
moiety of the enzyme might, in turn, cause a rearrangement of the
protein molecule with subsequent modification of the tertiary
structure, as observed by our recent work in diabetic
Na+,K+-ATPase (12). This
hypothesis is also supported by previous observations reporting a
significant correlation between
Na+,K+-ATPase activity and
membrane fluidity in erythrocytes from diabetic subjects (31). These
phenomena are consistent with the supposed role of increased LPC
concentrations in diabetic plasma, which have been hypothesized to
exert an inhibitory action on the
Na+,K+-ATPase (4).
The effects exerted by LPC incubation on
Na+,K+-ATPase purified from
healthy subjects seem to mimic the action of diabetic plasma on the
same preparations: inhibition of enzymatic activity, lack of action on
the ouabain-binding site, and similar effects on the microenvironment
where TMA-DPH is embedded. These findings, therefore, support the
hypothesis that increased plasma LPC levels might be responsible for
the decreased Na+,K+-ATPase
activity in IDDM (4). Although the circulating levels of LPC measured
in our previous work (4) seem too high in healthy subjects compared
with the levels causing
Na+,K+-ATPase inhibition
in vitro, it might be hypothesized that the in
vitro dilution of plasma with distilled water causes a reduced
binding of LPC to plasma proteins, so that the interaction of LPC with
the Na+,K+-ATPase
preparations is amplified under these experimental conditions.
LPC can be rapidly incorporated into plasma membranes, and its
inhibitory action might be exerted directly within the membrane, as
suggested by the observation that the lipid annulus of
Na+,K+-ATPase purified from
IDDM women contains an increased percentage of LPC compared with that
from control women. The altered lipid profile in diabetic placental
preparations of the enzyme might also be caused by an increased
conversion of phosphatidylcholine to LPC within the membrane, with a
direct effect on
Na+,K+-ATPase activity.
However, the actions exerted by IDDM plasma on
Na+,K+-ATPase preparations
suggest the presence of a circulating inhibitor that is able to modify
the cellular membrane around the enzyme and the enzymatic molecule
itself.
Kiziltunc et al. reported an increase in
Na+,K+-ATPase activity in
erythrocyte membranes obtained from diabetic subjects after in
vitro incubation with increasing concentrations of LPC (32), which
is apparently in conflict with our previous work (4) and the present
results.
However, it must be underlined that our observations of an inhibitory
action of LPC on
Na+,K+-ATPase were made on
erythrocyte membranes and enzyme preparations obtained from healthy
subjects. It might be hypothesized that membranes from diabetic
subjects already contain inhibitory levels of LPC and that a further
in vitro LPC addition might affect the physicochemical
properties of the membrane in a different way than in healthy
subjects.
Although the present data suggest a role for LPC in
Na+,K+-ATPase inhibition in
diabetes, it cannot be excluded that the reduction in this activity in
diabetes is multifactorial, as other researchers reported decreased
enzyme units per cell in IDDM patients (33) and reduced enzymatic
activity caused by the exposure to in vitro experimental
glycation (34).
In conclusion, protein-free diabetic plasma exerts an inhibitory effect
on normal human
Na+,K+-ATPase without
affecting the ouabain-binding site. Further study of the relation of
Na+,K+-ATPase to the
modification in the lipid microenvironment is needed to clarify the
roles of lipid substances in the modulation of enzymatic activities in
diabetes mellitus.
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Acknowledgments
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The authors thank Dr. Nicole Dousset (Institute of Biochemistry,
University of Rangueil, Toulouse, France) for the
lysophosphatidylcholine measurements.
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Footnotes
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1 This work was supported by Grant 95.00923CT04 from the Italian
National Research Council (to L.M.). 
Received October 30, 1997.
Revised February 12, 1998.
Revised March 19, 1998.
Accepted March 30, 1998.
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Top
Abstract
Introduction
