Institute of Physiology, University of Fribourg, CH-1700 Fribourg, Switzerland
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ABSTRACT |
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Insulin resistance is associated with deficits in glucose metabolism. We tested whether the vascular and renal responses to insulin might contribute to insulin resistance. Generation of endothelial-derived vasodilator nitric oxide (NO), estimated after a 2-h period of insulin stimulation, was inhibited in the presence of high glucose. Immunoprecipitations indicated that insulin-induced endothelial signal transduction was mediated through an immediate complex formation of insulin receptor substrate (IRS) with phosphatidylinositol 3-kinase, which caused serine phosphorylation of a protein complex that was comprised of Akt kinase and endothelial NO synthase. The enzymatic complexes did not form when the endothelial insulin stimulation occurred in the presence of high glucose concentrations. By contrast, neither epithelial signal transduction nor sodium transport in renal epithelial cells was affected by high glucose. Hence, glucose does not appear to modulate either the epithelial IRS cascade or renal sodium retention. Dysfunction of the endothelial IRS cascade and NO generation, which suppresses efficient delivery of nutrients, may further exacerbate the metabolic syndrome of insulin resistance.
electrophysiology; insulin resistance; protein kinase C-; renal
epithelial cells; signal transduction
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INTRODUCTION |
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EXPOSURE TO HIGH LEVELS OF INSULIN leads to increased blood flow and peripheral supply of energy and nutrients. Insulin stimulates the vasculature to produce endothelial-derived vasodilator nitric oxide (NO). Administration of a NO synthase inhibitor increases, via blockade of vasodilation, the arterial blood pressure and increases vascular reactivity in vivo (18, 25). This endothelial NO synthase (eNOS) is constitutively expressed and accounts for the spontaneous and likely the postprandial generation of NO in the vasculature. NO synthase of the vascular smooth muscle has to be induced by inflammatory cytokine-mediated gene expression. The endothelial eNOS protein is abundant in large arteries (22, 36) and in venules of the kidney (40). In the pulmonary blood vessels of newborns (16), gene expression of eNOS has been demonstrated to be augmented by tissue growth factors, such as the hypoxia-induced vascular endothelial growth factor (vEGF) (17). In adult life, acute transient exposure to the neurotransmitter acetylcholine to shear stress accompanying physical exercise and to postprandial increases in insulin stimulates the enzyme activity of eNOS (11). Conversely, molecular mediators of eNOS inhibition, as in endothelial dysfunction, have not been thoroughly investigated (20). Whether hyperglycemic-type pathological conditions affect acute endothelial enzyme activation and NO generation upon insulin stimulation is not known.
Elevated glucose levels are normally compensated by elevations of insulin generation. This compensatory mechanism (hyperinsulinemia) fails when receptor resistance develops, as in metabolic insulin resistance syndrome. However, no such rise in blood sugar levels was found to occur in mice with a genetically defective insulin receptor in skeletal muscle. The mice have normal glucose tolerance and never develop diabetes (7). Hence, a defect of the insulin signal at the skeletal muscle and concomitantly at additional sites is assumed to cause the (pre)diabetic features (23). Other potential sites include the glucose metabolism of fat tissue and liver, but also the endothelium and the renal epithelium delineating the tubules through which urine is excreted. Renal epithelial cells are stimulated by insulin and retain sodium in the distal tubules and both sodium and water in the proximal tubules (3, 39). It is not known whether the renal epithelial system uses an inhibitory mechanism to reduce such insulin-induced sodium retention.
A hereditary defect of insulin sensitivity has been ascribed to genetic defects (polymorphisms) in specific molecules. The docking molecule insulin receptor substrate (IRS) can carry a genetic polymorphism at the site where it binds the lipid second messenger, phosphatidylinositol (PI) 3-kinase. This polymorphism was found in patients with coronary artery disease (2). Frequency of the genetic defect was elevated 7-fold in a subgroup of patients with disorders of lipid metabolisms, and a 27-fold increase in its incidence was measured in a subgroup of patients with the clinical features of insulin resistance. IRS is the immediate signal transduction molecule of insulin stimulation in skeletal muscle cells and adipocytes. IRS-independent signal transduction cascades, such as those through transcription factor STAT5, involve gene expression (13). The IRS cascade engages the PI 3-kinase, which mediates many of the rapid effects of insulin. PI 3-kinase also plays a functional role in cell types other than muscle cells and adipocytes. PI 3-kinase has been shown to be essential for the endothelial and renal epithelial functions of insulin (30, 43). The involvement of IRS in the endothelial and renal epithelial cell types has not been investigated. The purpose of the present study is to delineate and compare the rapid cellular and molecular effects of insulin in endothelial and renal epithelial cells.
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METHODS |
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Cell cultures. Human umbilical vein endothelial cells (HUVEC) were isolated and used up to four passages (33). Cells were cultivated in culture dishes coated with a layer of fibronectin (Winiger, Wohlen, Switzerland), a member of a family of proteins of the large extracellular matrix circumventing the endothelial cells and the blood vessels in vivo (34). The culture medium was medium 199 (GIBCO Laboratories, Grand Island, NY), 15 mM HEPES pH 7.4, supplemented with 90 µg/ml Na-heparin (10,000 U; Novo Industries, Copenhagen, Denmark), 25 µg/ml endothelial cell growth supplement (Collaborative Research, Waltham, MA), 15% human heat-inactivated serum, 2 mM L-glutamine, and 20 µg/ml gentamicin.
Distal tubule epithelial cell line A6, originating from Xenopus laevis kidney, was provided by American Type Culture Collection (Manassas, VA) (ATCC CCL-102). Nitrate cellulose filters of 0.45 µm (Sartorius, Edgewood, NY) were coated with a collagen gel extracted from rat tail and overlaid by a layer of A6 cells. The culture medium was Waymouth (GIBCO), supplemented with 15% sterile water (osmotic adjustment to the amphibian cellular environment), 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (GIBCO). Human proximal tubule epithelial cells were provided by Clonetics (San Diego, CA) and used up to three passages. Cells were cultured in medium for epithelial cells supplemented with 15 mM HEPES, pH 7.4 (Clonetics), and with 1% human heat-inactivated serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. Human colorectal-tumor epithelial cell line HT-29 was provided by American Type Culture Collection (ATCC HTB-38). Cells were cultured in a 1:1 mixture of DMEM medium and Ham's F-12 nutrient mixture, supplemented with 5% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. The cellular constructs were cultured to confluence, and the culture medium was changed to serum-free medium 2 h before the experiment and treated with physiological concentrations of D-glucose, that is, a concentration of 5.5 mM D-glucose (Applichem, Darmstadt, Germany), and with high concentrations of D-glucose (as indicated, or 25 mM final concentrations) for 15-30 min before and during stimulation with human insulin (ICN, Eschwege, Germany). Stimulations lasted for 7 min for signal transduction experiments and up to 2 h for functional analysis. Insulin concentrations were 100 nM, or as indicated.Immunoprecipitation and immunoblot analysis.
Cells were lysed in buffer containing 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, supplemented with 1% Brij35 detergent and protease inhibitor cocktail from Calbiochem (La Jolla, CA) and fresh 1 mM
Na3VO4. Polyclonal antibodies raised against
IRS-1, threonine (T308)-phosphorylated Akt (UBI, Lake Placid, NY),
protein kinase C (PKC)-1 and -
2 (Santa
Cruz Biotechnology, Santa Cruz, CA), or serine phosphorylated residues
(Zymed Lab, San Francisco, CA) were used for immunoprecipitation from
the soluble part of the cell lysate for 2 h at 4°C. Samples were
separated on 4-15% polyacrylamide gradient gel by SDS-PAGE and
transferred to 0.45 µm polyvinylidene difluoride membranes by use of
10 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer (pH 11)
with 0.5 mM dithiothreitol and 10% methanol. Coimmunoprecipitated
proteins were detected by indirect immunoblot analysis with polyclonal
antibodies against the p85 subunit of PI 3-kinase, against the
threonine (T308)-phosphorylated Akt kinase, and against eNOS (UBI). To
verify equal loading on the gel of IRS-1 precipitates, immunoblot
analysis was performed with the same antibodies as used for the
immunoprecipitation. Incubations of the membranes with respective
first-step antibodies for 6 h were followed by incubation with
horseradish peroxidase-conjugated secondary-step antibodies (Santa
Cruz) for 1 h. Bands were visualized by applying the developing
kit for enhanced chemiluminescence according to instructions of the
manufacturer (Amersham, Uppsala, Sweden).
Determination of NO from endothelial cells.
During stimulation with insulin, the serum-free and phenol red-free
medium 199 (GIBCO) was complemented with 100 µM
L-arginine and 1 µM of 4,5-diaminofluorescein (DAF-2;
Alexis, San Diego, CA), which can be used for real-time imaging of NO
(4). The cell-permeable diacetate derivative DAF2-DA is
used to load the cells. Subsequent hydrolysis by cytosolic esterases
releases DAF-2, which reacts with the oxidized form of NO and converts
to the fluorescent triazole derivative DAF-2T (excitation 492 nm,
emission 515 nm), thereby increasing the quantum yield of
fluorescence. Endothelial cells were detached by short trypsinization
(phenol red-free solution) and yields of mean fluorescence intensity
(MFI) recorded directly by fluorescence-activated cell scanning (FACS), using the Cell-Quest Software Program of Becton-Dickinson caliber flow-cytometric equipment (Mountain View, CA). The markers for the
monovariant histogram were set on the basis of a staining control that
was of cells with inhibited NO synthase [by
nitro-L-arginine methyl ester (L-NAME)]. The
staining of NO synthase-inhibited cells was only one- to twofold
elevated when compared with the autofluorescence of HUVEC cells
(absence of fluorescent dye). The MFI of unstimulated cells was
subtracted from MFI fluorescence of insulin-stimulated cells (in the
presence or absence of high glucose) and given as an increase in MFI in
Fig. 1.
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Electrophysiological analysis of sodium transport in epithelial
cells.
Transport of Na+ across epithelial cells is mediated by the
serial arrangement of an apical, amiloride-sensitive Na+
channel, through which Na+ enters the cells, and the
basolateral Na+-K+-ATPase, which extrudes
Na+ and provides the driving force. For measurement of
modification of transepithelial ion transport, the A6 cells overlain on
transwell rings were placed in a modified Ussing chamber, as we have
described earlier (8). Briefly described, the
tight-junction cell constructs were optimal for experiments when the
transepithelial electrical resistance (Ohm × cm2),
calculated according to Ohm's law, was 1,000 Ohm over the filter
surface of 1.5 cm2. The measurements were performed at room
temperature in serum-free medium and ~240 mosmol/kgH2O
culture medium (0.85 × Waymouth medium) buffered by
CO2 gazing. The Ussing chamber was equipped with a calomel
voltage electrode pair for monitoring the transepithelial potential
difference and the Ag/AgCl current electrode pair (short-circuit current) for clamping the transepithelial potential difference to 0 mV
(30). The different currents requested for clamping to
zero mV of resting cells and cells stimulated basolaterally by insulin
for 90 min in the presence of 5.5 mM and 25 mM glucose were monitored
and recorded (Pharmacia, Uppsala, Sweden). By convention, positive
current corresponds to an apical to basolateral movement of positive
charges across the epithelium. Na+ transport as the major
positive charge was identified by susceptibility of the transport to
the Na+ channel blocker 10 µM amiloride.
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RESULTS |
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The present study largely focused on primary effects of acute
treatments, circumventing eventual secondary effects caused by
long-term exposure to stimuli glucose and insulin. In vivo insulin
functions of NO generation and vasodilation occur rapidly within
1-4 h (41, 24). Hence, we analyzed insulin
stimulation and NO generation in endothelial cell cultures within
2 h. Real-time generation of endothelial NO was assessed by FACS
with the fluorescent dye DAF-2 (4). The fluorescence
signal was set to zero, and the fluorescent dye was added to the HUVEC
cells simultaneously with L-NAME, a NO synthase inhibitor.
In Fig. 1, the change in the recorded MFI was calculated by subtracting
the MFI of resting endothelial cells from that of stimulated cells (see
the increase in MFI shown on the ordinate). The increase in MFI was
abolished when the insulin stimulation occurred in the presence of the
NO synthase inhibitor L-NAME (data not shown), confirming
specificity of dye DAF-2 for NO assessment. NO fluorescence was dose
dependently triggered by insulin stimulation of HUVEC for 2 h. It
was detectable at 10 nM insulin and saturating at 100 nM insulin (Fig.
1A). Generation of NO in response to 100 nM insulin was
detectable within a few minutes, as also described earlier (44,
20), and was detectable for 3 h (data not shown). Endothelial
cells that had been exposed for longer than 5 h to the
serum-depleted medium (including the 1- to 2-h serum depletion before
insulin stimulation) were not considered for analysis, because the
cells started to show morphological changes. The insulin-induced NO
fluorescence was abolished when elevated glucose concentrations were
added to the endothelium 15-30 min before the insulin stimulation.
Inhibition was detectable at twofold normal concentrations of glucose
(11 mM), it was partial at 16 mM, and it was maximal at 25 mM glucose
concentrations (Fig. 1B).
The insulin receptor signaling that causes intracellular serine
phosphorylation events was analyzed in cell lysates by
immunoprecipitation. For the immunoprecipitation, antibodies were used
that recognized protein serine residues in the phosphorylated but not
in the nonphosphorylated state. The immunoblot analysis shows eNOS
protein only in the immunoprecipitate that originates from
insulin-stimulated (Fig. 2A,
lane 2), but not from resting, endothelial cells (lane
1). The immunosignal of eNOS was remarkably weaker when the
insulin stimulation of the cells occurred in the presence of 25 mM
glucose (lane 3). A proportion of the serine
phosphorylations included the phosphorylated serine residue (Ser) 1177, an activation site of eNOS. This was assessed by immunoblot analysis
with specific antibodies against phosphorylated Ser1177,
which demonstrated a faint band (data not shown) upon insulin stimulation of the endothelial cells. The immunoblot analysis in Fig.
2A further identified threonine-phosphorylated Akt kinase in
the immunoprecipitate of serine-phosphorylated proteins. The kinase was
exclusively immunoprecipitated in cells stimulated with insulin
(lane 2). The immunosignal of the Akt kinase was not
detected in glucose-treated, insulin-stimulated cells (lane 3) or in resting cells (lane 1). Akt is a
serine/threonine-specific kinase that is known to bind to and to
serine-phosphorylate the eNOS system in response to an increased flow
of fluid [a model of shear stress (38)] and in response
to vEGF (27).
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Phosphorylation and activation of Akt are enzymatically driven by PI 3-kinase, and PI 3-kinase is recruited to the site of activity by interaction with IRS-1. Endothelial cell lysates were applied to immunoprecipitation by specific antibodies against IRS-1. The immunoblot in Fig. 2B analyzes the presence of PI 3-kinase in the immunoprecipitates of IRS-1. Coimmunoprecipitation of PI 3-kinase was detected in 7-min insulin-stimulated cells (Fig. 2B, lane 2) but not in resting cells (lane 1). The PI 3-kinase immunosignal was absent when the insulin stimulation occurred in the presence of 25 mM glucose (lane 3). Immunoblot analysis using IRS antibodies confirmed identical amounts of IRS-1 in the immunoprecipitates that originated from resting and from stimulated endothelial cells. Glucosamine is a metabolite of glucose, and it is also named the biochemical, nutrient-sensing signal. Addition of 100 µM glucosamine 30 min before the insulin stimulation abolished the PI 3-kinase immunosignal (data not shown) in a manner similar to the pretreatment with high glucose.
The presence or absence of development of insulin resistance in renal
epithelium was tested, first in an established model of A6 cells
[originating from the distal tubule of Xenopus laevis (30)] and second in a freshly isolated, human cell
culture (originating from the proximal tubule). The ion transport in
the A6 cells was estimated with electrodes of an Ussing chamber that
measure the transcellular electrical potential. A second pair of
electrodes provided the electrical current that was necessary to clamp
the transepithelial potential to 0 mV (termed short-circuit current) (8). The spontaneous short-circuit current of 5 µA/cm2 was reduced to 0-1 µA/cm2 when
amiloride was added (10 µM, data not shown). Amiloride is a blocker
of the epithelial sodium channel. The sensitivity to amiloride
indicates that a significant proportion of the vectorial ion transport
across the A6 cells represents sodium transport (ordinate in Fig.
3). Figure 3A shows
a dose-dependent increase in short-circuit current in response to
addition of insulin to the basolateral side of the cells. The increase
was initiated at 0.01 nM insulin, was partial at 0.1 nM, and reached
the maximum at 1 nM (open bars in Fig. 3A). The increase
started within 5 min, was saturated after 20 min, and lasted for 90
min. Bilateral or unilateral treatment of the cells with high glucose
concentrations did not impair the insulin-induced increase in
short-circuit current (solid bars in Fig. 3A). Successful
penetration of glucose into the A6 cells has previously been
ascertained (42).
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Addition of the PI 3-kinase inhibitor LY-294002 at 25 µM concentrations significantly inhibited the insulin-induced increase in short-circuit current by 80% (Fig. 3B, open bars). The inhibitory effect of LY-294002 found after 20 min of insulin stimulation was absent when cells were stimulated for 80 min with insulin (Fig. 3B, solid bars).
Functional involvement of PI 3-kinase in the renal epithelial cells
provided impetus to further investigate the epithelial signal
transduction pathway of insulin. Stimulatory regulation of epithelial
insulin signals has recently been reviewed (32). Inhibitory regulation of epithelial insulin stimulation is far less
often investigated. In essentially all insulin-responsive cell types
and tissues that have been tested so far, the insulin-induced complex
formation of PI 3-kinase and IRS-1 was found impaired under
hyperglycemic and diabetic conditions. Lysates of epithelial cells that
originated from human renal tubules underwent immunoprecipitation with
specific antibodies against IRS-1. The immunoblot in Fig. 4A analyzes the presence of PI
3-kinase in the immunoprecipitates of IRS-1. Coimmunoprecipitation of
PI 3-kinase was detected in insulin-stimulated cells, both in the
absence (lane 2) and in the presence of 25 mM glucose
(lane 3). It was exclusively absent in resting cells
(lane 1).
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Insulin regulates the paracellular permeability of cultured colon-derived epithelial cell monolayers (26). For this reason, the human colon-derived epithelial cell line HT-29 served to ascertain the absence of insulin resistance in an additional epithelial cell type. Identical results were obtained in the human colon epithelial cells (Fig. 4B) and in the human renal epithelial cells. Coimmunoprecipitation of PI 3-kinase was detected in insulin-stimulated colon epithelial cells, both in the absence (Fig. 4B, lane 2) and in the presence of 25 mM glucose (lane 3). Coimmunoprecipitation was exclusively absent in resting cells (lane 1). Immunoblot analysis confirmed identical amounts of 170-kDa IRS-1 in the immunoprecipitates originating from resting and from stimulated colon epithelial cells (data not shown). Pretreatment with high glucose for several hours also did not inhibit the epithelial insulin signals in both renal and colon-derived cells (data not shown).
The epithelial Akt kinase was contrasted with the endothelial Akt kinase, which had been affected by high glucose (insulin resistance). Human renal epithelial cell lysates were analyzed by immunoprecipitation with specific antibodies against threonine-phosphorylated Akt. The immunoblot analysis in Fig. 4C shows phosphorylated Akt in insulin-stimulated epithelial cells, both in the absence (lane 2) and in the presence of 25 mM glucose (lane 3). The immunosignal was absent in resting cells (lane 1).
The concentration of 25 mM glucose was not cytotoxic to the endothelial
and epithelial cells. The responsiveness of the cells to various agents
other than insulin (e.g., cytokines) was not affected by high glucose
(data not shown). Both cell types were left untreated or treated with
25 mM glucose for 30 min and lysed by hypotonic shock. After
fractionation by centrifugation, the cell precipitates (cytoskeleton
fraction) were solubilized by detergents and subjected to
immunoprecipitation and immunoblot analysis of PKC. A significant
recruitment of PKC- (
1 and
2) to the
cytoskeleton was detected in glucose-treated endothelial cells but not
in glucose-treated epithelial cells. PKC-
was not translocated in
response to glucose (data not shown). Figure
5 represents resting and high
glucose-stimulated human endothelial cells (Fig. 5A) and
resting and glucose-stimulated renal epithelial cells (Fig.
5B). The histogram indicates the arbitrary densitometric intensity of the PKC-
immunosignal (immunoblot analysis) on the ordinate.
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DISCUSSION |
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We have shown in the current study that insulin initiates a
cascade of endothelial protein phosphorylations, transferring the
signal through IRS-1 to cause activation of the eNOS system. Insulin-induced NO generation was increased within minutes and sustained for 2 h; thus it did not require gene expression. The physiological relevance of this finding in cell culture is suggested by
the similar time course reported in insulin-stimulated large blood
vessels in vivo (24, 41). It is often assumed that the insulin-induced generation of endothelial-derived NO is due to an
increase in expression of eNOS protein (12). Several
studies have demonstrated such insulin-induced increases in eNOS
protein (22, 36). In fact, we also found an increase in
eNOS protein by stimulation of HUVEC with insulin for 16 h (data
not shown). It is unclear whether there is an additional regulation by
high glucose after long-term insulin treatment. In particular, the matter is complicated by our finding that the presence of sustained high glucose levels is sufficient by itself to increase eNOS protein in
HUVEC (data not shown). A glucose-mediated increase in eNOS was shown
for aortic endothelial cells as well (9) but was not found
in coronary artery endothelial cells (12). Hence, our
current finding of acute insulin effects is consistent with the
assumption that NO is rapidly generated postprandially. This clearly
occurs through a signal transduction cascade and enzymatic eNOS
activation, rather than by the slow machinery of gene expression. The
acute insulin stimulation of eNOS potentiates vasorelaxation in vivo,
in particular when superimposed on
2- and
-adrenergic vasorelaxation (24). A transient entry of calcium into
endothelial cells is classically achieved in response to acetylcholine,
an activator of eNOS and a vasorelaxant (38). It has
previously been shown that serine-phosphorylated eNOS is active at
minimal amounts of calcium (one-tenth of normal concentrations)
(11, 15). Therefore, the acute phosphorylation signals of
insulin represent an alternative means to activate eNOS
(27).
It has been shown that endothelial dysfunction accompanies insulin resistance. The entry of calcium and activation of eNOS-mediated vasorelaxation (after muscarinic receptor activation by acetylcholine) are absent in insulin resistance (28). The question arises whether high glucose is able to inhibit the insulin stimulation of acute endothelial functions. We show that simulated hyperglycemia leads to an impaired molecular (IRS-1, PI 3-kinase) and functional insulin signal in vascular endothelial cells that may lead to impaired vasorelaxation. It appears from previous studies that physical exercise leads to increased blood flow (and flow-mediated shear stress) and is beneficial in insulin resistance (10, 21). This can be explained by the fact that shear stress causes the PI 3-kinase-mediated phosphorylation and activation of eNOS, independent of IRS. Therefore, unlike physical exercise, endothelial insulin signals do not substitute for a defect in calcium-mediated relaxation in insulin resistance. Endothelial dysfunction of the insulin signal may further exacerbate the metabolic syndrome of insulin resistance, affecting regional blood flow and the distribution and ultimate storage of glucose.
Absence of insulin resistance in renal epithelial cells is in agreement with studies of type 2 diabetes. In our cell cultures, as well as in type 2 diabetes patients, the renal sodium-retaining response to insulin was maintained in the presence of high glucose (35, 37). The consequently greater degree of sodium retention and the accompanying sodium- and osmosis-driven expansion of the extracellular volume are, however, not sufficient to induce a cardiovascular phenotype. Only when the expansion of extracellular volume is superimposed on endothelial dysfunction (endothelial insulin resistance) may a cardiovascular phenotype manifest itself. This may explain why a hypertensive phenotype frequently accompanies the metabolic insulin resistance syndrome (14).
Physiologically relevant conditions were used to experimentally study
the stimulatory effect of insulin and the inhibitory effect of high
glucose. Postprandial concentrations of ~1 nM (or 200 µU/ml)
insulin are found in vivo in the circulating blood. Regionally, in the
venous effluent of the isolated perfused human pancreas, levels of
insulin as high as 3,000 µU/ml are present (29, 31). In
our experiments, endothelial insulin resistance (after high glucose
treatment) was not overridden by insulin concentrations of 10-100
nM. Concentrations of insulin as low as 0.01-1 nM were effective
in renal epithelium, even with five times normal glucose concentrations, underlining the lack of renal epithelial insulin resistance. Several cell type-specific characteristics may contribute to the distinct effects of glucose on insulin actions. First, glucose
molecules can diffuse through the vascular cell layers in a
paracellular manner without significantly entering the endothelial cells. In contrast, glucose is actively transported across renal epithelium. Where tight junctions seal the paracellular transport, glucose crosses the apical membrane of the epithelial cells through ion-coupled cotransporters and is released basolaterally through glucose transporters, or GLUT. Resulting fluctuations of intracellular glucose may explain why the renal epithelial cells do not use a
glucose-mediated mechanism that limits the insulin signal and the ion
transport. Second, one intracellular target of high glucose is thought
to be PKC. Glycolytic intermediates provide a major source of
substrates for diacylglycerol generation. In turn, diacylglycerol activates PKC (5). Neither insulin signals nor PKC type
were affected by high glucose in epithelial cells. In endothelial
cells, we detected a high glucose-induced subcellular translocation of the
-isoforms of PKC. The rapid stimulation of PKC-
was
accompanied by the rapid inhibitory effect on insulin signal
transduction and function in the endothelium. PKC can trigger
inhibition of the insulin receptor and receptor substrates
(5). Especially, overexpression of PKC-
has caused
endothelial dysfunction (22). Conversely, PKC-
blockers
have previously been shown to improve endothelial functions of insulin.
In vivo, a treatment with the blockers that antagonize the PKC-
isoforms ameliorated vascular functions of diabetic animals
(19). PKC-
blockers are currently in phase II clinical investigations.
The experiments with regard to malfunction of IRS in the glucose-treated endothelial cells provide an explanation of in vivo approaches. Endothelial dysfunction (as well as hypertension) has been discovered in mice where the IRS-1 gene is deleted, mimicking dysfunction of IRS (1). The gene-deficient animals (mice and drosophila) commonly exhibit prediabetic insulin resistance and retardation of growth (6). In combination with the present results, these studies lead to the following model. Endothelial IRS-1 is coupled to its associates in response to insulin under normal concentrations of glucose. This IRS-1 signal triggers a subsequent phosphorylation of serine residues on eNOS and associated proteins. However, increasing NO generation would be reduced after elevations in glucose, which block the insulin signal transduction cascade. The suppression of the vasodilatory actions of insulin, which normally contributes to the distribution and ultimate storage of glucose, may further exacerbate the metabolic insulin resistance syndrome.
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ACKNOWLEDGEMENTS |
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We are grateful for Dr. Christopher Fiorillo's presubmission editorial help.
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FOOTNOTES |
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This work was supported by a grant from the Swiss National Science Foundation (no. 3200-054145.98 to B. Schnyder) and a grant by the Foundation Rentenanstalt (to J. Durand).
Address for reprint requests and other correspondence: B. Schnyder, Univ. of Fribourg, Institute of Physiology, Rue du Musée 5, CH-1700 Fribourg, Switzerland (E-mail: bruno.schnyder{at}unifr.ch).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 May 2001; accepted in final form 10 September 2001.
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