1 Department of Medicine, The effects of
tail-vein insulin injection (2 U/kg) on the regulation of
protein-serine kinases in hindlimb skeletal muscle were investigated in
hyperinsulinemic hypertensive fructose-fed (FF) animals that had been
fasted overnight. Basal protein kinase B (PKB) activity was elevated
about twofold in FF rats and was not further stimulated by insulin.
Phosphatidylinositol 3-kinase (PI3K), which lies upstream of PKB, was
increased ~3.5-fold within 2-5 min by insulin in control rats.
Basal and insulin-activated PI3K activities were further enhanced up to
2-fold and 1.3-fold, respectively, in FF rats. The 70-kDa S6 kinase
(S6K) was stimulated about twofold by insulin in control rats. Both
basal and insulin-stimulated S6K activity was further enhanced up to
1.5-fold and 3.5-fold, respectively, in FF rats. In control rats,
insulin caused a 40-50% reduction of the phosphotransferase
activity of the
insulin signaling; protein-serine/threonine kinases
THE FRUCTOSE-HYPERTENSIVE RAT has been used extensively
to examine the relationship among insulin resistance, hyperinsulinemia, and hypertension (6, 23-25). We and others have previously
demonstrated that feeding normal rats a fructose-enriched diet results
in marked insulin resistance in the animals (6, 23). To compensate for
the decreased insulin-stimulated glucose disposal, the rats secrete
more insulin, which results in compensatory hyperinsulinemia (6). This
compensatory hyperinsulinemia offsets the insulin resistance and allows
the animals to maintain normal plasma glucose levels despite the
presence of severe insulin resistance. Results from several studies
indicate that these metabolic defects may be intrinsically linked to
the development of hypertension in the fructose-hypertensive rat (6,
23, 24). Although numerous studies have examined the association
between insulin resistance and hypertension, the molecular mechanisms
underlying insulin signaling in hypertensive states remain
undetermined. In the present study, we have examined the regulation of
several postreceptor protein-serine kinases (now thought to be critical
to insulin-stimulated glycogen synthesis and glucose transport) in an
insulin-resistant, hyperinsulinemic, hypertensive rat model, the
fructose-fed rat.
Insulin-stimulated glucose utilization occurs primarily in the skeletal
muscle, where most of the glucose is converted to glycogen (4, 27, 34).
Insulin-stimulated glycogen synthesis has been shown to be markedly
impaired in both animal and experimental hypertension (17, 18, 41). One
of the protein kinases that has been implicated in the stimulation of
glycogen synthesis by insulin is the seryl/threonyl protein kinase B
(PKB) (12), also known as Rac-PK (26) or c-Akt (5). This enzyme, which
is the cellular homolog of the viral oncogene v-Akt, has been shown to
be activated by insulin in NIH-3T3 and Swiss 3T3 cells, rat adipocytes,
and L6 myotubes (1, 13, 28). Furthermore, it was demonstrated that PKB
inhibits glycogen synthase kinase-3 (GSK-3), an enzyme that
phosphorylates and inhibits glycogen synthase in vitro (13). This led
to the hypothesis that insulin activation of PKB and the subsequent
inhibition of GSK-3 may be one of the mechanisms that enhance glycogen
synthesis in vivo (13). PKB appears to lie downstream of the enzyme
phosphatidylinositol 3-kinase (PI3K) in the insulin signaling pathway,
because inhibitors of PI3K such as wortmannin inhibit the activation of
PKB (8, 13).
We previously demonstrated that an intravenous injection of insulin in
rats resulted in the activation of several seryl/threonyl protein
kinases that were resolved as multiple peaks of myelin basic protein
(MBP) phosphotransferase activity after fractionation by anion exchange
chromatography (21). Furthermore, these peaks of MBP phosphotransferase
activity remained unchanged in rats in which plasma glucose was clamped
at basal levels after insulin administration (21), indicating that
activation of these peaks was the direct result of insulin stimulation
and was not secondary to the hypoglycemia that occurs after insulin
injection. In the present study, we employed this model system to
examine in vivo basal and insulin-stimulated activities of PI3K,
PKB- Materials.
Regular insulin for intravenous injections was from Lilly;
Experimental protocol.
Sprague-Dawley rats were procured locally (body wt 130-150 g, age
5 wk). The animals were randomly assigned to either of the two
experimental groups: control (n = 28)
and fructose (n = 28). At 6 wk of age,
the animals in the fructose group were started on a 66% fructose diet
(66% fructose, 12% fat, and 22% protein), which had an electrolyte,
protein, and fat content very comparable to the standard rat chow. The
only difference was that the 60% vegetable starch present in normal
rat chow was replaced by 66% fructose in the fructose diet. The
fructose-induced metabolic changes become fully manifest 3-4 wk
after initiation of the fructose diet (6); therefore, 6 wk after the
initiation of the fructose diet, the rats were fasted overnight and
crude muscle extracts were prepared, as we will describe in the next
section. One week before termination, indirect systolic blood pressure
(BP) was measured in conscious rats by use of the indirect tail cuff
method without external preheating, as previously described (6). All experimental procedures were approved by the University of British Columbia Animal Care Committee.
Preparation of tissue extracts.
The procedure described by Gregory et al. (20) and Pelech and Krebs
(40) was modified and used to prepare tissue extracts. After
pentobarbital anesthesia, 20 rats in each of the control and fructose
groups were injected intravenously with insulin (2 U/kg dissolved in
saline given into the tail vein), whereas the remaining animals
(n = 8 in each group) were injected
with saline alone. In each of the control and fructose-injected groups,
the tissues were harvested 2 min (n = 7), 5 min (n = 7), or 15 min (n = 6) after the injection, and the
skeletal muscles from the hind legs were removed (white muscle,
primarily the gastrocnemius). The skeletal muscles were excised when
the animals were anesthetized completely (surgical anesthesia) but were
not dead. The muscles were removed rapidly, and the entire process
(which took <1 min) was timed very accurately so that each animal was
subjected to exactly the same procedure. The muscles were immediately
homogenized in ice-cold MOPS buffer (25 mM, pH 7.2) containing (in mM)
5 EGTA, 2 EDTA, 75 Anion-exchange chromatography.
Because of the existence of a multitude of protein kinases and other
proteins in crude skeletal muscle extracts that could interfere with
the various enzyme assays, the muscle extracts were fractionated to
partially purify the various MBP kinases before specific
immunoprecipitation assays were conducted. A fast protein liquid
chromatography system was used for all the chromatographic fractionations of muscle extracts, as previously described (21). Briefly, samples containing 5 mg of the protein were applied at a flow
rate of 0.8 ml/min to a MonoQ anion exchange column equilibrated with
buffer A (in mM: 10 MOPS, pH 7.2, 25 Determination of phosphotransferase activities.
MBP phosphotransferase activity from the MonoQ fractions was measured
by employing a filter paper assay. The reaction mixture was comprised
of 25 µg of substrate, 10 µl MonoQ fraction, 0.5 mM PKI, 50 µM
[
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-isoform of glycogen synthase kinase 3 (GSK-3
),
which is a PKB target in vitro. Basal GSK-3
activity was decreased
by ~40% in FF rats and remained unchanged after insulin treatment.
In summary, 1) the PI3K
PKB
S6K pathway was upregulated under basal conditions, and
2) insulin stimulation of PI3K and
S6K activities was enhanced, but both PKB and GSK-3 were refractory to
the effects of insulin in FF rats.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, p70 S6 kinase (p70 S6K), and GSK-3
in the
insulin-resistant, hypertensive, fructose-fed rats.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, EGTA, EDTA, MOPS,
-methylaspartic acid, sodium orthovanadate,
[
-32P]ATP,
phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, benzamidine, dithiothreitol (DTT), soybean trypsin inhibitor, pepstatin
A, and the peptide inhibitor of cAMP-dependent protein kinase (PKI)
were from Sigma. MBP was purified from bovine brain (15). The
anti-PKB-
-PH, anti-PKB-
-CT, and anti-p70 S6K-NT (all polyclonal
antibodies), as well as the monoclonal antibody raised against the
85-kDa subunit of rat PI3K, were purchased from Upstate Biotechnology
(Lake Placid, NY). The anti-p70 S6K-CT antibody was purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). The anti-GSK-3
antiserum
was a kind gift from Dr. J. Woodgett (Ontario Cancer Institute,
Toronto, ON, Canada); the GSK-3 substrate phosphopeptide and the
control GSK-3 (Ala21) peptide were from Upstate Biotechnology. Alkaline
phosphatase-conjugated goat anti-rabbit and goat anti-mouse IgGs and
horseradish peroxidase-conjugated goat anti-mouse IgG were from
Bio-Rad. Protein A-Sepharose, HR5/5 MonoQ, and MonoS columns were
purchased from Pharmacia. The fructose diet was bought from Teklad Labs
(Madison, WI). P-81 phosphocellulose filter paper was from Whatman.
Ribosomal 40S subunits were prepared from rat liver by a procedure
modified from that of Krieg et al. (29). All other chemicals and
reagents were of the highest grade commercially available.
-glycerophosphate, 1 sodium orthovanadate, 2 DTT, and various protease inhibitors (1 mM PMSF, 3 mM benzamidine, 5 µM
pepstatin A, 10 µM leupeptin, and 200 µg/ml trypsin inhibitor). The
homogenate was centrifuged at 10,000 g
for 15 min at 4°C (Beckman J2-21), and the pellet was
discarded. The supernatant was centrifuged at 100,000 g for 60 min (Beckman L8-60M),
and the resultant supernatant was stored at
70°C until
further analysis. At the time animals were killed, plasma samples were
collected for subsequent insulin and glucose assays, which were
performed as described previously (6).
-glycerophosphate, 5 EGTA, 2 EDTA, 2 sodium orthovanadate, and 2 DTT). The column was developed at the same flow rate with a 15-ml
linear NaCl gradient (0-800 mM) in buffer
A. Fractions (0.25 ml) were collected for assaying
protein kinase activities, for Western blots, and for specific
immunoprecipitation assays. For determination of GSK-3
activity,
samples containing 5 mg of the protein were applied at a flow rate of
0.8 ml/min to a MonoS cation exchange column equilibrated with
buffer C (20 mM HEPES, pH 7.0); the
column was developed at the same flow rate with a 15-ml linear NaCl
gradient (0-400 mM) in buffer C,
and 0.5-ml fractions were collected for subsequent assays.
-32P]ATP (specific
activity ~2,000 cpm/pmol) and assay dilution buffer, pH 7.2 (in mM:
20 MOPS, 25
-glycerophosphate, 20 MgCl2, 5 EGTA, 2 EDTA, 1 DTT, and
1 sodium vanadate). The reaction was allowed to proceed for 10 min at
30°C and then was terminated by spotting 25 µl of the reaction
mixture onto P-81 phosphocellulose paper. The papers were washed 5 times with 1% phosphoric acid to remove the free
[
-32P]ATP and were
then counted for radioactivity.
phosphotransferase activity was determined by incubation of 10 µl of the MonoS fractions with assay buffer containing 8 mM MOPS, 0.2 mM EDTA, 10 mM magnesium acetate, 0.1 mM ATP, and 125 µM of either the GSK-3 substrate phosphopeptide or the GSK-3 (Ala21) control peptide. The GSK-3 substrate phosphopeptide (GSK-3PP) contains serine residues at sites
3b, 3c, and phosphorylated site 4 from skeletal muscle glycogen synthase and is an excellent substrate for GSK-3. In contrast, the
GSK-3 (Ala21) control peptide does not contain the site 4 serine
residue, which is replaced with alanine; therefore, this peptide is not
a substrate for GSK-3, because GSK-3 requires a prephosphorylated
serine at site 4 to optimally phosphorylate sites 3b and 3c in the
enzyme glycogen synthase. The reaction was initiated by the addition of
50 µM [
-32P]ATP
(specific activity ~2,000 cpm/pmol) and allowed to proceed at
30°C for 30 min, after which it was terminated by spotting 25 µl
of the reaction mixture onto P-81 phosphocellulose paper. The papers
were washed 5 times with 1% phosphoric acid and counted for radioactivity.
Electrophoresis and immunoblotting. SDS polyacrylamide gel electrophoresis was performed (with 12.5% slab gels) as described by Laemmli (32). Gel electrophoresis was performed at 10 mA/gel overnight, and subsequently the proteins were transferred onto nitrocellulose membranes at 300 mA for 3 h. The membranes were blocked for 2 h with buffer containing 5% skim milk and sodium azide in 20 mM Tris · HCl, pH 7.4, and 0.25 M NaCl (TBS) and incubated with primary antibodies for 2 h or overnight. The membranes were then washed with TBS and Tween 20 (TTBS) and incubated for another hour with the secondary antibodies. After this, the membranes were washed again with TTBS and rinsed with TBS (without Tween 20), and the color reaction was performed for 5-30 min depending on the intensity of the bands. For procedures in which enhanced chemiluminescence was employed as the detection procedure, the secondary antibody was either goat anti-rabbit or the goat anti-mouse antibodies conjugated with horseradish peroxidase, and the incubation time was 45 min to 1 h.
Immunoprecipitation studies.
For PKB- immunoprecipitation assays, 200 µl of the particular
MonoQ fractions were incubated with an equal volume of 3% NETF (100 mM
NaCl, 5 mM EDTA, 50 mM Tris · HCl pH 7.4, 50 mM NaF,
5% glycerol, and 3% Nonidet P-40) and 35 µl of protein A-Sepharose beads and a combination of 5 µl of the anti-PKB-
-PH (Rac1-PH) and
5 µl of the anti-PKB-
-CT (Rac1-CT) antibodies. After a 3-h incubation period at 4°C, the protein A-Sepharose beads were
pelleted by centrifugation at 10,000 rpm for 2 min. The beads were
washed twice with 3% NETF and then twice with KII buffer (in mM: 12.5 MOPS, 12.5
-glycerophosphate, 20 MgCl2, 5 EGTA, 0.25 DTT, and 50 sodium fluoride), after which kinase assays were performed. The
reactions were initiated by the addition of 35 µl of KII buffer, 5 µl of 200 mM MgCl2, and 50 µM
of [
-32P]ATP
(specific activity ~2,000 cpm/pmol). The reaction was allowed to
proceed for 20 min at 30°C, after which it was stopped by the addition of 20 µl of 5× sample buffer. The reaction contents
were boiled and loaded onto 12.5% SDS polyacrylamide gels, and after transfer, the MBP bands were Ponceau stained, cut, and counted. The
membrane was then probed with specific antibodies to visualize the
protein of interest by use of immunoblotting procedures described in
Electrophoresis and
Immunoblotting. For the p70 S6K
immunoprecipitation assays, 200 µl of the particular MonoQ fractions
were incubated for 3 h with 10 µl of the anti-p70-CT antibody. After
this, the kinase reaction was performed in a manner identical to that
just described, except that S6 peptide was used as the substrate. For the GSK-3
immunoprecipitation assays, 200 µl of the particular MonoS fractions were incubated for 3 h with 3 µl of the anti-GSK-3
antiserum. The beads were washed twice with 3% NETF, twice with 100 mM
Tris, pH 7.4, and then once with 10 mM Tris, pH 7.4. After this, the
kinase reaction was performed as previously described for the GSK-3
MonoS fractions.
Statistical analysis. Data are presented as means ± SE unless indicated otherwise. Results were analyzed by an analysis of variance (ANOVA) procedure followed by a Newman-Keuls test. A probability of P < 0.05 was taken to indicate a significant difference between means.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
General characteristics of the animals. The fructose-fed rats were hypertensive compared with the control group (BP, 138 ± 3 mmHg vs. control 123 ± 2 mmHg, P < 0.05). The fructose-fed rats were also hyperinsulinemic compared with the Sprague-Dawley controls (fasted plasma insulin, 2.3 ± 0.6 ng/ml vs. control 0.9 ± 0.1 ng/ml, P < 0.05 by ANOVA). Basal plasma glucose levels were similar between the control and fructose-fed rats (control, 6.6 ± 0.2 vs. fructose, 7.1 ± 0.3 mmol/l). The fructose-fed rats gained weight at a similar rate compared with the control group (body weight at the time of euthanasia: control, 372 ± 20 g vs. fructose, 373 ± 8 g). These results are in accordance with earlier results (6), indicating that despite being severely resistant to the glucoregulatory effects of insulin, these animals were able to maintain normal glucose levels by secreting increased amounts of plasma insulin.
Insulin injection into the control animals caused a decrease in their plasma glucose concentration to 5.9 ± 0.3 mmol/l by 5 min postinjection and 2.5 ± 0.2 mmol/l by 15 min postinjection. Likewise, plasma glucose concentration dropped to 5.7 ± 0.1 mmol/l by 5 min and 3.2 ± 0.6 mmol/l by 15 min in the fructose-fed rats.Increased basal MBP kinase activities in hyperinsulinemic rats.
MonoQ fractionation on cytosolic extracts from the skeletal muscle
extracts revealed multiple peaks of MBP phosphotransferase activity in
the column fractions. Of the different peaks, peaks II, III, IV, and V
were activated about twofold in control rats (Fig.
1A).
These findings are in accordance with our previous studies, in which
the kinetics and time course of each of these MBP peaks were reported
(21). Interestingly, the basal activities of peaks II,
III, and V were
elevated about twofold in the hyperinsulinemic fructose-fed rats (Fig.
1B). Furthermore, insulin injection
did not cause any increase in the MBP kinase peaks II,
III, and V in
fructose-fed rats, whereas peak IV was
further activated by about twofold.
|
PKB is an insulin-activated kinase in MonoQ peaks II and III.
To determine the identity of the kinases that may contribute to MBP
peaks II and
III, we immunoblotted with antibodies
specific for PKB- (also known as Rac or c-Akt). Initial experiments
demonstrated that the
-isoform of PKB was one of the MBP kinases
that eluted in MBP peak fractions II
and III. Therefore, we performed
immunoprecipitation studies from the pooled MonoQ
fractions 25-34 using antibodies directed against the carboxy terminus and the pleckstrin homology domain of PKB-
. These experiments revealed that there was a
time-dependent increase in the activity of immunoprecipitated PKB-
in response to insulin in control rats (Fig.
2A). An
increase in PKB-
activity was evident as early as 2 min after the
insulin injection and was not accompanied by any change in the amount
of immunoprecipitated protein (Fig.
2B). In the fructose-fed
hyperinsulinemic rats, basal PKB-
activity was already increased
about twofold, which corresponded with the basal increase in the MBP
peaks II and
III. Insulin administration did not
cause any further increase in PKB-
activity in fructose-fed rats at
any of the time points studied.
|
Activation of insulin-modulated ribosomal S6 kinases in vivo.
To determine the activation of ribosomal S6 kinases in response to
insulin, muscle extracts were fractionated by MonoQ chromatography and
assayed using the 40S ribosomal protein as the substrate. Two major
peaks of S6 phosphotransferase activity were detected in normal rats,
which eluted at NaCl concentrations of ~100 and 350 mM, respectively
(Fig.
3A). In
fructose-fed rats, both peaks of S6 phosphotransferase activity were
elevated basally by about twofold. However, insulin activation of
peak I was decreased, whereas that of
peak II was markedly increased when
compared with control rats (Fig.
3B). Immunoblotting studies revealed
that the 70-kDa ribosomal S6K, which lies downstream of PKB, coeluted
with the second peak of S6 phosphotransferase activity (Fig.
3A,
inset), which also corresponded with
the fourth peak of MBP phosphotransferase activity (Fig.
1A). Immunoprecipitation studies
from the second peak of S6 phosphotransferase activity demonstrated
that the p70 S6K was activated about twofold by insulin in control rats
within 15 min after insulin injection (Fig.
4A).
This was accompanied by a gel mobility shift of the protein, which was
maximal at 15 min postinjection (Fig.
4B). Basal p70 S6K activity was
already elevated ~1.5-fold in the insulin-resistant rats (Fig.
4A). Surprisingly, insulin-stimulated p70 S6K activity could be further enhanced by
another twofold after insulin treatment, which was accompanied by a
corresponding gel mobility band-shift of the protein (Fig. 4B).
|
|
Increased PI3K activation in fructose-fed rats.
Because both PKB and p70 S6K activities exhibited intriguing changes in
the fructose-fed rats, we next examined the effects of insulin on PI3K,
which has been shown to function upstream of PKB in a signaling
pathway. Immunoprecipitation studies with the monoclonal anti-p85 PI3K
antibody demonstrated that PI3K was maximally stimulated within 5 min
after insulin injection by ~3.5-fold in control animals (Fig.
5, A and
B). An increase in enzyme activity could be observed as early as 2 min postinjection, and the activity returned to near basal levels by ~15 min postinjection. Basal PI3K
activity was elevated about twofold in the hyperinsulinemic rats, which
was similar to the changes observed with PKB and p70 S6K.
Insulin-stimulated PI3K activity was increased ~4.7-fold in the
insulin-resistant animals (when compared with control basal levels) and
peaked at 5 min after insulin injection (Fig. 5,
A and
B).
|
Regulation of glycogen synthase kinase-3 in
fructose-fed rats.
Recent reports have demonstrated that the enzyme glycogen synthase
kinase-3
(GSK-3
) is phosphorylated and inhibited by PKB in vitro
and that this enzyme may be a physiological substrate for PKB (13).
Crude muscle homogenates were subjected to MonoS chromatography and
assayed for phosphotransferase activity directed against GSK-3PP.
Results from these experiments revealed two basal peaks of GSK-3PP
phosphotransferase activity, which eluted at a NaCl concentration of
~150-225 mM (Fig.
6A).
When the same fractions were assayed using the control (Ala21) peptide
as the substrate (not a substrate for GSK-3), no detectable
phosphotransferase activity was observed. Immunoblotting studies
identified GSK-3
only within MonoS fractions
26-29 (Fig.
6C). Insulin administration in
control rats caused a decrease in the activity of both the peaks, the
first of which has been previously shown to contain the
-isoform of
GSK-3 (44). In fructose-fed rats, basal activity in the second GSK-3PP
peak was depressed, although the first GSK-3PP peak remained unchanged
(Fig. 6B). Insulin injection
appeared to cause a slight increase in both the GSK-3PP peaks, although the change did not attain statistical significance. To further confirm
that the changes in the second peak of GSK-3PP phosphotransferase activity were due to GSK-3
, immunoprecipitation studies were performed on the specific MonoS fractions by use of antiserum against
this isoform of GSK-3. These experiments confirmed that insulin caused
a
50% inhibition of GSK-3
in control rats and that basal GSK-3
activity was already depressed in fructose-fed rats (Figs.
6D and 7).
No change was observed after insulin administration in fructose-fed
rats at any of the time points studied.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo insulin injection in normal rats activated PI3K, PKB-, and
p70 S6K in muscle lysates in a time-dependent fashion. There was a
temporal correlation between the activation of these kinases such that
the maximal activation of PI3K preceded that of PKB-
, the latter
being activated earlier than the p70 S6K. Furthermore, activation of
PKB-
was accompanied by a concurrent inhibition of GSK-3
,
supporting the notion that GSK-3
may be a physiological substrate
for PKB. One of the primary findings of the study was that basal
activities of PI3K, PKB-
, and p70 S6K were increased in fructose-fed
rats, indicating that this pathway was chronically upregulated in these
hyperinsulinemic rats. This is in marked contrast to results in
insulin-resistant, hyperinsulinemic models of experimental
non-insulin-dependent diabetes mellitus (NIDDM), where basal PI3K
activity is markedly decreased (2, 19). Similarly, PKB activation has
been reported to be reduced in muscle samples obtained from
insulin-resistant diabetic rats, which is in contrast to the present
findings (30, 48).
An important difference between the fructose-fed model and several other rodent models of diabetes is that the fructose-fed rats are not hyperglycemic. Hyperglycemia per se could lead to changes in the regulation of signal transduction pathways, because it leads to glucose insensitivity. Therefore, some of the differences in particular kinase activities between fructose-fed rats and those studied previously (19, 31) could be explained by the differences in circulating glucose concentrations between the different animal models. However, models such as the fa/fa Zucker rat are also either normoglycemic or mildly hyperglycemic, and yet they demonstrate very different changes in the activities of the enzymes studied (for example, insulin-stimulated PI3K activity is impaired in muscle from fa/fa rats, in contrast to the fructose-fed rats). Taken together, these data raise the possibility that the mechanisms underlying the insulin resistance of hypertension may differ from those seen in NIDDM. However, it is necessary to study the regulation of these enzymes in other insulin-resistant hypertensive models before any conclusions can be drawn.
Insulin injection further enhanced PI3K activity in fructose-fed rats,
indicating that the defect in insulin signaling in these rats resided
distal to the insulin receptor. Similarly, there was a marked increase
in p70 S6K activity after insulin administration in these rats,
although PKB- activity could not be further stimulated above basal
levels. This observation indicates that inputs other than the PI3K-PKB
pathway are responsible for the activation of p70 S6K in fructose-fed
rats. Several enzymes, including the classical PKC isoforms and the
p21-activated kinases (also called PAKs) have been demonstrated to
indirectly activate p70 S6K (10, 11). Which, if any, of these activate
p70 S6K in insulin-resistant animals remains to be determined. It has been reported that activation of p70 S6K is not essential for glucose
utilization, as rapamycin fails to inhibit glucose transport in
response to insulin (9). Our findings are consistent with these
results, because activation of p70 S6K was not impaired in animals that
were refractory to insulin's glucoregulatory effects.
GSK-3 is an enzyme that has been reported to regulate several of
insulin's physiological effects, including glycogen and protein synthesis (13, 47). Recent studies have demonstrated that GSK-3 is a
substrate of the enzyme PKB in vitro and in vivo (13). Intriguingly,
although basal PKB- activity was increased, basal GSK-3 activity was
chronically inhibited in fructose-fed rats, which is consistent with
the notion that the PKB- GSK-3 pathway is upregulated in these
hyperinsulinemic rats. What is perhaps more interesting is the
observation that, although both PKB-
and GSK-3
activities
remained unchanged after insulin injection in fructose-fed rats, the
animals still displayed a fall in plasma glucose concentration after
insulin injection. This indicates that activation of enzymes other than
the PKB
GSK-3 pathway is involved in mediating insulin's
glucoregulatory effects in fructose-fed rats. It should be noted that
PKB-
is one of the three isoforms of PKB and that insulin has been
shown to have differential effects on the activation of different PKB
isoforms (46). Only the PKB-
isoform was examined in the present
study, which is the primary isoform that is activated by insulin in
skeletal muscle (46). The
-isoform of PKB has been shown to be
minimally activated by insulin in muscle, although in adipocytes, both
the
- and
-isoforms are stimulated to a similar extent.
These data support recent observations that inhibition of GSK-3 by
insulin may not be sufficient to explain insulin-induced activation of
glycogen synthase. For example, the
3-receptor agonists BRL-37344
and isoproterenol decreased the GSK-3 activity in epididymal fat cells
without having any effect on the activity ratio of glycogen synthase
(38). Furthermore, the mechanisms underlying the glucoregulatory
effects of insulin may be distinct in different insulin-target tissues.
For example, in rat skeletal muscle, the stimulation of glycogen
synthase by insulin was shown to be partly sensitive to inhibition by
rapamycin (3), whereas in rat adipocytes rapamycin had no effect on the
glycogen synthase activity ratio (37).
The observation that, despite being insulin resistant, the fructose-fed rats demonstrated an apparently normal response to an acute insulin injection, deserves mention. When the fructose-fed rats are subjected to an acute insulin injection (2 U/kg), they attain plasma insulin concentrations that are at least two- or threefold higher than those seen during the normal postabsorptive state in these hyperinsulinemic rats. This amount of insulin, combined with the endogenous insulin already present in the animals, is sufficient to offset any insulin resistance and, hence, these animals show a normal decrease in plasma glucose levels.
In conclusion, we have demonstrated that the PI3K PKB
p70 S6K pathway is chronically upregulated in insulin-resistant, hyperinsulinemic, fructose-hypertensive rats. In addition, although insulin stimulation of PI3K and p70 S6K activities is further enhanced
in fructose-fed rats, both PKB and GSK-3
are resistant to insulin's
effects. To our knowledge, this is the first report that has examined
the regulation of the PI3K pathway in hyperinsulinemic hypertensive
rats. Further studies are required to elucidate insulin signal
transduction in other models of experimental hypertension and to link
the activation of the various enzymes to the final biological effects
of insulin.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Violet Yuen for expert technical assistance during
preparation of the tissue extracts, Dr. J. Woodgett for the GSK-3 antiserum, and Dr. Jasbinder Sanghera for valuable advice during the
course of this study.
![]() |
FOOTNOTES |
---|
This study was supported in part by grants from the Medical Research Council of Canada (MRCC) (to J. H. McNeill and S. L. Pelech). S. Bhanot was the recipient of a Heart and Stroke Foundation of Canada Fellowship, S. Verma was the recipient of a MRCC Fellowship, and S. L. Pelech was the recipient of a MRCC Industrial Scientist Award.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. L. Pelech, Dept. of Medicine, Rm. S125, 2nd floor, Koerner Pavilion, 2211 Wesbrook Mall, Univ. of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada (E-mail: spelech{at}home.com).
Received 29 October 1998; accepted in final form 20 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alessi, D. R.,
M. Andjelkovic,
B. Caudwell,
P. Cron,
N. Morrice,
P. Cohen,
and
B. A. Hemmings.
Mechanisms of activation of protein kinase B by insulin and IGF-1.
EMBO J.
15:
6541-6551,
1996[Abstract].
2.
Anai, M.,
F. Makato,
O. Takehide,
T. Jungo,
I. Kouichi,
K. Hideki,
F. Yasushi,
Y. Yoshio,
K. Masatoshi,
O. Yoshitomo,
and
A. Tomoichiro.
Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker Fatty rats.
Diabetes
47:
13-23,
1998[Abstract].
3.
Azpiazu, I.,
A. R. Saltiel,
A. A. DePaoli-Roach,
and
J. C. Lawrence, Jr.
Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal muscle involves mitogen-activated protein kinase-independent and rapamycin-sensitive pathways.
J. Biol. Chem.
271:
5033-5039,
1996
4.
Baron, A. D.,
G. Brechtel,
P. Wallace,
and
S. V. Edelman.
Rates and tissue sites of non-insulin- and insulin-mediated glucose uptake in humans.
Am. J. Physiol.
255 (Endocrinol. Metab. 18):
E769-E774,
1988
5.
Bellacosa, A.,
J. R. Testa,
S. P. Staal,
and
P. N. Tsichlis.
A retroviral oncogene, Akt, encoding a serine-threonine kinase containing a SH2-like region.
Science
254:
244-247,
1991.
6.
Bhanot, S.,
J. H. McNeill,
and
M. Bryer-Ash.
Vanadyl sulfate prevents fructose induced hyperinsulinemia and hypertension in rats.
Hypertension
23:
308-312,
1994[Abstract].
7.
Blenis, J.
Signal transduction via the MAP kinases: proceed at your own RSK.
Proc. Natl. Acad. Sci. USA
90:
5889-5892,
1993[Abstract].
8.
Burgering, B. M. T.,
and
P. J. Coffer.
Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction.
Nature
376:
599-602,
1995[Medline].
9.
Chang, P. Y.,
Y. LeMarchand-Brustel,
L. A. Cheatham,
and
D. E. Moller.
Insulin stimulation of mitogen-activated protein kinase, p90rsk, and p70 S6 kinase in skeletal muscle of normal and insulin-resistant mice. Implications for the regulation of glycogen synthase.
J. Biol. Chem.
270:
29928-29935,
1995
10.
Chou, M. M.,
and
J. Blenis.
The 70 kDa S6 kinase: regulation of a kinase with multiple roles in mitogenic signalling.
Curr. Opin. Cell. Biol.
7:
806-814,
1995[Medline].
11.
Chou, M. M.,
and
J. Blenis.
The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1.
Cell
85:
573-583,
1996[Medline].
12.
Coffer, P. J.,
and
J. R. Woodgett.
Molecular-cloning and characterization of a novel putative protein-serine kinase related to the cAMP-dependent and protein-kinase-C families.
Eur. J. Biochem.
201:
475-481,
1991[Abstract].
13.
Cross, D. A. E.,
D. R. Alessi,
P. Cohen,
M. Andjelkovic,
and
B. A. Hemmings.
Inhibition of GSK-3 by insulin mediated by PKB.
Nature
378:
785-789,
1995[Medline].
14.
DeFronzo, R. A.,
and
E. Ferrannini.
Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia and atherosclerotic cardiovascular disease.
Diabetes Care
14:
173-194,
1991[Abstract].
15.
Deibler, G. E.,
L. F. Boyd,
and
M. W. Kies.
Proteolytic activity associated with purified myelin basic protein.
Prog. Clin. Biol. Res.
146:
249-256,
1984[Medline].
16.
Dent, P.,
A. Lavoinne,
S. Nakielny,
F. B. Caudwell,
P. Watt,
and
P. Cohen.
The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle.
Nature
348:
302-308,
1990[Medline].
17.
Ferrannini, E.,
R. D. Buzzigoli,
L. Giorico,
L. Oleggini,
L. Graziadei,
R. Pedrinelli,
L. Brandi,
and
S. Bevilacqua.
Insulin resistance in essential hypertension.
N. Eng. J. Med.
317:
350-357,
1987[Abstract].
18.
Ferrari, P.,
and
P. Weidman.
Insulin, insulin sensitivity and hypertension.
J. Hypertens.
8:
491-500,
1990[Medline].
19.
Folli, F.,
M. J. Saad,
J. M. Backer,
and
C. R. Kahn.
Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus.
J. Clin. Invest.
92:
1787-1794,
1993[Medline].
20.
Gregory, J. S.,
T. G. Boulton,
B. C. Sang,
and
M. H. Cobb.
An insulin-stimulated ribosomal protein S6 kinase from rabbit skeletal muscle.
J. Biol. Chem.
264:
18397-18401,
1989
21.
Hei, Y. J.,
J. H. McNeill,
J. S. Sanghera,
J. Diamond,
M. Bryer-Ash,
and
S L. Pelech.
Characterization of insulin-stimulated seryl/threonyl protein kinases in rat skeletal muscle.
J. Biol. Chem.
268:
13203-13213,
1993
22.
Hei, Y. J.,
S. L. Pelech,
X. Chen,
J. Diamond,
and
J. H. McNeill.
Purification and characterization of a novel ribosomal S6 kinase from skeletal muscle of insulin-resistant rats.
J. Biol. Chem.
269:
7816-7823,
1994
23.
Ho, H.,
and
B. B. Hoffman.
Somatostatin inhibition of fructose-induced hypertension.
Hypertension
14:
117-120,
1989[Abstract].
24.
Hwang, I. S.,
H. Ho,
B. B. Hoffman,
and
G. M. Reaven.
Fructose induced insulin resistance and hypertension in rats.
Hypertension
10:
512-516,
1987[Abstract].
25.
Hwang, I. S.,
W. C. Huang,
J. N. Wu,
L. R. Shian,
and
G. M. Reaven.
Effect of fructose-induced hypertension on the renin-angiotensin-aldosterone system and atrial natriuretic factor.
Am. J. Hypertens.
2:
424-427,
1989[Medline].
26.
Jones, P. F.,
T. Jakubowicz,
F. J. Pitossi,
F. Maurer,
and
B. A. Hemmings.
Molecular-cloning and identification of a serine-threonine protein kinase of the 2nd messenger subfamily.
Proc. Natl. Acad. Sci. USA
88:
4171-4175,
1991[Abstract].
27.
Jue, T.,
D. L. Rothman,
G. I. Shulman,
B. A. Tavitian,
R. A. DeFronzo,
and
R. G. Shulman.
Direct observation of glycogen synthesis in human muscle with 13C NMR.
Proc. Natl. Acad. Sci. USA
86:
1439-1442,
1989[Abstract].
28.
Kohn, A. D.,
K. S. Kovacina,
and
R. A. Roth.
Insulin stimulates the activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase.
EMBO J.
14:
4288-4295,
1995[Abstract].
29.
Krieg, J.,
J. Hofsteenge,
and
G. Thomas.
Identification of the 40S ribosomal protein S6 phosphorylation sites induced by cycloheximide.
J. Biol. Chem.
263:
11473-11477,
1988
30.
Krook, A.,
Y. Kawano,
X. M. Song,
S. Efendic,
R. A. Roth,
H. Wallberg-Henriksson,
and
J. R. Zierath.
Improved glucose tolerance restores insulin-stimulated Akt kinase activity and glucose transport in skeletal muscle from Goto-Kakizaki (GK) rats.
Diabetes
36:
2110-2114,
1997.
31.
Krook, A.,
R. A. Roth,
X. J. Jiang,
J. R. Zierath,
and
H. Wallberg-Henriksson.
Insulin-stimulated Akt kinase activity is reduced in skeletal muscle from NIDDM subjects.
Diabetes
47:
1281-1286,
1998[Abstract].
32.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
33.
Lavoinne, A.,
E. Erikson,
J. L. Maller,
D. L. Price,
J. Avruch,
and
P. Cohen.
Purification and characterisation of the insulin-stimulated protein kinase from rabbit skeletal muscle: close similarity to S6 kinase II.
Eur. J. Biochem.
199:
723-728,
1991[Abstract].
34.
Lawrence, J. C., Jr.
Signal transduction and protein phosphorylation in the regulation of cellular metabolism by insulin.
Annu. Rev. Physiol.
54:
177-193,
1992[Medline].
35.
Lazar, D. F.,
R. J. Wiese,
M. J. Brady,
C. C. Mastick,
S. B. Waters,
K. Yamauchi,
J. E. Pessin,
P. Cuatrecasas,
and
A. R. Saltiel.
Mitogen activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin.
J. Biol. Chem.
270:
20801-20807,
1995
36.
Moule, S. K.,
and
R. M. Denton.
Multiple signalling pathways involved in the metabolic effects of insulin.
Am. J. Cardiol.
80:
41A-49A,
1997[Medline].
37.
Moule, S. K.,
N. J. Edgell,
G. I. Welsh,
T. A. Diggle,
E. J. Foulstone,
K. J. Heesom,
C. G. Proud,
and
R. M. Denton.
Multiple signalling pathways involved in the stimulation of fatty acid and glycogen synthesis by insulin in rat epididymal fat cells.
Biochem. J.
311:
595-601,
1995[Medline].
38.
Moule, S. K.,
G. I. Welsh,
N. J. Edgell,
E. J. Foulstone,
C. G. Proud,
and
R. M. Denton.
Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and -adrenergic agonists in rat epididymal fat cells.
J. Biol. Chem.
272:
7713-7719,
1997
39.
Moxham, C. M.,
A. Tabrizchi,
R. J. Davis,
and
C. C. Malbon.
Jun N-terminal kinase mediates activation of skeletal muscle glycogen synthase by insulin in vivo.
J. Biol. Chem.
271:
30765-30773,
1996
40.
Pelech, S. L.,
and
E. G. Krebs.
Mitogen-activated S6 kinase is stimulated via protein kinase C-dependent and independent pathways in Swiss 3T3 cells.
J. Biol. Chem.
262:
11598-11606,
1987
41.
Reaven, G. M.
Insulin resistance, hyperinsulinemia, hypertriglyceridemia and hypertension: parallels between human disease and rodent models.
Diabetes Care
14:
195-202,
1991[Abstract].
42.
Sturgill, T. W.,
L. B. Ray,
E. Erikson,
and
J. L. Mailer.
Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II.
Nature
334:
715-719,
1988[Medline].
43.
Sutherland, C.,
D. G. Campbell,
and
P. Cohen.
Identification of insulin-stimulated protein-kinase-1 as the rabbit equivalent of rskmo-2. Identification of two threonines phosphorylated during activation by mitogen activated protein kinase.
Eur. J. Biochem.
212:
581-588,
1993[Abstract].
44.
Sutherland, C.,
and
P. Cohen.
The alpha-isoform of glycogen synthase kinase-3 from rabbit skeletal muscle is inactivated by p70 S6 kinase or MAP-kinase-activated protein-kinase 1 in vitro.
FEBS Lett.
338:
37-42,
1994[Medline].
45.
Verma, S.,
S. Bhanot,
and
J. H. McNeill.
Antihypertensive effects of metformin in fructose induced hypertensive rats.
J. Pharmacol. Exp. Ther.
271:
1334-1337,
1994[Abstract].
46.
Walker, K. S.,
M. Deak,
A. Paterson,
K. Hudson,
P. Cohen,
and
D. R. Alessi.
Activation of protein kinase B beta and gamma isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase B alpha.
Biochem. J.
331:
299-308,
1998[Medline].
47.
Welsh, G. I.,
and
C. G. Proud.
Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B.
Biochem. J.
294:
625-629,
1993[Medline].
48.
Yamauchi, T.,
K. Tobe,
H. Tamemoto,
K. Ueki,
Y. Kaburagi,
R. Yamamoto-Honda,
Y. Takahashi,
F. Yoshizawa,
S. Aizawa,
Y. Akanuma,
N. Sonenberg,
Y. Yazaki,
and
T. Kadowaki.
Insulin signaling and insulin actions in the muscles and livers of insulin-resistant insulin receptor substrate-1 deficient mice.
Mol. Cell. Biol.
16:
3074-3084,
1996[Abstract].