1 Department of Medicine, Research Center, Centre Hospitalier de l'Université de Montréal, University of Montreal, Montreal H2W 1T8; and 2 Department of Medicine, Clinical Research Institute of Montreal, University of Montreal, Montreal, Canada H2W 1R7
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ABSTRACT |
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Type 1 and type 2 diabetic patients often show elevated plasma ketone body
concentrations. Because ketone bodies compete with other energetic
substrates and reduce their utilization, they could participate in the
development of insulin resistance in the heart. We have examined the
effect of elevated levels of ketone bodies on insulin action in primary
cultures of adult cardiomyocytes. Cardiomyocytes were cultured with the
ketone body -hydroxybutyrate (
-OHB) for 4 or 16 h, and
insulin-stimulated glucose uptake was evaluated. Although short-term
exposure to ketone bodies was not associated with any change in insulin
action, our data demonstrated that preincubation with
-OHB for
16 h markedly reduced insulin-stimulated glucose uptake in
cardiomyocytes. This effect is concentration dependent and persists for
at least 6 h after the removal of
-OHB from the media. Ketone
bodies also decreased the stimulatory effect of phorbol 12-myristate
13-acetate and pervanadate on glucose uptake. This diminution could not
be explained by a change in either GLUT-1 or GLUT-4 protein content in
cardiomyocytes. Chronic exposure to
-OHB was associated with
impaired protein kinase B activation in response to insulin and
pervanadate. These results indicate that prolonged exposure to ketone
bodies altered insulin action in cardiomyocytes and suggest that this
substrate could play a role in the development of insulin resistance in
the heart.
insulin resistance; ketone bodies; glucose uptake; protein kinase B; heart
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INTRODUCTION |
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DIABETIC SUBJECTS have an increased morbidity and mortality from cardiac disease. In men and women, diabetes raises the risk of developing heart disease by 2.4 and 3.5 times, respectively (40). Recent studies suggest that abnormal regulation of glucose uptake in the heart plays a role in cardiac dysfunction. In humans, a reduction in cardiac glucose uptake has been observed in obese and type 2 diabetic subjects (3). Diminished glucose utilization and contractile dysfunction have been also observed in the heart of db/db mice, a model of type 2 diabetes (2). These abnormalities were normalized by selective overexpression of a glucose transporter (GLUT-4) in the heart (2).
In addition to glucose, the heart also uses ketone bodies as an energy source. Studies have shown that acetoacetate reduces the oxidation of both fatty acids and lactate in the heart (25, 36). Potentially, ketone bodies could modulate glucose utilization and insulin action in the heart. Various pathophysiological situations such as diabetes and starvation are associated with hyperketonemia (25). Significant increases in ketone body concentration are also observed in chronic heart failure (27, 28) and after a high-fat diet (25). Interestingly, perfusion of the rat heart with ketone bodies as the sole substrate provoked a severe decline in contractile function, a condition that was normalized with the inclusion of glucose in the perfusate (41). In congestive heart failure, the increase in plasma ketone body concentration correlates with the severity of cardiac dysfunction (28).
The major glucose transporters expressed in the heart are GLUT-1 and GLUT-4. In this tissue, >80% of GLUT-1 resides at the plasma membrane, and it has been suggested that this transporter is primarily responsible for basal glucose uptake (13). In contrast, GLUT-4 is found primarily in intracellular vesicles (4, 13). Insulin stimulation of glucose uptake is accomplished by recruiting GLUT-4 and to a lesser extent GLUT-1 from their intracellular sites to the plasma membrane (13, 37, 46, 48).
Our understanding of the insulin-signaling cascade has increased
dramatically in recent years, and many additional proteins involved in
insulin action have been identified. One of these proteins is the
serine/threonine kinase protein kinase B (PKB) or Akt. Overexpression
of a constitutively active PKB increases GLUT-4 translocation in both
muscle cells (15, 45) and adipocytes (22,
45). In addition, microinjection of either a PKB substrate peptide or PKB antibody reduces GLUT-4 in response to insulin by 50%
(18). On the other hand, transfection with dominant
negative PKB mutants has yielded contradictory results
(45). Recent studies have demonstrated, however, that in
mice lacking the gene for PKB-, the ability of insulin to reduce
plasma glucose concentration is reduced (6). Thus the bulk
of the data suggest that PKB plays a role in insulin-mediated glucose transport.
Because of the potential role of ketone bodies in the regulation of substrate utilization, we determined whether elevated concentrations of this substrate induce insulin resistance in the heart. To address this question, we have examined the effect of ketone bodies on insulin-stimulated glucose uptake and PKB activation in primary cultures of adult cardiomyocytes.
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EXPERIMENTAL PROCEDURES |
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Chemicals.
All cell culture solutions, supplements, and DNase I were from
Sigma-Aldrich Canada (Oakville, ON). Collagenase was obtained from
Worthington Biochemical (Lakewood, NJ). Human insulin (Humulin R) was
from Eli Lilly Canada (Toronto, ON). Anti-GLUT-1 and GLUT-4 antibodies
were from Research Diagnostics (Flanders, NJ) and Santa Cruz
Biotechnology (Santa Cruz, CA), respectively. Polyclonal antibodies for
phospho-PKB (Ser473) and PKB- were purchased from New
England Biolabs (Beverley, MA). 2-[3H]deoxyglucose (2-DG)
was purchased from NEN Research Products (Boston, MA). The enhanced
chemiluminescence detection system was from Amersham Pharmacia
Biotechnology (Baie d'Urfée, QC, Canada). The
bicinchoninic acid (BCA) protein assay kit was purchased from
Pierce (Rockford, IL). All electrophoresis reagents were obtained from
Boehringer-Mannheim (Laval, QC, Canada). Potassium diperoxovanadate was
synthesized according to Ravishankar et al. (34).
Isolation of adult rat cardiomyocytes.
All experiments conformed to guidelines of the Canadian Council of
Animal Care and were approved by the Animal Care Committee of the
Centre Hospitalier de l'Université de Montréal. Male Sprague-Dawley rats weighing 175-200 g were injected with heparin sulfate (500 units ip) 15 min before anesthesia with pentobarbital sodium (60 mg/kg ip). The heart was excised, and calcium-tolerant cardiomyocytes were isolated by the Langendorff method (retrograde perfusion), as described previously (43). During the whole
procedure, the cells were maintained at 37°C. Briefly, the hearts
were rinsed (4 ml/min) for 5 min in Krebs-Ringer (KR) buffer containing
(in mM) 119 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 11 dextrose, and
25 HEPES, pH 7.4. The heart was then perfused with a calcium-free KR
solution for 5 min to stop spontaneous cardiac contractions. This was
followed by perfusion with KR buffer supplemented with 0.05%
collagenase and 15 mM 2,3-butanedione monoxime for 20 min, after which
the ventricles were separated from the atria. Ventricles were minced in
KR supplemented with 0.05% collagenase, 15 mM 2,3-butanedione monoxime, and 0.2 mg/ml DNase I. The resulting cell suspension was
filtered through a nylon mesh and centrifuged at 1,000 g for 45 s. The cells were then diluted and allowed to sediment in the washed solution two times. Freshly isolated cells were diluted in
culture medium 199 (5.5 mM glucose) supplemented with 0.2% BSA (fatty
acid free), 5 mM creatine, 2 mM L-carnitine, 5 mM taurine, 107 M insulin, 0.1 M ascorbic acid, 100 IU/ml penicillin,
25 µg/ml gentamicin, and 100 µg/ml streptomycin. Cell viability was
determined by the percentage of rod-shaped cells and averaged
90-95%. Cardiomyocytes (1 × 105 cells/ml) were
plated on laminin-coated dishes. After 4 h, the cells were
washed to remove damaged cells and debris. The cells were
then incubated with media containing 5.5 mM glucose alone or were
supplemented with 5 mM
-hydroxybutyrate (
-OHB) for 16 h or
as stated in legends for Figs. 1-7.
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Glucose uptake in primary cultures of cardiomyocytes.
On the day of the study, the cells were washed two times with 2 ml of
KR buffer to remove insulin, glucose, and -OHB. The cells were then
incubated in 1 ml of fresh KR buffer containing no glucose, insulin, or
-OHB for 30 min to restore basal glucose uptake. We then added 0.5 µCi/ml 2-DG and 10
8 M insulin, and glucose uptake was
measured over a period of 30 min as described previously
(7). The cells were also stimulated with 100 or 500 nM
phorbol 12-myristate 13-acetate (PMA) or 100 µM pervanadate. Under
these conditions, 2-DG uptake was linear over 45 min. The uptake was
terminated by two rapid washes with 2 ml of ice-cold KR buffer. Cells
were disrupted with 1 ml of 0.05 M NaOH (60 min at 37°C), and
cell-associated radioactivity was determined by scintillation counting.
Glucose uptake was expressed as becquerels per milligram of protein.
Protein concentration was quantified using the BCA assay with BSA as a standard.
PKB activation.
Cells were pretreated with 5 mM -OHB for 16 h. On the day of
study, the cells were washed two times with 4 ml of media containing no
insulin or
-OHB. The cells were then incubated in 2 ml of media
containing no insulin or
-OHB for 1 h. They were then treated with saline, 10
8 M insulin, or 100 µM pervanadate for 5 min. The reaction was stopped by two rapid washes with ice-cold KR
buffer. Cells were lysed in buffer containing 25 mM Tris · HCl,
pH 7.4, 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM sodium fluoride,
10 mM sodium pyrophosphate, 2 mM EGTA, 2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 2 mM benzamidine, 10 µg/ml aprotinin,
0.5 µg/ml leupeptin, 1% Triton X-100, and 0.1% SDS. The lysate was
then centrifuged for 10 min at 12,000 g at 4°C to remove
insoluble material, and the resulting supernatant was used for immunoblotting.
Gel electrophoresis and immunoblotting.
Samples were electrophoresed on a 10% SDS polyacrylamide gel and
transferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA) for Western blotting. The membranes were blocked for 1 h in 5% (wt/vol) nonfat dry milk in PBS. They were then incubated with an appropriate primary antibody. For the PKB experiments, the blots were first probed with anti-phosphorylated (Ser473) PKB antibody and then reprobed with anti--PKB.
This was followed by a second incubation with the appropriate secondary
antibodies conjugated to horseradish peroxidase, and the
antigen-antibody complex was detected with the enhanced
chemiluminescence method. Quantitative analysis was performed using a
scanning densitometer.
Statistical analysis. Statistical analysis was performed using one-way ANOVA for multiple comparisons. This was followed by a Tukey's post hoc test. All data are reported as means ± SE. Glucose uptake data are means of at least five independent experiments. For the PKB experiments, densitometric analysis was performed on three independent experiments. A value of P < 0.05 was considered significant.
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RESULTS |
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Induction of insulin resistance by ketone bodies.
Because ketone bodies are increased under various pathophysiological
situations, we examined whether an elevation in their concentration
would decrease glucose uptake and create a state of insulin resistance
in the heart. To address this question, primary cultures of adult
cardiomyocytes were incubated with the ketone body -OHB for either 4 or 16 h, and insulin-stimulated glucose uptake was evaluated. We
chose
-OHB because it is the primary ketone body produced during
hyperketonemia (25). Addition of 10
8 M
insulin increased glucose uptake from to 7.2 to 15.2 Bq/mg protein in
cardiomyocytes (P < 0.05; Fig.
1A). The strength of this
effect was comparable to that in previous reports in the literature for
these cells (9). Although exposure for 4 h to 5 mM
-OHB did not affect basal or insulin-mediated glucose uptake, our
results demonstrated that incubation with
-OHB for 16 h
inhibited the stimulatory effect of insulin on glucose uptake in
cardiomyocytes (P < 0.05; Fig. 1B).
The development of insulin resistance induced by ketone bodies is
dose dependent.
Plasma ketone body concentrations vary greatly depending on the
nutritional or pathophysiological state of the animal. In healthy
individuals, ketone body concentrations are usually 0.2 mM. During
hyperketonemia, however, these levels can increase up to 1 mM, while
during ketoacidosis they reach levels between 3 and 20 mM
(25). Similarly, in the rat, diabetes and starvation are
associated with concentrations of
-OHB >1 mM (19). We
determined the concentration of ketone body required to induce insulin
resistance in cardiomyocytes. Cardiomyocytes were incubated with
increasing concentrations of
-OHB for 16 h, and glucose uptake
was evaluated (Fig. 2). Exposure to
either 1 or 5 mM
-OHB inhibited insulin action in these cells
(P < 0.05). Although not significant, a 20% decrease in
the maximal insulin effect was observed at 0.5 mM
-OHB. Pretreatment
with 0.2 mM
-OHB has no effect on insulin action. In addition,
incubation with increasing concentrations of
-OHB was associated
with a progressive reduction in basal glucose transport. Although never
significant, this effect was seen in most of the experiments (Fig.
3 and see Fig. 5). Thus the decreased
response to insulin after pretreatment with ketone bodies was dose
dependent and occurred at concentrations that are associated with
physiological and pathological states in vivo. All subsequent
experiments were done at 5 mM
-OHB.
Role of insulin in the reduction of glucose uptake induced by
ketone bodies.
Our preparations of adult cardiomyocytes were cultured in the presence
of 107 M insulin to maintain cell survival.
Desensitization of the glucose uptake process by high insulin
concentrations has been observed in both adipocytes (14,
42) and skeletal muscle (16) and could play a role
in the development of ketone body-induced insulin resistance. Thus the
reduction in insulin-stimulated glucose uptake that we observed with
ketone bodies could potentially result from a combination of
elevated concentrations of insulin and
-OHB. Adult
cardiomyocytes were cultured with either 10
11 or
10
7 M insulin in the presence or absence of ketone bodies
for 16 h, and insulin-mediated glucose transport was examined. We
chose these concentrations because complete removal of the hormone from the media for long periods of time decreased insulin action in primary
cultures of adult cardiomyocytes (data not shown and Ref. 9). As shown in Fig. 3, reducing the concentration of
insulin in the media from 10
7 to 10
11 M
caused a slight increase in the effect of insulin on cardiomyocytes. This protocol did not, however, prevent the development of insulin resistance, and the effect of the hormone was reduced by 50% both at
10
7 and 10
11 M insulin (P < 0.05).
Resensitization of insulin-mediated glucose uptake after ketone
body exposure.
To better understand the mechanism underlying the development of
insulin resistance by ketone bodies, we next investigated the time
course necessary to reestablish insulin action in insulin-resistant cells after removal of ketone bodies. Cardiomyocytes were exposed to 5 mM -OHB for 16 h, as described above. The cells were washed two
times with media containing no
-OHB and then incubated for a period
of 4, 6, or 8 h before evaluation of glucose uptake. Our results
demonstrated that removal of
-OHB for either 4 or 6 h did not
improve insulin action in these cells. A period of 8 h was
required to restore insulin responsiveness in
-OHB-pretreated cardiomyocytes (Fig. 4). These results
indicate that insulin resistance persists for an extended period of
time, even after the removal of
-OHB from the media.
Effect of ketone bodies on the activation of glucose uptake by
PMA and pervanadate.
We next examined whether ketone bodies alter the response to other
glucose transport agonists. It has been shown that, in skeletal muscle,
phorbol esters mediate glucose uptake by a mechanism distinct from
insulin (17). Phorbol esters are functional analogs of
diacylglycerol that are able to activate classical and novel protein
kinase C (PKC). Acute activation of PKC was achieved with the phorbol
ester PMA. As shown in Fig.
5A, exposure to 100 and 500 nM
PMA increased glucose uptake from 12.3 to 17.5 and 20.7 Bq/mg protein,
respectively. In adult cardiomyocytes, the effect of insulin and PMA on
glucose uptake was not additive. Pretreatment with -OHB for 16 h reduced the effect of 100 and 500 nM PMA on glucose uptake by 55 and
64%, respectively (P < 0.05).
Effect of ketone bodies on GLUT-1 and
GLUT-4 expression.
The reduction in insulin-stimulated glucose uptake by ketone bodies
could result from changes in the expression of the glucose transporter
in cardiomyocytes. To address this possibility, we evaluated both
GLUT-1 and GLUT-4 content in control and -OHB-treated cardiomyocytes. As shown in Fig. 6,
ketone bodies did not modify either GLUT-1 or GLUT-4 protein content in
these cells.
Impairment in PKB activation in response to ketone
bodies in adult cardiomyocytes.
PKB is a serine/threonine kinase that has been implicated in insulin
action. Studies by Tsiani et al. (44) have shown that pervanadate is a powerful activator of PKB. Because PKB activation also
plays a key role in insulin action, we were interested in determining
whether the activation of this enzyme by either insulin or pervanadate
was altered in -OHB-pretreated cardiomyocytes. To address this
question, cardiomyocytes were pretreated with ketone bodies for 16 h, after which PKB activation in response to either insulin or
pervanadate was evaluated. To ensure that these differences were not
the result of unequal PKB expression, the relative levels of PKB were
determined by immunoblotting. Ketone bodies did not affect PKB
expression in cardiomyocytes (Fig.
7B). In control cells, insulin
and pervanadate induce a four- to six-fold increase in PKB
phosphorylation (Fig. 7A). Ketone body pretreatment
decreases PKB phosphorylation in response to insulin and pervanadate by
84 and 69%, respectively (Fig. 7C). In addition to its
effect on insulin and pervanadate action,
-OHB-treated cardiomyocytes show a tendency toward lower basal PKB phosphorylation.
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DISCUSSION |
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Diabetic ketoacidosis is a major complication of type 1 diabetes.
Less well known, however, is that plasma ketone body concentrations are
also increased in poorly controlled type 2 diabetic patients, particularly in African-Americans (1, 25). Higher ketone body levels have also been observed during chronic heart failure and
after consumption of a high-fat diet (25, 27, 28).
Therefore, there exist a number of physiological and pathological
situations where plasma ketone body concentrations are augmented.
Because ketone bodies compete with various metabolic substrates such as fatty acids, lactate, or glucose for utilization by the heart (31, 36, 47), their contribution to the energy supply of the myocardium will increase significantly during hyperketonemia. Our
results support a role for ketone bodies in the regulation of substrate
utilization and demonstrate for the first time that chronic exposure to
-OHB induces insulin resistance in primary cultures of adult
cardiomyocytes. We have demonstrated that 1) prolonged
exposure to
-OHB decreases insulin responsiveness as evaluated by
glucose transport and PKB activation, 2) the development of
insulin resistance by
-OHB is concentration dependent, and 3) in ketone body-treated cardiomyocytes, impaired glucose
uptake in response to insulin persists for at least 6 h after the
removal of
-OHB. Together, these data suggest that ketone bodies can play an important role in the regulation of glucose uptake in cardiomyocytes.
Plasma ketone body concentrations can rise dramatically during
diabetes, reaching levels up to 20 mM (25). Our results
demonstrated that a diminution in insulin-mediated glucose transport
occurs after prolonged exposure to ketone bodies (16 h) at
concentrations of 1 mM and greater. This could have clinical
significance because it has been shown that, during diabetic
ketoacidosis, plasma levels of ketone bodies usually remain elevated
for >12 h after the beginning of treatment to reduce their levels
(25). Thus the reduction in insulin responsiveness
observed in -OHB-pretreated cardiomyocytes in vitro occurs at
concentrations and within a time frame that are consistent with a role
for this substrate in the regulation of glucose transport during the
etiology of diabetic complications. Because our measurements were done
at supraphysiological insulin concentrations, we do not know whether
ketone bodies also alter insulin sensitivity in these cells. Studies by
Fischer et al. (12) have demonstrated that the inhibitory
effect of various cardiac substrates on glucose transport are more
pronounced at submaximal than at maximal insulin concentration. It
would thus be very interesting to determine whether prolonged exposure
to ketone bodies also alters insulin sensitivity in addition to its effect on insulin responsiveness.
Our results show that short-term exposure to -OHB (4 h) does not
modify insulin action in cardiomyocytes. Therefore, the inhibition of
insulin-stimulated glucose uptake observed in these cells is not
consistent with a direct competition between glucose and
-OHB as
energy sources. However, the regulation of glucose uptake could
potentially result from the intracellular metabolism of
-OHB. It has
been shown that ketone bodies modulate the production of a number of
metabolites in the heart. Perfusion of the isolated heart with the
ketone body acetoacetate increases the concentration of acetyl-CoA,
acetoacetyl-CoA, and citrate (31-33, 36). Randle and
coworkers (33) have proposed that an increase in citrate and acetoacetyl-CoA levels could inhibit phosphofructokinase and pyruvate dehydrogenase activity, respectively. This would lead to an
increase in glucose 6-phosphate concentrations that could then inhibit
hexokinase activity and reduce glucose uptake in the heart. An increase
in glucose 6-phosphate has been observed in hearts perfused with
acetoacetate and could play a role in the decreased glucose uptake seen
in
-OHB-treated cardiomyocytes (36). A second
possibility is that ketone bodies increase glycogen and/or triglyceride
content in cardiomyocytes and could reduce glucose transport by a
feedback mechanism. An increase in glycogen accumulation could be
caused either by the stimulation of glycogen synthase activity by
glucose 6-phosphate or by an increase in
-OHB utilization during
hyperketonemia, which could divert some of the glucose taken up
by the cardiomyocytes toward either glycogen or lipid synthesis. This
would also explain the 8-h period that is necessary to restore insulin
action in
-OHB-treated cardiomyocytes. In skeletal muscle, an
increase in glycogen or triglycerides levels has been associated with
reduced insulin-stimulated glucose uptake (16, 20, 21,
35). Similar mechanisms could also operate in the heart and
induce insulin resistance in this tissue. We are currently examining
these various possibilities.
In addition to its effect on insulin action, -OHB induces a
nonsignificant decrease in basal glucose uptake compared with control
cells. Because
-OHB induces a similar decrease in basal glucose
transport at both 10
7 and 10
11 M insulin,
we believe that this reduction is not the result of incomplete removal
of the hormone during the washes. Reductions in basal and
insulin-stimulated glucose uptake have been observed in cardiomyocytes
exposed to extracellular ATP because of a redistribution of glucose
transporters from the plasma membrane to their intracellular site
(11). Potentially, ketone bodies could promote a similar internalization of these transporters and thus lower basal glucose transport in these cells.
Our results demonstrated that neither GLUT-4 nor GLUT-1 protein content
was altered by ketone body treatment. Thus a change in glucose
transporter expression cannot explain the reduction in
agonist-stimulated glucose uptake observed in cardiomyocytes. In the
heart, glucose uptake results from glucose transport into the cells and
its subsequent phosphorylation by the enzyme hexokinase. It has been
shown that, during insulin stimulation, the rate-limiting step for this
process switches from glucose transport to glucose phosphorylation
(29). Recent studies suggest that, in the presence of
-OHB, glucose transport may not be the rate-limiting step for
glucose uptake (5). Thus ketone body-induced insulin
resistance could be the result of a decrease in GLUT-4 translocation, a
decrease in hexokinase activity, or a combination of both. Ketone
bodies could also impair glucose transporter trafficking or their
fusion/insertion in the plasma membrane independently of the
insulin-signaling cascade. A defect at either of these steps would
diminish the effect of glucose transport agonists in
-OHB-pretreated cardiomyocytes.
Our results show that, in addition to insulin, ketone bodies also
impair the activation of glucose uptake in response to both phorbol
esters and pervanadate. This suggests that more than one signaling
pathway is affected by ketone body treatment or alternatively that they
regulate a common step of the three glucose uptake agonists or a
combination of both. Recent data support a role for PKB in the
regulation of insulin-stimulated glucose uptake. Impaired PKB
activation in response to insulin has been observed in skeletal muscle
from type 2 diabetic subjects (24) and diabetic
Goto-Kakizaki rats (39). Furthermore, improvement in
glucose uptake in these rats was associated with normalization of PKB
activation in response to insulin treatment (23). For
these reasons, we investigated the effect of -OHB on both insulin
and pervanadate activation of PKB. Our results demonstrated that
-OHB impairs PKB phosphorylation by both agonists in adult
cardiomyocytes. It has been shown that PKB lies downstream of the
insulin receptor substrates (IRS)-phosphatidylinositol (PI)-3-kinase
signaling pathway. The induction of insulin resistance caused
by ketone bodies could therefore arise from impaired activation of this
signaling cascade, resulting in decreased PKB activation. Alternatively, the block in insulin action after ketone body treatment could directly target PKB. In skeletal muscle, both mechanisms have
been implicated in the development of insulin resistance by different
fatty acids. Infusion of a mixture of fatty acids reduced both IRS-1
tyrosine phosphorylation and PI 3-kinase activity in human skeletal
muscle (8). On the other hand, Schmitz-Peiffer et al.
(38) have shown that the fatty acid palmitate inhibits PKB
activation by insulin without altering IRS-1 phosphorylation and PI
3-kinase activation in response to the hormone. Although we have
established that ketone bodies act at the level of PKB, it remains to
be determined whether this effect involves alteration in PI 3-kinase
activation and/or direct inhibition of PKB. Potentially, ketone bodies
could activate a phosphatase, resulting in enhanced PKB
dephosphorylation and thus inactivation of the enzyme. Selective dephosphorylation and inhibition of PKB by protein phosphatase 2A has
been observed after osmotic shock (30).
Interestingly, hyperosmotic stress also prevents PKB activation in
response to pervanadate (30). It is therefore possible
that ketone bodies antagonize both insulin and pervanadate action in
cardiomyocytes by promoting PKB dephosphorylation.
In conclusion, our results demonstrate that chronic exposure to ketone bodies inhibits insulin-stimulated glucose uptake in primary cultures of adult cardiomyocytes. This effect is both time and concentration dependent. Ketone bodies also alter the ability of both phorbol esters and pervanadate to stimulate glucose uptake in these cells. Our data also indicate that ketone body pretreatment is associated with impaired PKB activation in response to insulin and pervanadate.
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ACKNOWLEDGEMENTS |
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We thank Marie-Claude Guertin for statistical analysis and Dr. T. Ramasarma from the Indian Institute of Science for the generous gift of potassium diperoxovanadate.
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FOOTNOTES |
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This work was supported by the Canadian Diabetes Association and the Association Diabète Québec. L. Coderre is a scholar of the Canadian Diabetes Association.
Address for reprint requests and other correspondence: L. Coderre, Research Center, Hôtel-Dieu-CHUM, 3850 St. Urbain, Montréal, Québec, Canada H2W 1T8 (E-mail: lise.coderre{at}umontreal.ca).
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 15 November 2000; accepted in final form 12 July 2001.
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