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Exercise modulates myocardial protein kinase B/Akt in Zucker obese rats

Authors:
Barbara Huisamen at Stellenbosch University
Barbara Huisamen
Amanda Lochner at Stellenbosch University
Amanda Lochner
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SCIENTIFIC LETTER
Exercise modulates myocardial protein kinase B/Akt in
Zucker obese rats
B Huisamen, A Lochner
...............................................................................................................................
Heart 2005;91:227–228. doi: 10.1136/hrt.2003.027094
In type 2 diabetes, heart muscle is insulin resistant. Protein
kinase B (PKB)/Akt) acts as a mediator of the metabolic
effects of insulin, including translocation of the insulin
sensitive glucose transporter, GLUT4, in a manner dependent
on phosphatidylinositol-3-kinase (PI-3-K).
1
We have pre-
viously described dysregulation of PKB/Akt in hearts from a
rat model of type 2 diabetes, the Zucker fa/fa rat.
2
Exercise or contraction of heart muscle does not activate
PKB/Akt or PI-3-K but can induce GLUT4 translocation and
glucose uptake.
3
However, exercise alleviates peripheral
insulin resistance at a currently unknown, post-receptor
level. To better understand the mechanism whereby this may
be accomplished, we investigated whether chronic exercise
will induce beneficial changes in the regulation of PKB/Akt
that translates into improved myocardial insulin stimulated
glucose uptake.
METHODS
To accomplish this, we subjected Zucker fa/fa rats (20 weeks
old at the beginning of training) and a control group of age
matched Wistar rats, to an exercise training programme, and
documented changes in glucose uptake, myocardial GLUT4,
and PKB/Akt expression and phosphorylation, in comparison
to their sedentary counterparts. Rats were trained by
swimming for four weeks, six days per week, on set times
each day, starting at five minutes per day, escalating to 90
minutes per day, to avoid stress. The water temperature was
34˚
C and for half the exercise time, rats were weighted with
2% of their body weight, carried on their tails. Animals were
sacrificed by intraperitoneal injection of sodium pentobarbi-
tone (0.1 mg/g) after which ventricular myocytes were
harvested via standard methods.
4
2-Deoxy-D-glucose (2-DG) uptake by cardiomyocytes was
measured as previously described
4
while PKB/Akt phosphor-
ylation and GLUT4 expression were determined via standard
Western blotting techniques and suitable antibodies. PKB/
Akt kinase activity was determined using a commercial non-
radioactive assay system (Cell Signaling Technology). Laser
scanning densitometry and suitable software (UN-SCAN-IT,
Silkscience) were used to analyse the Western blots.
Comparisons of datasets were made either by analysis of
variance (ANOVA) followed by a Bonferroni correction or a
Student’s ttest for unpaired data. A probability value of
p,0.05 was considered significant.
RESULTS
Blood glucose determinations (Boehringer Mannheim,
Accutrend glucose meter) at the time of euthanasia,
demonstrated that Zucker fa/fa rats were diabetic at this age
(mean (SEM) 10.3 (0.3) mmol/l (fa/fa)v7.8 (0.4) mmol/l
(Wistar), p ,0.01, n = 6). Training lowered the blood
glucose of the fa/fa group to 8.8 (0.6) mmol/l (p ,0.05,
n = 7).
2-DG accumulation by cardiomyocytes from sedentary
diabetic rats (15.3 (1.4) pmol/mg protein/30 mins) was lower
than the sedentary control group (25.5 (1.8) pmol/mg
protein/30 mins), while insulin could not elicit a significant
response in the former (19.9 (3.6) v99.6 (8.7)). Exercise had
no effect on basal glucose uptake (15.2 (1.7) pmol/mg
protein/30 mins), but improved the insulin stimulated
response to 38.5 (5.1) pmol/mg protein/30 mins in the
diabetic rats. This was, however, still less than controls after
exercise. Concurrently, we found a 30% lower GLUT4 content
in diabetic versus control hearts. Expression of this protein
was upregulated by exercise in the diabetic hearts only, to a
level similar to that of control hearts after exercise.
We furthermore documented raised serine-473 phosphor-
ylation of PKB/Akt in diabetic hearts compared to control
hearts, under basal conditions. This was coupled to equal
expression and kinase activity of the protein. Nevertheless, it
was less sensitive to stimulation by insulin. Following
training however, insulin stimulated phosphorylation of
PKB/Akt was equal to that found in control hearts (fig 1).
DISCUSSION
It is well established that exercise can improve insulin
resistance by sensitising muscle to insulin mediated glucose
Figure 1 (A) Profile of serine-473 phosphorylation of PKB/Akt.
(B) A representative Western blot of PKB/Akt serine-473
phosphorylation.
Abbreviations: 2-DG, 2-deoxy-D-glucose; PI-3-K, phosphatidylinositol-
3-kinase; PKB, protein kinase B
227
www.heartjnl.com
metabolism, and it has been used to treat or prevent type 2
diabetes in patients successfully. Glucose transport is the rate
limiting step in muscle glucose utilisation
5
and the capacity
for glucose transport may vary according to the total amount
of GLUT4 protein. Exercise raised GLUT4 expression in
diabetic hearts; however, despite this upregulation, insulin
stimulated glucose uptake was still only 50% of control
values, indicating either defective translocation of the
transporter or glucose uptake per se.
A high level of serine-473 phosphorylation of PKB/Akt is
usually associated with activation of the kinase and should
lead to enhanced glucose uptake. However, we found high
basal phosphorylation levels associated with low glucose
uptake in sedentary diabetic animals. To the best of our
knowledge, this is the first demonstration of such dysregula-
tion at the level of PKB/Akt in diabetes. Exercise normalised
the phosphorylation pattern of PKB/Akt in diabetic hearts, an
effect probably caused by improved insulin signalling to the
applicable phosphatases. This would mean that improved
PKB/Akt phosphorylation is not the mediator of improved
insulin resistance but an effect thereof. Furthermore, despite
normal phosphorylation of this kinase in cardiomyocytes
from trained diabetic rats and normalisation of GLUT4
expression, insulin stimulated glucose uptake was still
curtailed. This result is an important indication that a major
alteration in the diabetic heart, leading to faulty glucose
metabolism, must be in the functioning or translocation of
the GLUT4 protein. These results suggest the possibility that
this signalling pathway is a potential target of treatment for
type 2 diabetes.
Authors’ affiliations
.....................
B Huisamen, A Lochner, Department of Medical Physiology and
Biochemistry, Faculty of Medicine, University of Stellenbosch, Republic of
South Africa
Correspondence to: Dr Barbara Huisamen, Department of Medical
Physiology and Biochemistry, Faculty of Health Sciences, University of
Stellenbosch, PO Box 19063, Tygerberg 7505, Republic of South Africa;
bh3@sun.ac.za
Accepted 15 April 2004
REFERENCES
1Shepherd PR, Nave BT, Rincon J, et al. Involvement of phosphoinositide 3-
kinase in insulin stimulation of MAP-kinase and phosphorylation of protein
kinase B in human skeletal muscle: Implications for glucose metabolism.
Diabetologia 1997;40:1172–7.
2Huisamen B. Protein kinase B in the diabetic heart. Mol Cell Biochem
2003;249:31–8.
3Lund S, Pryor PR, stergaard S, et al. Evidence against protein kinase B as a
mediator of contraction-induced glucose transport and GLUT4 translocation in
rat skeletal muscle. FEBS Letters 1998;425:472–4.
4Fischer Y, Rosen H, Kammermeier H. Highly insulin-responsive isolated rat
heart muscle cells yielded by a modified isolation method. Life Sciences
1991;49:1679–88.
5Cline GW, Petersen KF, Krssak M, et al. Impaired glucose transport as a cause
of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes.
N Eng J Med 1999;341:240–6.
IMAGES IN CARDIOLOGY ............................................................................
doi: 10.1136/hrt.2004.037242
Value of the unipolar electrogram in the diagnosis of right ventricular perforation following
pacemaker implantation
A76 year old patient with parox-
ysmal atrial fibrillation and recur-
rent syncope caused by sinus
arrest was admitted for permanent
pacemaker implantation. This was
uncomplicated with satisfactory pacing
parameters (ventricular lead: threshold
0.7 V at 0.5 ms, R wave 18.1 mV). A
routine pacing check the following day
revealed complete loss of ventricular
pacing and abdominal twitching during
unipolar ventricular pacing. The sensed
unipolar ventricular electrogram demon-
strated a dominant, upright R wave
(panel A) and a postero-anterior chest
xray showed the lead tip pointing
superiorly, outside the right ventricular
boundaries. An epicardial position was
suspected and screening in the left
anterior oblique projection during sub-
sequent repositioning showed the lead
overlying the apical left ventricular free
wall (panel B). Repositioning was per-
formed without further complication
and a post-procedure unipolar electro-
gram was obtained (panel C).
Ventricular depolarisation normally
occurs from endocardium to epicar-
dium. Endocardial lead positions there-
fore produce a predominantly negative
unipolar electrogram (panel C) and
epicardial sites result in a predomi-
nantly positive electrogram (panel A).
The endocardial electrogram at the
repositioned site (panel C) shows a
small initial R wave, which is normal
for an apical endocardial right ventricu-
lar lead position. This is because during
normal intrinsic conduction the earliest
site of ventricular activation is the septal
aspect of the left ventricular endocar-
dium and septal activation then occurs
left to right.
Perforation of the right ventricular
free wall during pacemaker implanta-
tion is often clinically silent. Findings
may include chest pain, a pericardial
rub, and a right bundle branch block
(RBBB) pacing pattern. If hypotension
occurs cardiac tamponade must be
excluded.
J Macdonald
D Kelly
J Waktare
macdonald_je@hotmail.com
228 Scientific letter
www.heartjnl.com
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... Although exercise has been shown to markedly upregulate GLUT4 expression in the skeletal muscle [6], GLUT4 expression was found to be comparable between sedentary and exercised hearts in our study. No precise mechanism may be offered for this seemingly tissue-specific effect in GLUT4 expression following swim training. ...
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... Therefore, restoration of this pathway can significantly improve insulin sensitivity and elicit insulinassociated metabolic effects by modulating glucose disposal, gluconeogenesis, lipogenesis and FFA oxidation (reviewed in [70,73]). In animal models, endurance exercise has been shown to substantially improve insulin responsiveness through the phosphorylation of IRS-associated PI3K pathway, particularly IRS-1 and -2 proteins, Akt and its downstream substrate AS160 [169][170][171][172][173]. Other signalling pathways such as liverkinase B1 (LKB1)-mediated phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) pathway in skeletal muscle [174], adaptor protein phosphotyrosine interaction PH domain and leucine zipper containing 1 (APPL1)-mediated Akt pathway in liver [175] have also been reported to potentiate the metabolic actions of insulin. ...
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Insulin resistance, a major factor in the pathogenesis of type 2 diabetes mellitus, is due mostly to decreased stimulation of glycogen synthesis in muscle by insulin. The primary rate-controlling step responsible for the decrease in muscle glycogen synthesis is not known, although hexokinase activity and glucose transport have been implicated. We used a novel nuclear magnetic resonance approach with carbon-13 and phosphorus-31 to measure intramuscular glucose, glucose-6-phosphate, and glycogen concentrations under hyperglycemic conditions (plasma glucose concentration, approximately 180 mg per deciliter [10 mmol per liter]) and hyperinsulinemic conditions in six patients with type 2 diabetes and seven normal subjects. In vivo microdialysis of muscle tissue was used to determine the gradient between plasma and interstitial-fluid glucose concentrations, and open-flow microperfusion was used to determine the concentrations of insulin in interstitial fluid. The time course and concentration of insulin in interstitial fluid were similar in the patients with diabetes and the normal subjects. The rates of whole-body glucose metabolism and muscle glycogen synthesis and the glucose-6-phosphate concentrations in muscle were approximately 80 percent lower in the patients with diabetes than in the normal subjects under conditions of matched plasma insulin concentrations. The mean (+/-SD) intracellular glucose concentration was 2.0+/-8.2 mg per deciliter (0.11+/-0.46 mmol per liter) in the normal subjects. In the patients with diabetes, the intracellular glucose concentration was 4.3+/-4.9 mg per deciliter (0.24+/-0.27 mmol per liter), a value that was 1/25 of what it would be if hexokinase were the rate-controlling enzyme in glucose metabolism. Impaired insulin-stimulated glucose transport is responsible for the reduced rate of insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes mellitus.
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Protein kinase B in the diabetic heart
Article
  • Aug 2003
This paper summarizes data from different studies all aimed at elucidating regulation of protein kinase B in the diabetic heart. Two rat models of type 2 diabetes mellitus ((i) elicited via neonatal streptozotocin injection (Stz) and (ii) Zucker fa/fa rats), were used as well as different experimental models viz isolated, Langendorff perfused hearts as well as adult ventricular myocytes. Glucose uptake was elicited by a variety of stimuli and the activation of PKB measured in tandem. Basal glucose uptake was impaired in both diabetes models while basal phosphorylation of PKB differed, showing lower levels in the Stz model but higher levels in the Zucker rats. Neither 100 nM insulin nor 10(-8) M isoproterenol could stimulate PKB phosphorylation to the same extent in the diabetic myocardium as in controls, regardless of the method used, but a combination of these stimuli resulted in an additive response. Concurrent glucose uptake however, was not additive. Wortmannin abolished both insulin and isoproterenol stimulation of glucose uptake as well as PKB phosphorylation. In contrast to the above-mentioned results, the protein tyrosine phosphatase inhibitor vanadate, alone or in combination with insulin, elicited PKB phosphorylation to the same extent in diabetic cardiomyocytes as in controls. Despite this, glucose uptake stimulated by vanadate or insulin in combination with vanadate was attenuated. The combination of insulin and vanadate may however be beneficial to the diabetic heart as it resulted in improved glucose transport. Results from the different studies can be summarized as follows: (i) dysregulation of PKB is evident in the diabetic myocardium, (ii) PKB activation is not always directly correlated with glucose uptake and (iii) insulin resistance is associated with multiple alterations in signal transduction, both above and below PKB activation.
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