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Cardiovascular Research 2004 61(2):297-306; doi:10.1016/j.cardiores.2003.11.027
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Copyright © 2004, European Society of Cardiology

The development of myocardial insulin resistance in conscious dogs with advanced dilated cardiomyopathy

Lazaros A Nikolaidisa, Anthony Sturzua, Carol Stolarskia, Dariush Elahib, You-Tang Shenc and Richard P Shannona,*

aCardiovascular Research Institute and the Department of Medicine, Allegheny General Hospital, 320 East North Ave., Pittsburgh, PA 15212, USA
bUniversity of Massachusetts Medical Center, Worcester, MA 02114, USA
cMerck Research Laboratories, West Point, PA 19486, USA

* Corresponding author. Tel.: +1-412-359-3022; fax: +1-412-359-8152. rshannon{at}wpahs.org

Received 16 July 2003; revised 17 November 2003; accepted 24 November 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: The failing heart demonstrates a preference for glucose as its metabolic substrate. Advanced, severe DCM is characterized by depletion of adenosine triphosphate (ATP) stores, which may be a consequence of impaired insulin mediated glucose uptake and oxidation at a time when the myocardium prefers glucose as its substrate. We examined the time course and magnitude of myocardial insulin resistance during the evolution of dilated cardiomyopathy. Methods and results: Thirty-four conscious, chronically instrumented dogs were studied at four stages during the evolution of dilated cardiomyopathy (DCM) induced by rapid RV pacing [control, early, late and advanced severe]. Transmyocardial glucose, lactate, and non-esterified fatty acid (NEFA) concentrations were measured in the fasting state. The cellular insulin signaling cascade and ATP levels were measured on myocardial samples. NEFA and insulin concentrations increased early and progressively in DCM in association with increased norepinephrine concentrations and progressive hemodynamic impairment. In advanced DCM but not earlier stages, myocardial ATP levels were decreased by 34%. There was decreased myocardial glucose uptake evident under both basal (–29±5%) and insulin stimulated (–32±4%) conditions in advanced, severe DCM, associated with a 31% reduction in GLUT-4 translocation. Importantly, there were no alterations in proximal steps in insulin signaling, but significant reductions in serine (Ser473) phosphorylation of Akt-1. Conclusions: Advanced, severe DCM is associated with the development of myocardial insulin resistance. There is impaired myocardial glucose uptake and altered myocardial insulin signaling, involving decreased Ser 473 phosphorylation of Akt-1. Myocardial insulin resistance in advanced, severe DCM was also associated with reduced myocardial ATP levels.

KEYWORDS Insulin resistance; NEFA; Norepinephrine; Dilated cardiomyopathy; Akt-1; GLUT-4


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
There remains considerable controversy as to whether the development of dilated cardiomyopathy (DCM) is accompanied by a shift in myocardial metabolic substrate preference from non-esterified fatty acid (NEFA) to glucose [1–9]. The intent is to take advantage of the efficiency of glucose as a substrate for high-energy phosphate generation. Recent evidence using sophisticated imaging and tracer elements has again suggested an increased dependence of myocardium on glucose uptake and utilization in DCM in both humans [7] and experimental animals [8]. This shift is transcriptionally regulated and recapitulates the fetal cardiac metabolic profile by down-regulating adult gene transcripts, particularly those involved in NEFA uptake and transformation to acyl CoA [10]. However, these studies have largely examined compensated states of DCM in humans [7] or moderate, but not severe cardiomyopathy in dogs with pacing induced DCM [8], where ATP levels and myocardial oxygen consumption (MVO2) are maintained. However, as DCM progresses from a compensated to a decompensated state, there is depletion of ATP, despite further increases in oxygen consumption that appear uncoupled from oxidative phosphorylation [11]. We hypothesized that the eventual loss of energetic balance may occur as a consequence of impaired myocardial glucose uptake and oxidation, mediated via insulin resistance, at a critical time when the myocardium is dependent on glucose metabolism.

Accordingly, the purpose of the present study was to determine whether LV dysfunction induced by rapid pacing in conscious dogs is associated with the development of myocardial insulin resistance. A second goal was to determine if the observed myocardial insulin resistance affected myocardial glucose uptake. A third goal was to determine alterations in myocardial insulin signaling associated with impaired insulin action in advanced, severe DCM.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Instrumentation
Thirty-four mongrel dogs of either sex weighing 15–20 kg were instrumented as described previously [11,12]. The dogs were allowed to recover from the surgical procedure for two weeks, during which time they were trained to lie quietly on the experimental table in a conscious, unrestrained state. Hemodynamic measurements were made with the dogs fully awake, lying quietly on their right side. Animals used in this study were maintained in accordance with the "Guide for the Care and Use of Laboratory Animal Resources" [DHHS Publication No. (NIH) 86-23, Revised 1996] and the guidelines of the Institutional Animal Care and Use Committee at Allegheny General Hospital.

2.2. Hemodynamic measurements
Control experiments consisted of systemic and coronary hemodynamic recordings [11–14] and arterial and coronary sinus blood sampling, as described previously from our laboratory [13,14]. Dilated cardiomyopathy (DCM) was induced by rapid right ventricular (RV) pacing (240 min–1) for 5–6 weeks, as described previously [11–14]. A 30-min stabilization period following deactivation of the pacemaker preceded all hemodynamic and metabolic measurements in DCM. In addition, all catheters were flushed with saline and all instruments were calibrated daily. Heparin was avoided due to its known lipolytic effects.

2.3. Metabolic determinations
All dogs were fed a diet with fixed carbohydrate and fat content. Body weights were monitored weekly. Metabolic parameters were measured at 8 am, following an overnight fast. Arterial and coronary sinus blood samples were obtained in all dogs at control, 24, 48, and 72 h and at 7, 14, 21, 28, and 32–34 days following initiation of rapid pacing. Transmyocardial substrate balance was calculated as the difference between arterial and coronary sinus content. Myocardial substrate uptake was calculated as the product of myocardial substrate balance and coronary blood flow.

Using a custom-made HPLC column and mobile phase purchased from Chromsystems (Munich, Germany), the norepinephrine and epinephrine levels were measured using electrochemical detection. The results were analyzed and quantitated using Millennium Chromatography Manager Software (Waters, Milford, MA). The measurements of non-esterified fatty acids (NEFA) in the plasma were carried out using the NEFA C test kit purchased from Wako Diagnostics (Richmond, VA) [15]. The plasma glucose levels were measured using a Beckman Glucose Analyzer II. The measurements of lactate levels in the plasma were carried out using a kit purchased from Sigma Diagnostics (St. Louis. MO). The measurements of plasma insulin levels were carried out using the Human Insulin Specific RIA Kit from Linco Research (St. Charles, MO). Myocardial ATP levels were measured on snap-frozen biopsy samples from five control dogs and six dogs with advanced, severe DCM at the time of euthanasia as described previously [11].

2.4. Hyperinsulinemic–euglycemic clamps
Hyperinsulinemic–euglycemic clamps were performed in the same six dogs at control and then in advanced, severe DCM to determine whole body and myocardial insulin sensitivity under conditions of supraphysiologic insulin concentrations. Under fasting conditions and following a 30-min period of metabolic stabilization, all animals underwent a prime-constant infusion of insulin (480 pmol m–2 min–1), designed to create steady state plasma insulin levels of 1000–1100 pmol/l. Plasma glucose levels were sampled every 5 min and 10% glucose solution was infused to maintain plasma glucose levels at 5 mmol/l±10%. Plasma glucose levels were clamped for 120 min. Whole body glucose disposal was measured as the average glucose infusion rate over the final 90 min of the clamp. Myocardial glucose uptake was measured as the product of coronary blood flow (ml/min) and myocardial substrate balance measured every 15 min during the clamp.

2.5. Western blotting
Samples of LV myocardium from six controls and eight dogs with advanced, severe DCM were snap-frozen in liquid nitrogen and stored at –70 °C. Purified sarcolemmal membranes were prepared as described previously [16] using density gradient centrifugation.

LV myocardium was homogenized in a buffer free of phosphatase inhibitors and subjected to electrophoretic separation by SDS-PAGE [17]. Resolved proteins were transferred onto PVDF membrane (ImmobilonTM-PSQ, Millipore, Bedford, MA) at a constant voltage (100 V) for 1–2 h at 4 °C [18]. Nonspecific membrane protein binding sites were blocked for at least one h at room temperature and then membranes were probed with the specific primary antibody overnight at 4 °C. The blots were washed in appropriate secondary antibody–horseradish peroxidase conjugate. The immunoreactive proteins were detected by use of an enhanced horseradish peroxidase/luminol chemiluminescence reaction kit (Perkin Elmer Life Sciences, Boston, MA) and exposed to X-ray film (Hyperfilm ECLTM, Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric analysis of the bands was carried out using a Personal Densitometer SI and ImageQuaNTTM Software (Molecular Dynamics, Sunnyvale, CA). Adjustments for protein loading were accomplished by normalizing bands based upon Coomassie staining of the blots.

Anti-insulin receptor-β subunit (IR-β), anti-phosphatidylinositol-3' kinase (PI3-kinase) p85, rabbit anti-sheep IgG-horse radish peroxidase (HRP) conjugate, and normal rabbit IgG were purchased from Upstate Biotechnology (Lake Placid, NY). Goat anti-rabbit IgG–HRP conjugate, anti-phosphotyrosine (RC20)–HRP conjugate, anti-Akt-1, anti-phospho-Akt-1 (Ser 473), and anti-insulin receptor substrate-1 (IRS-1), were purchased from BD Transduction Laboratories (San Diego, CA). Anti-PI3-kinase p110{alpha}, anti-phosphatase and tensin homolog deleted on chromosome 10 (PTEN), anti-GLUT4 and Protein A/G-PLUS Agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-Akt-1 (Ser 473) specificity was confirmed using phosphorylated and non-phosphorylated NIH/3T3 cell extracts purchased from Cell Signaling Technology (Beverly, MA).Immunoprecipitation of IRS-1 followed by anti-phosphotyrosine and anti-phosphoserine Western blotting was performed with 200 µg of tissue lysate and pre-cleared with 0.05 µg normal rabbit IgG together with 20 µl of re-suspended Protein A/G PLUS-Agarose by mixing 2 h at 4 °C. The immunocomplex was captured by adding 100 µl (25 µl packed beads) Protein A/G-PLUS Agarose and incubating at 4 °C for at least 3 h. The supernatant was discarded and the beads were washed three times in ice-cold PBS followed by one wash with 0.5 M Tris–HCl, pH 6.8. Beads were re-suspended in Laemmli buffer and boiled for 5 min. Samples were then subjected to Western Blot analysis as described above.

2.6. RNA preparation and competitive RT-PCR for GLUT-4
Total RNA was prepared from frozen LV myocardium from four control dogs and eight HF dogs using the TRIzol® Reagent protocol and reverse-transcribed into cDNA using SuperScriptTM First-Strand Synthesis System for RT-PCR with gene specific priming according to the manufacturer's protocol (Invitrogen Life Technologies, Carlsbad, CA). The RT-PCR strategy employed in these studies is based upon the competitive co-amplification of a known amount of an internal standard and endogenous RNA of unknown quantity. The competitor RNA was designed, isolated, and quantitated using the RT-PCR Competitor Construction Kit (Ambion, Austin, TX). For each sample, 1 µg of total RNA and varying known amounts of competitor RNA were reversed-transcribed and then subjected to PCR using a primer set specific for GLUT4 which amplified both the target (196 bp) and competitor (176 bp) strand (forward: 5'-GATGACCATAGCCCTGCTTC-3'; reverse: 5'-GTTGCTTGTCCAGTTGCAGA-3').

The PCR products were separated by electrophoresis in a 4% agarose gel and visualized by ethidium bromide staining. The PCR products, which were radioactively labeled with 32P, were quantified using phosphoimaging on a Storm 840 and analyzed using ImageQuant software version 4.2A (Molecular Dynamics). Data analysis to quantitate the target RNA was carried out using linear regression plotting of log[(competitor density x 196/176)/target density] vs. log[known concentration of starting GLUT4 competitor RNA]. The 196/176 ratio was corrected for the difference in length between the target and competitor product.

2.7. Statistical analysis
Data are expressed as the mean value±S.E.M. Differences between hemodynamic and metabolic parameters among the groups were determined by repeated measures ANOVA. Densitometric data between control and advanced DCM samples were compared by a two-tailed t-test. A level of p<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Table 1 depicts the hemodynamic alterations in the conscious dogs (n = 12) studied during the progression to advanced, severe DCM. There was a significant decrease (–42±6%) in LV contractile function (LV dP/dt) and increases in heart rate (+29±5%) and LVEDP (+55±3%) early in DCM (4–7 days of pacing). However, cardiac output was maintained and there was little evidence of LV dilatation. During late DCM (24–26 days), there were progressive increases in LVEDP, heart rate, and LV end diastolic dimension, but maintenance of cardiac output, consistent with a compensated clinical state at rest. With the progression to advanced, severe DCM, contractile function deteriorated further and there were progressive increases in heart rate and LVEDP. There was evidence of progressive LV dilatation and cardiac output decreased by 29% while MVO2 increased by 22%.


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  		Table 1 LV, systemic, and coronary hemodynamic effects of rapid pacing in conscious dogs

 
Fig. 1 reveals the time course of the alterations in fasting metabolic parameters in 12 dogs. Plasma NEFA rose progressively together with increases in circulating norepinephrine beginning early in the evolution of DCM. Plasma insulin levels also rose within 24 h and increased threefold during the transition to advanced, severe DCM. In contrast, plasma glucose levels rose only modestly during the transition to advanced, severe DCM.


Figure 1
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  		Fig. 1 Time course of the changes in plasma norepinephrine, non-esterified fatty acids (NEFA), insulin and glucose in 12 dogs during the evolution from a normal state to advanced, severe dilated cardiomyopathy.

 
Table 2 illustrates the alterations in substrate availability, glucoregulatory hormones, and myocardial substrate uptake under basal conditions during the evolution of DCM in conscious dogs. There were no significant differences in myocardial uptake for any of the three substrates during early DCM compared to control. There were significant decreases in NEFA uptake in late DCM, while myocardial glucose uptake was maintained. However, there were progressive declines (p<0.05) in myocardial NEFA, glucose and lactate uptake during the evolution to advanced, severe DCM. The decline in uptake in the respective substrates in advanced, severe DCM was not attributable to differences in coronary blood flow or substrate availability, but was associated with a significant twofold increase in plasma epinephrine levels. The impairment in myocardial substrate uptake was temporally associated with a significant reduction in ATP levels in the myocardium in advanced, severe DCM [Control: 41±3 nmol/mg protein (n = 6); Advanced DCM: 27±3 nmol/mg protein (n = 6), p≤0.05].


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  		Table 2 The effects of evolving DCM on systemic metabolic parameters

 
Fig. 2 illustrates the time course of the hyperinsulinemic–euglycemic clamps in 6 dogs studied in control and then in advanced, severe DCM following 35 days of rapid pacing. Plasma insulin levels rose promptly to comparable levels (1100 pmol/l) in control and advanced, severe DCM. However, the glucose infusion rate required to maintain plasma glucose levels at 5 mM was significantly less in the dogs with advanced, severe DCM. Importantly, hyperinsulinemia was associated with a marked suppression of plasma NEFA within 30 min after initiating insulin infusion.


Figure 2
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  		Fig. 2 The time course of the changes in plasma insulin levels, glucose infusion rates and plasma NEFA during hyperinsulinemic–euglycemic clamps in six dogs studied in control and then again in advanced, severe DCM (*p<0.05 DCM compared to control).

 
Fig. 3 summarizes the effects of hyperinsulinemia on whole body and myocardial glucose disposition. There was a significant 24% reduction in whole body glucose disposal and a 32% reduction in myocardial glucose uptake in advanced, severe DCM compared to the same animals studied in control. There was also a blunting in the coronary blood flow response to hyperinsulinemia in advanced, severe DCM compared to control.


Figure 3
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  		Fig. 3 The effects of hyperinsulinemia on whole body glucose disposition, myocardial glucose uptake and coronary blood flow while maintaining euglycemia in six dogs studied in control and then again in advanced, severe DCM (*p<0.05 DCM compared to control and hyperinsulinemia compared to baseline). p<0.01 is the response in DCM to hyperinsulinemia compared to the response in control.

 
Fig. 4 illustrates the alterations in insulin signaling in the myocardium from normal dogs and dogs with advanced, severe DCM. There was no significant difference in the density of the β-subunit of the insulin receptor or the protein levels of IRS-1 and phosphatidylinositol-3' kinase (PI-3 K). The tyrosine and serine phosphorylation of IRS-1 were not different between groups. However, in advanced, severe DCM, there were significant decreases in serine (473) phosphorylation of Akt-1, despite normal levels of the protein (Fig. 5). The decrease in serine (473) phosphorylation of Akt-1 occurred in association with an increase in PTEN abundance in advanced DCM. The cumulative consequence of impaired Akt-1 phosphorylation was a significant impairment in a downstream target of activated Akt-1, namely the translocation of the insulin sensitive GLUT-4 transporter in severe DCM. Overall, cytosolic GLUT-4 protein levels were unchanged and the GLUT-4 mRNA, determined by RT-PCR, was not different between control and advanced DCM (Fig. 6). However, the purified membrane fraction was reduced by 31%, despite threefold increases in plasma insulin levels under basal circumstances. We also measured insulin stimulated GLUT-4 translocation in purified sarcolemmal membrane preparations prepared from biopsy samples prior to and after insulin stimulation (Insulin 100uU IV). There was significant insulin stimulated translocation of GLUT-4 in control, but the response was blunted in advanced DCM (Fig. 6). Thus, the decrease in GLUT-4 translocation appeared to be a consequence of altered insulin action.


Figure 4
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  		Fig. 4 Changes in protein levels of proximal components in the insulin signaling cascade. IR-1β=β subunit of insulin receptor; IRS-1=insulin receptor substrate -1; phospho-tyr-IRS-1; phospho-ser-IRS-1, PI3 kinase=phosphatidylinositol 3'-kinase (p85 regulatory subunit/p110{alpha} catalytic subunit). Control (n = 6); severe DCM (n = 8).

 

Figure 5
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  		Fig. 5 Changes in distal components of the insulin signaling cascade. Akt-1 = total protein abundance; Phosphoserine 473-activated Akt-1; PTEN=phosphatase and tensin homolog deleted on chromosome 10 (*p<0.05 compared to control). Control (n = 5); advanced DCM (n = 6).

 

Figure 6
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  		Fig. 6 RT-PCR for GLUT-4 mRNA performed on sarcolemmal membranes from control (n = 6) and advanced DCM. Cytosolic GLUT-4 protein abundance from five controls and six dogs with advanced DCM. GLUT-4 translocation determined in purified sarcolemmal membrane preparations under basal conditions and following 100 mU of regular insulin (IV) in five control dogs and six dogs with advanced DCM. Myocardial samples were harvested at the time of euthanasia (*p<0.05 compared to control; {dagger}p<0.05 compared to response in control).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we observed for the first time that the progression of LV dysfunction to advanced, severe DCM in conscious dogs was accompanied by the development of myocardial and whole body insulin resistance. The evidence of myocardial insulin resistance is manifest in the both diminished basal and insulin stimulated glucose uptake during hyperinsulinemic–euglycemic clamp studies. Finally, there was impaired myocardial insulin signaling as evidenced by altered activation of Akt-1 and impaired GLUT-4 translocation.

While insulin resistance and Type 2 diabetes are known to predispose to LV dysfunction [19], there is little consensus as to whether LV dysfunction contributes to the development of insulin resistance. Several studies have demonstrated fasting hyperinsulinemia and insulin resistance using minimal modeling, hyperinsulinemic–euglycemic clamps, or PET scanning in patients with heart failure [20–23]. However, these correlative studies do not exclude the possibility that these patients had insulin resistance prior to developing LV dysfunction. In our study, conscious, chronically instrumented dogs were found to have normal fasting glucose, NEFA and insulin levels prior to the development of DCM, but developed myocardial insulin resistance and insulin signaling abnormalities in advanced, severe DCM, but not earlier stages. These data suggest that advanced, severe DCM is a newly recognized insulin resistant state; the consequences of which may be considerable, given the evolving dependence on glucose oxidation as heart failure progresses.

There is controversy as to whether there are shifts in myocardial substrate preference toward glycolysis in non-ischemic DCM. Prior work from our laboratory [13,14] and Recchia et al. [24] in the model of pacing induced DCM demonstrated an increase in the respiratory quotient (RQ), indicative of a shift toward glucose oxidation. However, we observed an associated decrease in NEFA uptake [22], raising questions as whether the observed increase in RQ reflects decreased NEFA oxidation or increased glycolysis. Our findings of myocardial insulin resistance and decreased glucose uptake are at odds with recent reports in humans with idiopathic DCM in which myocardial glucose uptake was enhanced [7]. However, these apparent differences are reconciled by the fact that these patients had mild to moderate heart failure, were fully compensated on conventional heart failure treatment, and had normal fasting insulin levels (30 pmol/l). We observed decreased myocardial glucose uptake in the setting of myocardial insulin resistance in advanced, severe DCM, but not in earlier, compensated stages of DCM, where our findings are similar to those of Davilla-Roman et al. [7]. Osorio et al. [8] examined NEFA uptake and utilization in conscious dogs with pacing induced heart failure and found reduced myocardial NEFA uptake and markedly enhanced glucose uptake and oxidation. These differences are reconciled by the fact that our dogs were paced continuously at a faster rate (240 min–1), for a longer duration (35 days), and consequently had more severe heart failure as evidenced by greater LVEDP, higher resting heart rates, increased MVO2 and depressed cardiac output. Osorio et al. [8] did not measure plasma insulin levels or myocardial insulin signaling, but did report myocardial glucose uptake (>15 µmol/min/100 g) in a fasting, basal state in excess of that demonstrated during hyperinsulinemia in our study or that reported in humans following intracoronary infusion of insulin to concentrations of 600 pmol/l [25]. We did find decreased myocardial NEFA uptake, but preserved as opposed to enhanced, glucose uptake in late DCM. This apparent discrepancy is reconciled by the fact that MVO2 was reduced in late DCM in our study, suggesting that reduced energy demands did not require enhanced, but simply preserved glucose uptake in these conscious dogs. In contrast, MVO2 in DCM was comparable to control values in both the Daviila-Roman [7] and Osorio [8] studies, necessitating increased glucose uptake to compensate for decreased NEFA uptake, in order to maintain energetic balance. One important consequence of decreased myocardial NEFA, glucose, and lactate uptake in advanced, severe DCM is myocardial ATP depletion. The magnitude of the decrease in ATP stores in advanced, severe DCM is comparable to that reported previously [11], but here we demonstrated that decreases in ATP stores occurs in temporal association with the development of myocardial insulin resistance, confirming that this metabolic perturbation has significant energetic consequences. The decrease in ATP stores in advanced, severe DCM occurs despite increased MVO2, suggesting uncoupling of oxidative phosphorylation, which has also been reported in advanced DCM [26–28].

There is also controversy as to whether insulin resistance when present is manifest in all insulin sensitive tissues. Jagasia et al. [25] found no evidence of myocardial insulin resistance in Type 2 diabetics with coronary artery disease, despite significant impairment in whole body glucose uptake. Importantly, these subjects had normal LV function. The extent to which these differences are attributable to organ specific abnormalities in insulin action or the complicating effects of hyperglycemia is unknown. In our study, we performed hyperinsulinemic–euglycemic clamps in conscious dogs at control and then following the development of advanced, severe DCM, confirming whole body and myocardial insulin resistance. Prior studies [29,30] have demonstrated that this technique in dogs is comparable to responses seen in humans. Notably, the increased plasma NEFA and insulin levels in conscious dogs with advanced, severe DCM are comparable to those reported by Paolisso et al. [1,22,23] in patients with symptomatic, non-ischemic DCM, who were confirmed to have whole body insulin resistance by the euglycemic–hyperinsulinemic clamp. Subsequently, these investigators also demonstrated that myocardial glucose uptake was impaired in DCM [1].

Alterations in the insulin signaling cascade have been well characterized in skeletal muscle in both states of insulin resistance [31] and Type 2 diabetes [32], but little is known with respect to insulin signaling in the failing myocardium. While the proximal signaling cascade was unaltered in advanced, severe DCM, there was a marked decrease in serine (Ser473) phosphorylation of Akt-1. The Ser 473 site is located in the non-regulatory hydrophobic C-terminal and is important in promoting substrate interaction and activation [33,34]. Consequently, there were significant decreases in GLUT-4 translocation in severe DCM, consistent with the observed decrement in glucose uptake. Phosphorylation at Thr 308 is another important activation site for Akt-1. We were unable to measure accurately Thr 308 phosphorylation due to the lack of antibody specificity. The observation of a decrease in GLUT-4 translocation under both basal and insulin-stimulated conditions is substantiated by decreased purified sarcolemmal membrane GLUT-4 without alterations in cytosolic GLUT-4 levels or GLUT-4 gene transcription and is consistent with decreased Akt-1 activation. The observation that protein levels of the phosphatase, PTEN, were increased in myocardium from dogs with advanced, severe DCM is consistent with the impaired Ser473 phosphorylation of Akt-1. PTEN is known to inhibit intregrin-linked kinase-2 (ILK-2) mediated phosphorylation of Akt-1 by hydrolyzing tris-phosphatyl-inositol (Ptdn 3',4',5')P3 and preventing the binding and activation of these key serine/threonine kinases [31].

These findings are in contrast to abnormalities that have been reported in skeletal muscle and adipose tissue in insulin resistant states and Type 2 diabetes [32,33] where the principal findings involved decreased tyrosine and increased serine phosphorylation of IRS-1 or decreases in IRS-1 protein levels with concomitant decreases in downstream PI-3 kinase activity. While we did not measure PI-3 kinase activity, neither the IRS-1 protein abundance nor the phosphorylation at tyrosine or serine residues was altered. Thus, the reduced glucose uptake and the apparent decrease in glucose oxidation in advanced, severe DCM are likely consequences of altered insulin signaling through decreased Akt-1. Akt-1 is known to facilitate the translocation of GLUT-4 [34,35] and to phosphorylate two key regulatory sites on phosphofructokinase [35], a proximal step in glycolysis.

In conclusion, advanced, severe DCM in conscious dogs is associated with myocardial insulin resistance and basal and insulin stimulated impaired myocardial glucose uptake. The associated abnormalities in insulin signaling in the myocardium in severe DCM involve decreased serine 473 phosphorylation of Akt-1. The implications of myocardial insulin resistance at a time of crucial dependence on glucose uptake and utilization in advanced, severe DCM are reflected in decreased myocardial ATP stores. Whether restoring myocardial insulin responsiveness improves LV performance in DCM remains to be determined.


   Acknowledgements
 
This work has been supported in part by US Public Health Service Grants DA-10480 and AG-023125 and AHA Fellowship Grant (PA-DE Affiliate, 0020248 U).


   Notes
 
Time for primary review 00 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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