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Published as doi: 10.1096/fj.07-102269.
 (The FASEB Journal. 2008;22:2161-2167.)
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HIV protease inhibitors that block GLUT4 precipitate acute, decompensated heart failure in a mouse model of dilated cardiomyopathy

Paul W. Hruz*,{dagger},1, Qingyun Yan*, Heidi Struthers* and Patrick Y. Jay*,{ddagger}

* Department of Pediatrics,

{dagger} Department of Cellular Biology and Physiology, and

{ddagger} Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA

1Correspondence: 660 South Euclid Ave., Campus Box 8208, St. Louis, MO 63110, USA. E-mail: hruz_p{at}kids.wustl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The clinical use of HIV protease inhibitors is associated with insulin resistance and other metabolic changes that increase long-term cardiovascular risk. Since the failing heart has increased reliance on glucose, the influence of drug exposure on glucose homeostasis, myocardial glucose uptake, cardiac function, and survival was determined in TG9 mice, an established transgenic model of dilated cardiomyopathy generated by cardiac-specific overexpression of Cre-recombinase, as these animals progressed to overt heart failure. Beginning on day of life 75, TG9 mice and nontransgenic littermate controls were given a daily 10 mg/kg intraperitoneal injection of HIV protease inhibitors (ritonavir, lopinavir/ritonavir 4:1, atazanavir, atazanavir/ritonavir 4:1) or vehicle. Glucose tolerance testing, measurement of in vivo myocardial 2-deoxyglucose uptake, and echocardiography were performed before and 30 min following drug administration. The progression of dilated cardiomyopathy in TG9 animals was accompanied by impaired glucose tolerance, which was acutely exacerbated by exposure to ritonavir. Ritonavir and lopinavir precipitated acute, decompensated heart failure and death from pulmonary edema in TG9 mice. However, atazanavir, which does not inhibit glucose transport, had no effect. These studies demonstrate that, in the presence of dilated cardiomyopathy, HIV protease inhibitors that impair glucose transport induce acute, decompensated heart failure. The potential for HIV protease inhibitors to contribute to or exacerbate cardiomyopathy in human patients warrants further investigation.—Hruz, P. W., Yan, Q., Struthers, H., Jay, P. Y. HIV protease inhibitors that block GLUT4 precipitate acute, decompensated heart failure in a mouse model of dilated cardiomyopathy.


Key Words: insulin resistance • glucose transport • diastolic dysfunction • ritonavir • atazanavir • pulmonary edema


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HIV PROTEASE INHIBITORS (PIs) directly contribute to the development of insulin resistance and other metabolic changes that increase the risk of cardiac-related morbidity in the setting of chronic exposure (1) . Recent studies in HIV-negative healthy human volunteers have demonstrated that PIs can induce acute insulin resistance following a single dose of the drug (2 3 4 5 6) . A great deal of attention has been focused on the mechanisms by which long-term, highly active antiretroviral therapy (HAART) contributes to coronary artery disease but direct effects on the myocardium remain largely unexplored. Although the introduction of effective antiretroviral drug regimens has led to improvements in HIV-associated cardiomyopathy, heart failure continues to occur in a significant number of patients (7 , 8) . Patients who develop cardiomyopathy have significantly increased morbidity and mortality (9 , 10) .

The adult heart relies predominantly on fatty acid metabolism for basal energy production. In contrast, the failing heart shifts toward greater glucose utilization even in the presence of insulin resistance (11) . While the functional significance of these changes in substrate preference remains incompletely understood, the impaired ability to use glucose is deleterious to the stressed heart. In particular, impaired glucose tolerance can contribute to the progression of chronic heart failure in patients with dilated cardiomyopathy (12) . Since several PIs directly inhibit GLUT4 (13) , the predominant glucose transporter in the adult heart, we hypothesized that PI-mediated inhibition of myocardial glucose uptake would be detrimental to the failing heart.

The acute effects of ritonavir on glucose tolerance, myocardial GLUT expression, glucose uptake, morbidity, and mortality were therefore investigated in an established mouse model of dilated cardiomyopathy (14 , 15) . TG9 mice, which express high levels of the Cre recombinase under the {alpha}-MHC promoter, predictably die of dilated cardiomyopathy between 11 and 13 wk of life. The animals express common molecular markers of heart failure and respond positively to standard heart failure drugs like beta-blockers and ACE inhibitors (14) .

We demonstrate that acute, intermittent exposure of TG9 mice to ritonavir leads to transient deterioration in overall glucose homeostasis, including an acute reduction in myocardial uptake of glucose. This condition is accompanied by the development of acute pulmonary edema and death. Lopinavir, another protease inhibitor that blocks GLUT4, similarly causes acute cardiac decompensation, whereas atazanavir, which does not inhibit glucose transport, has no effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Reyataz (atazanavir) was obtained from Bristol-Myers Squibb (Princeton, NJ, USA). Kaletra (lopinavir/ritonavir) and Norvir (ritonavir) were obtained from Abbott (Chicago, IL, USA). Insulin (Humulin-R) was purchased from Eli-Lilly (Indianapolis, IN, USA). For all of the PIs, reference standards were obtained from the U.S. National Institutes of Health (NIH) AIDS reference and reagents program. Blood glucose levels were determined using a Glucometer Elite XL (Bayer, Tarrytown, NY, USA). 1-[3H]-2-deoxyglucose and β-lactosyl-FITC labeled bovine albumin were purchased from Sigma (St. Louis, MO, USA). GLUT4 antibody was custom produced by Invitrogen (Carlsbad, CA, USA) using a peptide corresponding to the 16 amino acids at the transporter carboxy terminus. GLUT1 antibody was a gift from Dr. Mike Mueckler (Washington University, St. Louis, MO, USA).

Drug assays
Serum PI levels were determined by the HPLC method of Foisy (16) using a Waters 626 HPLC system with a Microsorb C-8 column (Waters Corp, Milford, MA, USA). Samples were run in duplicate on 50 µl of serum. Standard curves were generated by adding pure PI standards directly to control mouse serum.

Animal procedures
All animal procedures were approved by the animal studies committee at Washington University School of Medicine. Mice were housed in the animal facility at Washington University and fed a standard mouse chow diet and water ad libitum. Beginning on day of life 75, female TG9 and nontransgenic FVB/N littermate controls (NTG) were given daily intraperitoneal (i.p.) injections of PI or vehicle control (10% ethanol, 9 µl/g body weight). Animals were closely monitored for activity, respiratory rate, and general signs of distress during the 90–120 min interval where measurable levels of the administered PI were present in serum. In animals that died within this 2 h window, blood, heart, and skeletal muscle were collected. Harvested tissue was placed immediately in liquid nitrogen and stored at –80°C pending further analysis.

I.p. glucose tolerance tests
Following a 4-h fast, HIV protease inhibitors (in 10% ethanol) were administered by i.p. injection. After 15 min, the mice received an i.p. injection of 50% dextrose (2 g/kg). Approximately 5 µl of blood was sampled from tail veins at regular intervals over the following 2 h. In a subset of animals, ~100 µl of blood was obtained for serum drug level determination 30 min after dosing.

Myocardial 2-deoxyglucose uptake
[3H]-2-deoxyglucose (DOG) was administered via a tail vein catheter. Blood was collected at 5 min intervals for determination of the tracer specific activity. Immediately following euthanasia by sodium pentobarbital administration, the left ventricular myocardium was harvested and placed in liquid nitrogen for subsequent analysis. The frozen samples (20–50 mg) were ground with a mortar and pestle, boiled in 1.2 ml of water for 10 min, and spun in a microcentrifuge at 15,000 g for 10 min. Accumulated 2-deoxyglucose-6-phosphate in the supernatant was separated from 2-DOG by ion-exchange chromatography using a Dowex 1X-8 (100–200 mesh) anion exchange column (Sigma) as described previously (17) . The tissue glucose metabolic index (Rg') was calculated as described by Smith et al. (18) .

Quantification of GLUT expression
Left ventricular myocardium was harvested from mice immediately following euthanasia and placed in liquid nitrogen. Lysates were prepared by homogenization in buffer containing 20 mM Tris-HCl, pH 7.4; 137 mM NaCl; Sigma protease inhibitor cocktail; 1% nonidet P-40; and 10% glycerol. Lysates were kept on ice for 15 min and cleared by centrifugation at 1500 g for 20 min at 4°C. Protein concentration was determined by the Bradford method (Bio-Rad, Hercules, CA, USA). Western blot analysis was then performed on 5 µg of total protein per lane using GLUT1 or GLUT4 rabbit polyclonal antibody recognizing the C terminus of the transporters.

Pulmonary edema
Mice were euthanized 30 min after administration of drug or placebo with an i.p. injection of ketamine/xylazine (80:16 mg/kg). After measuring whole body weight, the lungs were immediately removed and weighed. In a subset of animals, 25 mg/kg of β-lactosyl-FITC labeled bovine albumin in a 10 mg/ml PBS stock solution was administered via tail vein injection. Ritonavir (10 mg/kg) or vehicle control was then administered by i.p. injection. After 30 min the mice were euthanized, and 300 µl of blood was collected via a retro-orbital bleed. Trachea were then cannulated with a 24-gauge angiocatheter, and the thoracic cavity was exposed. Lungs were lavaged twice with 1 ml PBS. The bronchoalveolar lavage fluid was centrifuged at 2100 g for 15 min at 4°C (19) . FITC-labeled albumin in the lavage fluid supernatant was then quantified by fluorescence spectroscopy (Model 680 XR Microplate Manager, Bio-Rad).

Echocardiography
Transthoracic echocardiographic images were obtained on conscious 75-day-old TG9 and NTG mice using an Acuson Sequoia 256 Echocardiography system with a 15 MHz transducer (Acuson Corp., Mountain View, CA, USA) before and 30 min after a single 10 mg/kg dose of ritonavir. Quantification of left ventricular chamber dimensions and function by M-mode echocardiography was done as described previously (20) . The internal diameter of the left ventricle was measured in end-diastole and end-systole (LVEDD and LVESD). Systolic function was quantified by fractional shortening, FS = (LVEDD–LVESD)/LVEDD.

Statistical analysis
Results are reported as the mean ± SE of at least 4 independent measurements per experimental group. For single-group comparisons, the Student’s t test was used to determine significance at P < 0.05. For multiple comparisons, one-way ANOVA was used with the post hoc Bonferroni t test. For survival analyses, the log-rank test was performed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Glucose and lipid homeostasis in the transgenic model of dilated cardiomyopathy
To characterize glucose homeostasis during the progression of dilated cardiomyopathy, fasting blood sugars were measured in TG9 and in nontransgenic FVB/N control animals (NTG) from 5 to 10 wk of age. TG9 mice at age 5 wk have normal left ventricular size and function. Echocardiographic signs of dilated cardiomyopathy manifest at 6 wk and worsen until the mice develop overt heart failure and death at 11–13 wk (14) . Over this interval, fasting blood sugars became progressively elevated in parallel with deteriorating cardiac function (Fig. 1 A). Serum-free fatty acid levels did not change during the evolution of heart failure and worsening glucose homeostasis (data not shown).


Figure 1
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  		Figure 1. Glucose homeostasis during the evolution of dilated cardiomyopathy. A) Blood sugars were determined weekly following a 4-h fast. B) I.p. glucose tolerance tests (2 g/kg) were performed on 75-day-old NTG or TG9 mice 15 min following the injection of ritonavir (10 mg/kg) or vehicle control. Results represent the mean ± SE of 4 animals/group. *P < 0.05.

Similar to our previous observations in healthy Wistar rats (21) , a single 10 mg/kg dose of ritonavir administered 15 min prior to a glucose tolerance test caused an acute reduction in whole-body glucose disposal in NTG mice as shown by the doubling of the peak glucose level (Fig. 1B ). This ritonavir dose was sufficient to transiently achieve peak serum ritonavir levels of 6.2 ± 0.6 µM. Consistent with the short half-life of ritonavir in rodents (22) , 2 h glucose levels were not different between vehicle and ritonavir-treated animals. Glucose tolerance tests performed on 75-day-old TG9 animals showed baseline glucose intolerance with a further exacerbation in glucose response following a single 10 mg/kg ritonavir dose. Peak glucose levels were also shifted ~60 min later. Thus, ritonavir acutely and reversibly worsens glucose homeostasis in this animal model of dilated cardiomyopathy.

Glucose transporter expression
Since the diabetic adult heart shifts toward the almost exclusive utilization of fatty acids, whereas the failing heart increases glucose utilization, even in the setting of altered glucose homeostasis (11) , we examined changes in facilitative glucose transporter (GLUT) expression. GLUT1 and the insulin-responsive GLUT4 are the predominant transporters in the fetal and adult hearts, respectively. GLUT1 is up-regulated in the setting of myocardial stress or injury (23) . In agreement with past observations, GLUT1 protein in the failing TG9 heart increased 2-fold relative to age-matched nontransgenic mice (Fig. 2 A). Although it is not possible to directly compare the relative amounts of these two transporter isoforms by Western blot analysis, GLUT4 protein was found to be unchanged in the failing TG9 heart.


Figure 2
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  		Figure 2. Glucose transporter expression and myocardial glucose uptake in TG9 mice. A) Western blot analysis was performed on total cell homogenates of left ventricular myocardium harvested from 75-day-old TG9 mice. Top panel: the signals represent the broad transporter band migrating between 55–60 kDa, as expected for the glycosylated mature GLUT proteins. Bottom panel: the intensities of these protein bands were quantified using ScionImage software; values are means ± SE. *P < 0.05, NTG vs. TG9 groups. B) Following a 4-h fast, 75-day-old NTG and TG9 mice were given a single dose of ritonavir (10 mg/kg) or vehicle 15 min prior to the administration of [3H]-2-deoxyglucose via a tail vein catheter. After 30 min, the mice were euthanized and the hearts were immediately removed and analyzed for accumulation of radiolabeled 2-deoxyglucose-6-phosphate into the left ventricular myocardium. Rg', tissue glucose metabolic index; *P < 0.05, vehicle-treated vs. ritonavir-treated groups.

Cardiac glucose uptake
The continued expression of GLUT4 in the setting of dilated cardiomyopathy suggested that myocardial glucose transport in TG9 mice may be significantly affected by PIs known to target this protein. Measurement of 2-deoxyglucose uptake into left ventricular myocardium in age-matched vehicle-treated TG9 vs. NTG mice revealed a 64% reduction in glucose transport in the transgenic animals (Fig. 2B ), reflecting the combined effects of insulin resistance, which decreases glucose uptake, and myocardial stress, which increases it. A single dose of ritonavir administered 30 min prior to the measurement of 2-DOG uptake resulted in a 46% reduction in glucose uptake in the NTG animals and a 25% decrease in uptake in the TG9 animals. Since GLUT1 is not inhibited by ritonavir (13) , these results are consistent with the relative differences in GLUT expression in these two mouse strains.

Pulmonary edema
To determine whether PI exposure affects cardiac function in the TG9 mice, each PI was administered by intraperitoneal injection. Ritonavir had no clinically observable effect on the mice prior to the development of echocardiographic signs of dilated cardiomyopathy (data not shown). However, once the animals had developed dilated cardiomyopathy (as evidenced by echocardiography without observable clinical changes), ritonavir exposure resulted within minutes in the onset of respiratory distress and inactivity, signs of end-stage heart failure (14) . Under identical conditions, atazanavir did not produce any observable effect. Consistent with drug metabolism, the clinical appearance of the ritonavir-treated mice returned to baseline by 90 min. Since the clinical changes in the ritonavir-treated TG9 mice were suggestive of acute pulmonary edema, we measured the change in the ratio of lung to body weight, an established marker of pulmonary edema (24) , at baseline and 30 min following ritonavir administration in 75-day-old animals. PI exposure did not affect the lung/body weight ratio in the NTG mice (Fig. 3 A). Baseline lung weights in the TG9 animals were slightly higher than in the nontransgenic mice (P<0.05). Ritonavir caused an acute 23% increase in the lung/body weight ratio (P<0.02). As an independent measure of pulmonary edema, transudation of FITC-albumin from the vascular to alveolar space was assessed (19) . In agreement with the changes in lung weights, ritonavir did not cause FITC-albumin transudation in NTG animals (Fig. 3B ). However, alveolar FITC-albumin rose by 67% in TG9 mice within 30 min of exposure to ritonavir (P<0.005). These results demonstrate that ritonavir induces acute pulmonary edema in this adult mouse dilated cardiomyopathy model.


Figure 3
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  		Figure 3. Development of acute pulmonary edema. A) Lung/body weight ratio of NTG and TG9 mice 30 min following a single dose of ritonavir (10 mg/kg) or vehicle. *P < 0.05 vs. vehicle-treated NTG mice; Figure 3P < 0.05 vs. vehicle-treated TG9 mice. B) Alveolar transudation of FITC-albumin. Mice were given i.v. FITC-albumin followed by a 10 mg/kg dose of ritonavir or vehicle. After 30 min, bronchioalveolar lavage was performed on anesthetized mice.

Echocardiography
Since the induction of pulmonary edema in TG9 mice acutely exposed to ritonavir is indicative of diastolic dysfunction, we investigated the effect of this PI on cardiac function via echocardiography in 75-day-old animals immediately before and 30 min following a single 10 mg/kg dose of ritonavir. As shown in Fig. 4 , ritonavir produced an acute and reproducible bradycardia immediately following administration of ritonavir. Bradycardia is a commonly observed phenomenon in rodents with severe heart failure and is thought to be a compensatory mechanism to increase left ventricular filling time in mice with normally fast heart rates (25) . Ritonavir also caused a dramatic decrease in the left ventricular end-diastolic diameter, suggesting a defect of diastolic relaxation in the PI-treated heart. Fractional shortening was increased by nearly 50%. While this most likely reflects the smaller end-diastolic diameter, a paradoxical improvement in systolic function cannot be excluded.


Figure 4
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  		Figure 4. Echocardiography of TG9 and NTG mice. Transthoracic M-mode echocardiography was performed on conscious 75-day-old mice before (basal) and 30 min following a single 10 mg/kg dose of ritonavir; TG9 (solid lines), NTG (dashed lines). LVEDD represents the left ventricular internal diameter at end-diastole. Fractional shortening is calculated as (LVEDD-LVESD)/LVEDD, where LVEDD and LVESD are the left ventricular diameter at end-diastole and end-systole, respectively. Each line represents an individual mouse experiment (n=3/group); P < 0.05 for each comparison, NTG vs. TG9 mice.

Survival analyses
To determine whether the acute cardiac decompensation induced by PI exposure affected survival, we continued daily administration of ritonavir. As shown in Fig. 5 A, vehicle-treated animals predictably died at an age of 83.0 ± 0.8 days, consistent with past results (14) . Ritonavir significantly accelerated mortality from heart failure (78.9±0.6 days). Since ritonavir is frequently added to PI treatment regimens to boost serum drug levels through its effect on cytochrome P450 metabolism, we also tested the effect of low ("boosting") doses of ritonavir on survival in TG9 mice with dilated cardiomyopathy (Fig. 5B ). The administration of 2.5 mg/kg of ritonavir alone (which produced transient peak drug levels of 2.2±0.2 µM) did not produce any observable change in activity or respiratory rate in the TG9 mice. Survival (81.6±0.2 days) was slightly but not significantly lower than that of vehicle-treated mice.


Figure 5
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  		Figure 5. Kaplan-Meier survival curves for TG9 mice exposed to HIV protease inhibitors. A single daily i.p. injection of drug was administered beginning on day of life 75 and continuing until the time of death. All plots represent experiments with TG9 mice except NTG-Ritonavir, which represents nontransgenic littermate control animals. A) Mice were dosed at 10 mg/kg for each PI. B) Mice were given a boosting dose (2.5 mg/kg) of ritonavir alone or together with the indicated PI. n ≥ 8 animals/group.

The correlation between the ability of PIs to acutely impair glucose transport and survival was further investigated by performing the survival study with additional PIs that have differing effects on GLUT4 activity. Atazanavir, which does not acutely alter glucose transport in the heart (26) , had no detrimental effect on survival (82.8±0.4 days) when administered at a daily dose of 10 mg/kg (peak drug levels 13.7±1.1 µM). The addition of a boosting dose of ritonavir to atazanavir-treated animals, which produced supratherapeutic levels of atazanavir (45.6±9.4 µM), had the same effect as low-dose ritonavir alone (mean survival 81.5±0.5 days). Consistent with the previously published effect of lopinavir/ritonavir (4:1) on glucose transport both in vitro and in vivo (26) , daily administration of this PI combination (which produced peak lopinavir levels of 9.1±0.3 µM) adversely affected survival to nearly the same degree as 10 mg/kg ritonavir (77.9±0.5 days). Thus, PI-induced changes in the survival of TG9 mice with dilated cardiomyopathy directly correlate with the ability of these drugs to alter glucose transport.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The failing heart shifts toward greater glucose utilization despite the presence of insulin resistance. This shift is hypothesized to be a compensatory mechanism to maintain energy homeostasis in a nutrient-deprived environment (27) . The healthy heart can shift between fuel sources and hence can tolerate transient reductions in glucose delivery induced by PI-mediated inhibition of glucose transport. Therefore, PIs do not cause acute changes in cardiac function when it is normal. In contrast, acute inhibition of facilitative glucose transport in the failing heart, which has reduced substrate flexibility, is likely responsible for the acute decompensation observed in this study.

The effect of certain PIs on glucose transport and cardiac function raises a number of pathophysiologic and clinical issues. While the correlation between the influence of PIs on myocardial glucose uptake (26) and decompensated heart failure in the TG9 mouse model strongly suggests a link between impaired glucose utilization and cardiac diastolic function, it remains possible that these drugs have additional systemic effects that contribute to the phenotype observed. Since the current studies were performed in the setting of intermittent semiacute drug exposure, it is unknown whether in chronic PI administration the heart would compensate for the impairment of GLUT4 by shifting toward an even greater reliance on GLUT1-mediated transport, which is resistant to PI-induced inhibition (28) . This study focused on the effect in the setting of dilated cardiomyopathy, but the effects of PIs in other forms of heart failure such as hypertrophic or ischemic cardiomyopathy clearly warrant investigation in animal models and patients. Furthermore, the results raise the concern that the concomitant use of non-nucleoside reverse-transcriptase inhibitors, which can impair mitochondrial function (29) , and PIs may increase the risk of cardiomyopathy or exacerbate heart failure symptoms in patients at risk.

The pulmonary edema induced by ritonavir and lopinavir in TG9 mice reflects diastolic dysfunction (30 , 31) . The established effect of PIs such as ritonavir and lopinavir on GLUT4 activity suggests that diastolic function in the setting of cardiomyopathy is critically sensitive to either the function of this transporter or the total amount of glucose transport. Thus, these drugs could serve as useful reagents to study the function of GLUT4 in the heart and diastolic dysfunction, which may be particularly relevant to elucidating pathophysiologic mechanisms in diabetic cardiomyopathy or heart failure in general (32) .

Although highly effective antiretroviral therapies have dramatically reduced HIV-associated morbidity and mortality (33) , the increasing life expectancy of patients, together with shifting expectations of therapy to span decades, has raised concern over the long-term effects of drug-induced insulin resistance. Ongoing longitudinal studies of the relationship between HIV therapies and cardiac-related morbidity have demonstrated that PI exposure does increase myocardial infarction risk (34) . The current demonstration that PIs can adversely alter myocardial energy homeostasis in the setting of dilated cardiomyopathy should motivate a careful assessment of the effect of individual PIs like ritonavir and lopinavir on cardiac function in patients who are at risk for or who have developed heart failure of any etiology. It remains to be determined whether similar effects occur in patients, but past studies have established a strong correlation between PI-mediated effects on peripheral glucose disposal in our rodent model systems and in HIV-negative human volunteers (2 , 6 , 21 , 26) . If similar pathophysiologic effects are observed in humans, it will be important to reconsider antiretroviral regimens in HIV-infected patients who develop dilated or other cardiomyopathy, with avoidance of PIs that alter glucose disposal. Assessment of diastolic function in patients receiving antiretroviral therapy could be useful in identifying those at risk for cardiac decompensation. Treatment of myocardial insulin resistance may also have a beneficial impact. Finally, aggressive management of PI-treated patients with comorbid risk factors for diastolic dysfunction, like diabetes or hypertension, warrants serious consideration (30 , 32) .


   ACKNOWLEDGMENTS
 
The authors thank Dr. Avihu Gazit, Anatoly Tzekov, Carolyn Crankshaw, and Dr. Joseph Koster for invaluable assistance and many helpful suggestions. This research was supported in part by grants from NIH (DK64572) and Bristol-Myers Squibb, along with the Clinical Nutrition Research Unit (P30DK052574) and the Digestive Diseases Research Core Center at Washington University (P30DK056341). P.Y.J. is a Scholar of the Child Health Research Center of Excellence in Developmental Biology at Washington University School of Medicine (K12-HD001487).

Received for publication November 20, 2007. Accepted for publication January 17, 2008.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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