Intramyocellular lipid accumulation and reduced whole body lipid oxidation in HIV lipodystrophy

Livio Luzi1,2,3, Gianluca Perseghin2,3, Giuseppe Tambussi4, Elena Meneghini2, Paola Scifo3,5,6, Emanuela Pagliato1, Alessandro Del Maschio3,5, Giulio Testolin1, and Adriano Lazzarin4

Divisions of 4 Infectious Diseases, 5 Diagnostic Radiology, and 6 Nuclear Medicine, 2 Clinical Research Unit II, and 3 Clinical Spectroscopy Unit, San Raffaele Scientific Institute, and 1 International Center for Assessment of the Nutritional Status, Universita' degli Studi di Milano, 20132 Milan, Italy


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Antiretroviral therapy in human immunodeficiency virus (HIV)-positive patients can induce a lipodystrophy syndrome of peripheral fat wasting and central adiposity, dyslipidemia, and insulin resistance. To test whether in this syndrome insulin resistance is associated with abnormal muscle handling of fatty acids, 12 HIV-1 patients (8 females/4 males , age = 26 ± 2 yr, HIV duration = 8 ± 1 yr, body mass index = 22.0 ± 1.0 kg/m2, on protease inhibitors and nucleoside analog RT inhibitors) and 12 healthy subjects were studied. HIV-1 patients had a total body fat content (assessed by dual-energy X-ray absorptiometry) similar to that of controls (22 ± 1 vs. 23 ± 2%; P = 0.56), with a topographic fat redistribution characterized by reduced fat content in the legs (18 ± 2 vs. 32 ± 3%; P < 0.01) and increased fat content in the trunk (25 ± 2 vs. 19 ± 2%; P = 0.03). In HIV-positive patients, insulin sensitivity (assessed by QUICKI) was markedly impaired (0.341 ± 0.011 vs. 0.376 ± 0.007; P = 0.012). HIV-positive patients also had increased total plasma cholesterol (216 ± 20 vs. 174 ± 9 mg/dl; P = 0.05) and triglyceride (298 ± 96 vs. 87 ± 11 mg/dl; P = 0.03) concentrations. Muscular triglyceride content assessed by means of 1H NMR spectroscopy was higher in HIV patients in soleus [92 ± 12 vs. 42 ± 5 arbitrary units (AU); P < 0.01] and tibialis anterior (26 ± 6 vs. 11 ± 3 AU; P = 0.04) muscles; in a stepwise regression analysis, it was strongly associated with QUICKI (R2 = 0.27; P < 0.0093). Even if the basal metabolic rate (assessed by indirect calorimetry) was comparable to that of normal subjects, postabsorptive lipid oxidation was significantly impaired (0.30 ± 0.07 vs. 0.88 ± 0.09 mg · kg-1 · min-1; P < 0.01). In conclusion, lipodystrophy in HIV-1 patients in antiretroviral treatment is associated with intramuscular fat accumulation, which may mediate the development of the insulin resistance syndrome.

human immunodeficiency virus


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A SYNDROME CHARACTERIZED by lipodystrophy (peripheral lipoatrophy and truncal fat accumulation), hyperlipemia, and insulin resistance was recently described in human immunodeficiency virus (HIV)-1-infected patients (5). Although the clinical and biochemical picture is rather consistent, little is known about the pathogenic events leading to abnormal fat deposition and insulin resistance in HIV-1-infected patients. The first hypothesis, proposed by Carr et al. (6), suggested a major role of protease inhibitors (PIs) in the induction of hypertriglyceridemia (and insulin resistance), since they can interact with retinoic acid receptors at the liver site (6). This initial hypothesis was subsequently modified by the demonstration of the lipodystrophic syndrome in HIV-1-infected patients, either on other antiretroviral drugs [mainly nucleoside analog reverse transcriptase inhibitors (NRTI); see Refs. 2 and 36] or naive to PIs (25). Thus it seems that both the viral infection per se or combined with different antiretroviral treatments plays a role in the pathogenesis of the lipodystrophic syndrome.

The abnormal fat distribution of HIV-1-infected patients is characterized by visceral and truncal obesity, adipose tissue atrophy of the limbs (mainly lower limbs), increment of adipose tissue in the mammary region, and "buffalo hump" (5). The metabolic alterations characterizing the insulin resistance syndrome include hyperlipemia (mainly hypertriglyceridemia), reduction of high-density lipoprotein (HDL) cholesterol, glucose intolerance, and hyperuricemia (5, 6). Among the most prominent hormone alterations, increased levels of insulin, C-peptide, prolactin, erithropoietin, and tumor necrosis factor (TNF)-alpha were described (9). An abnormal circadian cortisol pattern was also described (when this is the prevailing hormone alteration, the term "pseudo-Cushing" syndrome is used; see Ref. 34). A beneficial effect of growth hormone (GH) administration (indicated when an absolute or relative GH secretory defect is present) was suggested (44).

A clear-cut pathogenesis of HIV-1-related lipodystrophy is presently unknown. A disturbance of lipid metabolism (resulting from either the viral infection or the association virus-antiretroviral therapy-genetic background) seems to play a central role in the pathogenesis of the syndrome. The liver has been considered the main organ responsible for the defective lipid metabolism in lipodystrophic HIV-1-infected patients. The present study was undertaken to define a possible role of muscle in the pathogenesis of insulin resistance of HIV-1-related lipodystrophy. In particular, we asked the following questions: 1) is the HIV-1-related lipodystrophy associated with an alteration of whole body energy metabolism and substrates oxidation rate; and 2) is there a metabolic alteration at the skeletal muscle site? Our data indicate an impaired whole body lipid oxidation in HIV-1-infected patients and a strong association between insulin resistance and intramyocellular lipid accumulation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Subjects. Twelve HIV-1-infected patients were selected in the population of over 600 patients at the Outpatient Clinic of Metabolic Diseases in the Division of Infectious Diseases of San Raffaele Hospital. Table 1 represents the anthropometric parameters and the major clinical and biochemical data of the study patients. Twelve healthy volunteers, matched for major anthropometric parameters, were enrolled in the study and served as a control group. Antiretroviral therapy included both PIs and nucleoside analog reverse transcriptase inhibitors in all patients. Table 2 outlines the major inclusion and exclusion criteria followed in the selection of the 12 HIV-1-infected patients. Only patients who were on a stable antiretroviral therapy for at least 1 yr (both PI and NRTI) and had constant blood CD4 count and viral level for at least 4 mo were enrolled in the study. Patients who had a variation of body weight >10% of total body weight were excluded from the study. In all patients, fasting glucose and glycosylated Hb were assessed on day 1 (see Experimental protocol for details) and were found to be normal. Previous normal blood glucose measured within 12 wk before the study allowed us to rule out diabetes mellitus. In addition, all HIV-1-infected patients received a standard 75-g oral glucose tolerance test (found normal) on a separate day within 2 wk of the study period. In all patients, 24-h urinary cortisol excretion was assessed in the 24-h urinary volume collected starting at 0800 of the day before day 1. Triglycerides and total cholesterol were higher in HIV-1-infected patients than in control patients. The finding of higher postabsorptive levels of insulin and C-peptide, indicating insulin resistance, completed the biochemical picture of the lipodystrophic syndrome. All patients could be classified as having a type III, grade 2 lipodystrophy according to the "ART-associated Lipodystrophy European Comparative Study" group (33). All patients had increased truncal adiposity, reduced or absent lower-extremity subcutaneous fat, and hypertriglyceridemia. Both HIV-1-infected patients and healthy controls were informed of the possible risks involved in the participation in the present study and gave their consent. The experimental protocol was approved by the Ethics Committee of San Raphael Scientific Institute, and written informed consent was obtained from all patients.

                              
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Table 1.   Clinical and biochemical parameters of study subjects


                              
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Table 2.   Major inclusion and exclusion criteria for the participation of HIV-1 patients in this study

Experimental protocol. All subjects involved in this protocol were studied on three separate days. The subjects assumed an isocaloric diet in the 4 wk preceding the study, and they did not modify their usual life style. On day 1, they were admitted to the Metabolic Unit of the Department of Medicine of San Raffaele Scientific Institute after an overnight fast (from 2200 of the day before study, only water was allowed). At 0800, a polyethylene catheter was inserted in the antecubital vein of one forearm, as previously described (23). At 0830, a basal blood sample for the measurements of hormones and metabolites was drawn. On day 2, all subjects were readmitted to San Raffaele Hospital at the Division of Diagnostic Radiology, where a 1H NMR-spectroscopy scan was performed between 0700 and 0800 to assess the intramyocellular concentration of triglycerides, as previously described (30). On day 3, all subjects were admitted to the International Center for Assessment of Nutritional Status of the Universita' degli Studi di Milano to perform dual-energy X-ray absorption (DEXA) for the measurement of fat distribution in different body districts. The same day, respiratory gas exchange measurement by means of indirect calorimetry (model Vmax 29; Sensor Medics Italia, Milan, Italy) was started at 0830 after an overnight fast (9-11 h) and continued for ~30 min. The three tests were performed in random order in no more than a 1-wk period in all subjects.

Analytical determinations. Aliquots of blood for the measurement of metabolite [glucose, triglycerides, total cholesterol, HDL, low-density lipoprotein (LDL), and uric acid] levels were placed in heparinized tubes, as previously described (30). Plasma glucose was measured with a Beckman glucose analyzer (glucose oxidase). Blood aliquots for insulin, C-peptide, prolactin, TNF-alpha , and GH were collected in tubes for serum separation. Plasma insulin was measured with a microparticle enzyme immunoassay technology with no cross-reactions with proinsulin, C-peptide, and glucagon (Imx Insulin assay; Abbott Laboratories, Rome, Italy; see Ref. 30). Free fatty acids (FFA; Table 1) were collected and measured as previously described (30). Urine collection (24 h) was performed to quantify cortisol excretion. Glycosylated Hb, uric acid, TNF-alpha , urinary cortisol, and insulin-like growth factor (IGF)-I were not measured in normal subjects. All blood samples were placed on ice until the plasma or serum was prepared by centrifugation at 4°C (within 1.5 h of sampling). All plasma and serum aliquots were frozen at -60°C until later analysis.

Indirect calorimetry. Indirect calorimetry measurement was performed as previously described by means of a Vmax 29 Sensor Medics Instrumentation operated in the canopy/nutritional dilution mode (23). Respiratory exchanges were measured over a 30-min period with the patient resting in bed in a quiet room with an ambient room temperature ranging between 23 and 25°C. Only parameters obtained in steady-state conditions were used for final calculations (steady state defined as >= 3 min of VO2 within 10%, of respiratory quotient within 5%, and of air flow through the canopy system within 10%).

1H NMR spectroscopy. 1H NMR spectroscopy was performed on a Sigma 1.5 Tesla scanner (General Electric Medical Systems, Milwaukee, WI) using a conventional linear extremity coil. High-resolution, T1-weighted images of the right calf were obtained before spectroscopic acquisition to localize the voxel of interest for the 1H spectroscopy study. Voxel shimming was executed to optimize the homogeneity of the magnetic field within the specific volume of interest. Two 1H spectra were collected from a 15 × 15 × 15 mm3 volume within the soleus and tibialis anterior muscles. A PRESS pulse sequence (repetition time = 2,000 ms and echo time = 60 ms) was used, and 128 averages were accumulated for each spectrum, with a final acquisition time of 4.5 min. The water signal was suppressed during the acquisition, since it would dominate the other metabolite's peak signals of interest. A third 1H spectrum of a triglyceride solution inside a glass sphere, positioned within the extremity coil next to the calf, was also obtained during the same session to have an external standard acquired in the same conditions as the subject's spectra. Postprocessing of the data, executed with the Sage/IDL software, consisted of high-pass filtering, spectra apodization, zerofilling, Fourier transformation, and phasing of the spectra. The integral of the area under the peak was calculated using a Marquardt fitting with Lorentzian functions of the peaks of interest. The integral of the methylene signal (-CH2) at 1.35 parts/million was used to calculate intramyocellular triglyceride content expressed in arbitrary units (AU) as the ratio to the integral of the peak of the external standard × 1,000 (30, 31).

DEXA. DEXA was performed with a Lunar-DPX-IQ scanner (Lunar, Madison, WI). A different scan mode was chosen with respect to each subject's body size, as suggested by the manufacturer's operator manual. For regional analysis, three-compartment processing was performed in the arms, trunk, and legs (30). Fat content is expressed as kilograms of fat mass and as a percentage of tissues. Because all patients had truncal fat accumulation (rather than pure visceral obesity), the computed tomography scan of the abdomen was not performed.

Insulin action. Insulin action was assessed using a quantitative insulin sensitivity check index (QUICKI) (18). QUICKI hinges on the measurement of fasting insulin and glucose concentrations. It takes both the logarithm and the reciprocal of the glucose-insulin product (a, b)
QUICKI(G<SUB>b</SUB>, I<SUB>b</SUB>)<IT>=</IT><FR><NU>1</NU><DE>log(G<SUB>b</SUB> × I<SUB>b</SUB>)</DE></FR> = <FR><NU>1</NU><DE>log(G<SUB>b</SUB>) + log(I<SUB>b</SUB>)</DE></FR>
where Gb (mg/dl) is the fasting glucose concentration and Ib (µU/ml) is the fasting insulin concentration and was found to be the best predictor of insulin sensitivity on the basis of fasting blood sampling.

Statistical analysis. All data are expressed as means ± SE. Comparisons between the control and the HIV groups were performed with the Student's t-test for unpaired data when appropriate.


    RESULTS
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INTRODUCTION
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DISCUSSION
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Body composition. Table 3 presents data on absolute and percent fat composition in HIV-1-infected patients and in healthy volunteers in different body districts. No difference was observed in the absolute and percent fat content in the whole body and in the upper limbs between the two study groups. In contrast, both the absolute (P = 0.003) and percent (P = 0.002) fat content in the legs were markedly reduced with respect to the control group. The absolute (P = 0.02) and percent (P = 0.02) fat content of the trunk were higher in the HIV-1-infected patients with respect to controls.

                              
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Table 3.   Body composition of study subjects (DEXA)

Energy metabolism. Resting energy expenditure (REE) in HIV-1-infected patients was not statistically different compared with healthy volunteers either in absolute terms (P = 0.47) or when calculated per kilogram body weight (P = 0.54) or kilogram lean body mass (P = 0.21). In contrast, the respiratory quotient, calculated by dividing the VCO2 by the VO2, was markedly increased in HIV-1-infected patients (0.91 ± 0.02, P = 0.0001) with respect to healthy volunteers (0.81 ± 0.1), indicating an impaired lipid oxidation. Accordingly, whole body lipid oxidation (mg · kg-1 · min-1) was markedly impaired in HIV-1-infected patients with respect to controls (0.20 ± 0.06 vs. 0.88 ± 0.09, respectively, P = 0.00001). The reduction of lipid oxidation was matched by a complementary increment of glucose oxidation (mg · kg-1 · min-1) in HIV-1-infected patients (1.87 ± 0.21, P = 0.003) with respect to healthy subjects (1.03 ± 0.13; Fig. 1 and Table 3).


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Fig. 1.   Respiratory quotients (RQ) of human immunodeficiency syndrome (HIV)-1-infected patients (open bar) and of healthy control subjects (filled bar). *P = 0.0001.

Intramyocellular triglyceride concentration. The intramyocellular triglyceride content of the soleus muscle was significantly increased in HIV-1-infected patients compared with healthy controls (101 ± 15 vs. 44 ± 5 AU, P = 0.001). Similarly, the triglyceride content of the tibialis anterior muscle was higher in HIV patients compared with controls (26 ± 6 vs. 11 ± 2 AU, respectively, P = 0.02; Fig. 2).


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Fig. 2.   Intramyocellular triglyceride concentration expressed in arbitrary units (AU) in the soleus (filled bars) and tibialis anterior (open bars) muscles. HIV-1-infected patients show a higher triglyceride content in both muscles than healthy subjects. *P = 0.03 and **P = 0.001 with respect to controls.

Insulin action. The QUICKI was lower (P = 0.012) in HIV-1-infected patients (0.341 ± 0.011) compared with healthy controls (0.376 ± 0.007). An inverse correlation between insulin action and intramyocellular triglyceride concentration was shown in the study subjects (Fig. 3). Gan et al. (11) found a similar correlation between insulin action assessed via the clamp and intramyocellular lipid content.


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Fig. 3.   Inverse relationship between insulin sensitivity (QUICKI) and the soleus intramyocellular triglyceride concentration in HIV-1 () and control (open circle ) subjects.


    DISCUSSION
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INTRODUCTION
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DISCUSSION
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All patients included in the study were lipodystrophic (Table 2). In particular, the total body weight and the total percentage of body fat mass were not statistically different from the control group. Interestingly, the arm fat percentages were also similar in HIV-1-infected patients and in healthy subjects, confirming that the body districts mainly involved are 1) the lower extremities, where the apoptotic process is prominent (1), and 2) the trunk, where the fat accumulation is increased. Both the antiretroviral drugs and the virus itself have been hypothesized as possible causes of HIV-1-related lipodystrophy (25). The complete sequence of events by which PIs lead to insulin resistance, cause apoptosis, and eventually determine lipodystrophy is presently still unknown. PIs have two possible mechanisms by which they induce lipodystrophy and insulin resistance. First, PI drugs have a direct effect on the retinoic acid receptor (leading to apoptosis of adipocytes in specific body districts) and on the lipoprotein receptor-related protein; second, PIs have an indirect negative effect on insulin action by impairing triglyceride clearance (5, 6, 32). Because in the present work we did not measure the lipolytic rate of adipose tissue, we cannot draw any conclusion on a possible regional difference of PIs on lipolytic rate. In contrast, NRTI may, possibly, induce lipodystrophy via mitochondrial toxicity. In addition, non-virus-related mitochondrial toxic myopathies are known to induce lipid storage inside the skeletal muscle cells (10). Finally, the demonstration of lipodystrophy in patients naive for antiretroviral drugs (25) or in the very early stages of disease (4) may support the possibility that the retrovirus itself may induce the syndrome, without the concomitance of any drug treatment. Complex interactions are present among the retroviral infection, PIs, and NRTI drugs. Therefore, we selected patients on both classes of drugs (PI and NRTI for >= 1 yr), having the three known factors possibly involved in the pathogenesis of the lipodystrophy/insulin resistance syndrome (Table 2).

All patients were hypertriglyceridemic (Tables 1 and 3). No patient had hormone or metabolic factors that, per se, could induce or worsen an altered distribution of body fat or insulin resistance. First, diabetes mellitus was excluded, since hyperglycemia is known to induce insulin resistance. All HIV-1-infected patients had a normal urinary excretion of cortisol and plasma hormone levels in the normal range, excluding a major hormonal alteration leading to truncal adiposity. Interestingly, FFA concentration was not increased as in other forms of lipodystrophy, possibly because of the differential fat distribution between the abdomen (accumulation) and legs (atrophy; Table 1).

All patients were insulin resistant, with comparable glucose levels in the two groups. We made an effort to exclude a number of different diseases (diabetes mellitus, obesity, hyperuricemia, cancer, hereditary diseases, and sepsis) on the basis of known risk factors leading to a reduction of insulin action. We also ruled out a coexisting hyperuricemia by measuring the plasma uric acid concentration (normal in all subjects; Table 1). The normal hormone levels previously discussed excluded a hormonal cause of reduced insulin action, pointing to either the viral infection or the pharmacological interactions of antiretroviral drugs as isolated causes of insulin resistance. A possible mechanism of induction of insulin resistance by PIs was suggested by Murata et al. (27), who recently described a selective inhibition of GLUT4 activity by indinavir (a PI drug), with a normal phosphorylation of the tyrosine residues of insulin receptor and insulin receptor substrate-1.

REE is increased during sepsis (29) or in patients with advanced acquired immunodeficiency syndrome (37), leading eventually to cachexia. Our HIV-1-infected population does not show a significant increase in REE compared with the control group. Nonetheless, two previous reports demonstrate an increased REE in HIV-lipodystrophic patients on antiretroviral drug treatment (17, 38). Possible causal links between increased metabolic rate and either higher TNF-alpha blood levels or resistance to antiretroviral drug treatment were previously suggested. Shevitz et al. (38) recently suggested that, although highly active antiretroviral therapy (HAART) may decrease the metabolic rate by lowering viral load, it increases the metabolic rate by a distinct, independent, and unknown mechanism. It is important to note that lipodystrophy itself (not associated with HIV infection, but with other diseases) may increase the metabolic rate. We (24) and others (20, 21) have shown increased REE in diabetic patients with partial or total lipoatrophic diabetes mellitus. Nonetheless, REE was found to be increased in diseases associated with congenital/acquired, total/partial lipodystrophy, like Kobberling-Dunningan syndrome (42), Nasu-Hakola disease (8), and cystic angiomatosis (3).

It is noteworthy that our patients demonstrate a markedly reduced whole body lipid oxidation rate (and a complementary increase of carbohydrate oxidation) compared with the control group (Fig. 1 and Table 4). This finding is somewhat expected in a cohort of patients hypertriglyceridemic, insulin resistant, and with an impaired clearance of plasma lipids, although it is most consistent in HIV-infected patients treated with antiretroviral drugs. In contrast, an early defect of lipid oxidation was shown in obese and postobese subjects (26, 35). Once obesity or type 2 diabetes is established, there is not necessarily a decreased lipid oxidation. A previous report of Hommes et al. (17) found the lipid oxidation normal to increased in HIV-infected patients not on HAART, underlining the importance of antiretroviral drugs in the development of the lipid oxidation defect. Korach et al. (22) recently showed that only after the institution of antiretroviral therapy did HIV patients develop a decrease in lipid oxidation.

                              
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Table 4.   Energy expenditure, respiratory quotient, and substrate oxidation rates in HIV and Con

Both the liver and the skeletal muscle are major sites of lipid oxidation. It is reasonable to postulate that an impairment of beta -oxidation of fatty acids induces triglyceride accumulation at the organ sites. Liver steatosis (assessed by ultrasound echography) was detected in all HIV-1-infected patients included in this study (Table 1; and is commonly present in most lipodystrophic patients; see Ref. 16), although this finding might be indicative of a concomitant chronic hepatitis instead of primary hepatic lipid accumulation. For this reason, the report of a higher intramyocellular triglyceride concentration in the HIV-1-infected group is of crucial importance (Fig. 2). This is the first demonstration that HIV-1-infected patients with lipodystrophy have an increased level of lipids in the skeletal muscle cells. Because all HIV-1-infected patients were on both classes of drugs (PIs and nucleoside analog inverse transcriptase inhibitors), it is impossible to ascribe to one class of drug or to the other the increase of intramyocellular lipid content. Interestingly, many viral infections cause lipid (either cholesterol or triglyceride) accumulation in the liver parenchyma (7), heart (39), or skeletal muscle (15, 28). Therefore, the retroviral infection per se may be partially responsible for the higher triglyceride content in the skeletal muscle. Finally, intramyocellular triglyceride accumulation was described in other forms of lipodystrophy not characterized by cytokine production, either in rodent models (12) or in humans (40). Therefore, the lack of an HIV-nonlipodystrophic group does not allow us to dissect out the role of lipodystrophy and of viral infection itself in the development of intramuscular triglyceride accumulation.

In conclusion, the present data indicate a novel metabolic defect of HIV-1-infected patients treated with HAART, namely an intramyocellular accumulation of triglycerides associated with a reduced whole body lipid oxidation. The intramyocellular lipid concentration inversely correlates with insulin sensitivity. Probably, the retroviral infection in combination with HAART is responsible for the metabolic defect, which is associated with abnormal fat distribution. Recent publications point to a selective impairment of GLUT4 activity by PI drugs (27), and genetic manipulation of GLUT4 expression has shown a role of this protein in adipose tissue metabolism (13, 19, 41). Therefore, on the basis of our results and on in vitro experimental evidence, the use of pharmacological agents selectively enhancing insulin action could be suggested in HIV-1-infected patients (14, 43).


    ACKNOWLEDGEMENTS

This work was supported by Progetto Finalizzato del Ministero della Sanita', Italy, Grant no. R.F. 99 Unita' 03 and 04.


    FOOTNOTES

Address for reprint requests and other correspondence: L. Luzi, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy (E-mail: luzi.livio{at}hsr.it).

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.

First published October 8, 2002;10.1152/ajpendo.00391.2001

Received 29 August 2001; accepted in final form 17 September 2002.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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