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Original Article |
International Antiviral Therapy Evaluation Center (M.v.d.V., F.C.v.L.) and Departments of Infectious Diseases, Tropical Medicine, and AIDS (P.R.), Clinical Chemistry, Laboratory of Endocrinology and Radiochemistry (M.T.A., E.E.), and Endocrinology and Metabolism (H.S.), Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; Department of Endocrinology, Leiden University Medical Center (J.A.R.), 2333 ZA Leiden, The Netherlands; and Department of Internal Medicine, Hospital Gelderse Vallei (R.H.), 6710 HN Ede, The Netherlands
Address all correspondence and requests for reprints to: Dr. M. van der Valk, International Antiviral Therapy Evaluation Center, Academic Medical Center, T0-119, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. E-mail: m.vandervalk{at}amc.uva.nl.
Abstract
Combination antiretroviral therapy for the treatment of human immunodeficiency virus type 1-infected patients is associated with development of the lipodystrophy syndrome (LD). We previously showed that plasma levels of free fatty acids are higher in patients with lipodystrophy. The purpose of this study was to evaluate the postabsorptive rate of lipolysis, using [2H5]glycerol infusion, the resting energy expenditure (REE) measured by indirect calorimetry, and the responses of both to epinephrine infusion (
15 ng/kg·min) in patients with LD. Results were compared with those obtained in five matched human immunodeficiency virus type 1-infected patients. The postabsorptive rate of appearance of glycerol did not differ between the two groups. There was no difference in the lipolytic response to epinephrine, although the response in the LD group was delayed (P < 0.001). The postabsorptive REE adjusted for lean body mass was lower and remained lower during epinephrine infusion in the LD group. Postabsorptive norepinephrine concentrations were higher and remained elevated during epinephrine infusion in the LD group.
We conclude that the lipolytic response to epinephrine in the LD group was normal, albeit delayed. Norepinephrine concentrations were increased in patients with lipodystrophy, indicating increased sympathetic activity. Postabsorptive REE was lower in the patients with lipodystrophy. Our data suggest that highly active antiretroviral therapy-associated lipodystrophy normalizes the REE, but has only minor effects on lipolysis as a result of concomitant sympathetic stimulation of adipose tissue.
COMBINATION ANTIRETROVIRAL therapy, including protease inhibitors, for the treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients has been associated with the development of a fat redistribution syndrome [lipodystrophy (LD)], which may include both central fat accumulation and peripheral fat wasting (1). In patients with LD severe insulin resistance has been documented, which is associated with marked increases in plasma insulin concentrations. Insulin is the main inhibitor of lipolysis. In patients with and without LD using highly active antiretroviral therapy (HAART) the postabsorptive concentration of free fatty acids (FFA) is higher, and the suppression of FFA concentrations by insulin is inhibited (2, 3, 4).
The natural course of HIV-1 infection is associated with both an increase in whole body lipolysis and an increase in resting energy expenditure (REE) (5, 6, 7, 8). The cause of these changes is unknown. Catecholamines are the main stimulators of both metabolic processes, but are not increased in HIV-1-infected patients without concomitant opportunistic infection. Moreover, the lipolytic response to the administration of epinephrine is normal in such patients (7).
It is not known whether lipolysis is regulated differently and whether the REE is increased in HIV-1-infected subjects with LD compared with patients without LD. To answer these questions, we evaluated basal REE and lipolysis and the sensitivity of both metabolic processes to an epinephrine infusion, resulting in slightly increased, but still physiological, epinephrine plasma levels in HIV-infected patients with LD. We compared these with data obtained in untreated HIV-1-infected patients without concomitant opportunistic infections.
Subjects and Methods
Subjects
We studied nine HIV-1-positive men with LD, who were included in the Reverse study. This is an ongoing protocol in which HIV-1-infected patients with LD are examined for reversibility of the syndrome when replacing the protease inhibitor component in their regimen by the nucleoside reverse transcriptase inhibitor abacavir. Patients eligible for inclusion in this study have to use a protease inhibitor-containing regimen and have had a plasma HIV-1 RNA level below 400 copies/ml for at least 6 months. Patients with diabetes mellitus, defined by a postabsorptive plasma glucose concentration above 7.0 mmol/liter, were excluded (9).
After 6 wk of adding abacavir (300 mg, twice daily) to their current regimen, patients were randomized to either discontinue their protease inhibitors immediately or continue protease inhibitor use for another 12 wk and then stop. Patients referred for this protocol had LD in the opinion of their treating physician. Before inclusion in the study this was confirmed by physical examination and by obtaining the patients’ history by two study physicians. LD was defined as the presence of peripheral lipoatrophy, central fat accumulation, or both. All assessments reported for the LD group were performed 6 wk after adding abacavir to the current antiretroviral regimen, but before withdrawal of protease inhibitors. We included patients who reached this point between February 2000 and April 2001. In the final analysis, one of the nine patients was excluded because he had developed a left bundle branch block on electrocardiogram, a contraindication for the administration of epinephrine.
We compared the results with those obtained in our hospital in five HIV-infected patients who were not treated with HAART (HIV). All subjects from the HIV group were weight-stable and did not have any active opportunistic disease. Patients who had fever (temperature >37.5 C); diarrhea; renal, hepatic, or endocrine disease; malignancies other than Kaposi’s sarcoma of the skin; weight loss; or clinically active opportunistic infection in the 2 months before study entry were excluded. Two of five patients used zidovudine monotherapy, and the rest had never been treated for their HIV-1 infection. The results from this control group have been published previously (7). Both studies were approved by the institutional review board of the Academic Medical Center (Amsterdam, The Netherlands). Written informed consent was obtained from all subjects.
Study design
The subjects were admitted to the metabolic clinical research center and studied in the supine position. After a 12-h fast, a catheter was inserted antegrade in a deep antecubital vein of each arm. One catheter was used for sampling of arterialized blood using a heated handbox (60 C). The other catheter was used for infusion of [2H5]glycerol (1.6 µmol/liter priming dose and 0.11 µmol/kg·min) and epinephrine (15 ng/kg·min). After blood samples were taken for determination of background isotope enrichment, iv infusion of [2H5]glycerol was started and continued for 120 min. Epinephrine (15 ng/kg·min) was infused during the last 60 min of isotope infusion. This specific dose of epinephrine was chosen because it results in adequate stimulation of lipolysis at physiological plasma concentrations of epinephrine. Blood samples were obtained at 45, 50, 55, and 60 min of isotope infusion to determine basal lipid kinetics and every 5 min during epinephrine infusion to determine the lipolytic response to epinephrine. Blood samples for hormone concentrations were obtained every 15 min during epinephrine infusion. Blood pressure and heart rate were monitored every 10 min.
All samples were put on ice immediately. Plasma was separated by centrifugation at 4 C within 10 min and stored at -20 C.
Analytical procedures
Samples for catecholamine analysis were collected in 5-ml glass tubes containing reduced glutathione and EGTA. Plasma epinephrine and norepinephrine concentrations were determined by high performance liquid chromatography and electrochemical detection in controls (HIV) and fluorescence detection in the LD group (10). Plasma insulin concentration was determined by a RIA (Insulin RIA 100, Amersham Pharmacia Biotech, Uppsala, Sweden; intraassay coefficient of variation, 3–5%, interassay coefficient of variation: 6–9%; detection limit, 15 pmol/liter).
Blood for analysis of glycerol enrichment was collected in prechilled heparinized tubes. Isotope enrichment of glycerol in plasma was determined by gas chromatography-mass spectrometry using an MSD 5971 system in the HIV group (Hewlett-Packard Co., Palo Alto, CA) (11) and in the LD group as described previously (12).
Body composition
Body composition was measured with a body impedance analyzer (BIA 109, Akern, Florence, Italy) the morning before the start of the isotope infusion study.
Indirect calorimetry
Oxygen consumption and CO2 production were measured by indirect calorimetry using a ventilated hood system (model 2900, Sensormedics, Anaheim, CA). Oxygen consumption and CO2 production were measured continuously during the first 30 min of glycerol infusion without epinephrine infusion and during the last 30 min of epinephrine infusion. REE was calculated using formulas for substrate oxidation as proposed by Frayn (13).
Calculations statistics
Steele’s equation for steady state conditions as adapted for the use of stable isotopes (14) was used to calculate baseline glycerol rate of appearance (Ra). During epinephrine infusion, the glycerol Ra was calculated using the Steele equation for nonsteady state kinetics. The effective volume of distribution of glycerol was assumed to be 235 ml/kg. Enrichment and concentration data obtained during epinephrine infusion were smoothed by spline fitting (15), and substrate kinetics were calculated using these smoothed data.
Statistical analysis
The Ra glycerol data derived after spline fitting were analyzed using the SAS proc mixed procedure (version 8, SAS Institute, Inc., Cary, NC), which accommodates repeated measurements. The first level autoregressive covariate structure appeared to be the most appropriate after comparing several covariate structures through restricted maximum likelihood calculations. In all initial analyses the explanatory variables were group, time (categorical), and their interaction. For Ra glycerol an additional model was used to correct the outcome measurements for possible baseline differences. When medians between the two groups were compared, the Kruskal-Wallis method was used. The REE results were compared between the two groups using a linear regression model adjusted for the amount of lean body mass in kilograms. The overall level of significance was set at 5%.
Results
Patient characteristics
The HIV+LD subjects were comparable to the HIV group with respect to age and body mass index [age, 47 (range, 36–58) and 53 (range, 39–64) yr (P = 0.3); BMI, 24.9 (17.6–32.2) and 23.3 (range, 21.6–26.5) kg/m2 (P = 0.45) in the HIV+LD and HIV groups, respectively]. There also was no difference in amount of fat and fat-free mass as measured by body impedance analysis [BIA; fat mass, 13.9 (range, 7.2–20.8) and 16.4 (range, 8.5–24.4) kg (P = 0.7); fat-free mass, 63.5 (range, 47.2–84.5) and 54.2 (range, 50.1–61.5) kg (P = 0.06) in the HIV+LD and HIV groups, respectively]. All LD patients had HIV-1 viral loads of less than 50 copies/ml at the time of assessment. The mean CD4 cell count was 510 cells/mm3 (range, 270–980) in the LD group and 390 cells/mm3 (range, 10–1130) in the control group (P = 0.63). Antiretroviral drug history, details of the regimens used, and body fat changes at enrollment for each of the eight LD subjects are shown in Table 1
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Fasting plasma insulin and norepinephrine concentrations were significantly higher in the LD group compared with the HIV group (P = 0.03 and P = 0.005, respectively). There was no difference in plasma epinephrine concentrations between the groups (Table 2). The postabsorptive rate of appearance of glycerol per kg body weight did not differ between the HIV and LD groups (Fig. 1), nor was there a difference in the Ra glycerol expressed per kg fat mass between the two groups. There were no differences in systolic and diastolic blood pressure or pulse rate (data not shown). The median basal REE adjusted for lean body mass was 27% lower in the LD compared with the HIV group (P = 0.002; Fig. 2).
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In both groups Ra glycerol per kilogram body weight increased. The response over time in the LD group was delayed compared with that in the control group (P < 0.001; Fig. 1
). The prox mix model used showed no significant difference in overall response between the two arms, indicating that the areas under the curve were not different (P = 0.52). This same pattern was observed when we compared Ra glycerol expressed per kg fat mass between the two groups (P = 0.52). There was no difference in systolic and diastolic blood pressure over time between the two groups. The pulse rate increased slightly in both groups, but there was no difference in the response between the groups over time. Plasma norepinephrine concentrations remained higher in the LD group during the whole experiment compared with the control values (P = 0.009; Table 2 and Fig. 3). The REE increased equally by about 10% in both groups (P = 0.59).
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HAART-related LD is associated with subtle changes in endocrine regulation and substrate and energy metabolism. In a previous study we found that the suppressive effect of insulin on plasma FFA levels was decreased. In the current study basal plasma glycerol turnover was not decreased in the postabsorptive state despite higher insulin concentrations, also suggesting insulin resistance. In addition, plasma norepinephrine concentrations were increased. The lipolytic response to epinephrine was delayed, although the qualitative response per se was not affected. Finally, the subjects with LD had a lower REE compared with the HIV- control group.
We previously found that plasma FFA concentrations are increased in treatment-naive, HIV-infected patients compared with healthy volunteers (16). We chose to measure glycerol turnover in the current study to measure lipolysis more reliably. In the present study basal lipolysis was not different in HAART-treated subjects with LD compared with untreated HIV-infected subjects. The increased rate of basal glycerol turnover found in both groups in our study compared with healthy controls in whom we have previously shown that basal lipolysis was significantly lower (16) is similar to that previously found in HIV-negative subjects with severe obesity (BMI, 39 kg/m2). Basal lipolysis in our HIV-infected patients is similar to that previously found in HIV-negative subjects with severe obesity (BMI, 39 kg/m2) (17). Just as in our HAART patients, in obesity the lipolytic response to epinephrine seems to be delayed compared with that in lean subjects (18). Moreover, in obese subjects a pancreatic clamp that inhibits endogenous insulin production increases glycerol turnover by approximately 50% (19). The similarity of these findings in obese subjects and our LD patients suggests that HAART itself is not responsible for the delayed response in Ra glycerol. The higher concentrations of insulin found in the HIV+LD group during epinephrine infusion therefore may very well be directly or indirectly responsible for the observed delayed increase in lipolysis. One other important factor that might account for the delayed response is a difference in the perfusion of visceral adipose tissue between the groups. Adipose tissue blood flow plays an important role in the regulation of lipolysis (20). There is a difference between the blood flow-stimulating capacity of catecholamines in different fat compartments (21). This difference is influenced by various tissue parameters, such as the size of adipocytes and differences in the organization and permeability of the connective web surrounding the adipocytes. A rise in plasma catecholamines results in an increase in lipolysis and a decrease in local blood flow (21). Although never examined in LD, one can hypothesize, that an increased size of omental adipocytes and a reduced fluid circulation in the visceral fat compartment may result in a decreased delivery of epinephrine to the adipocytes and a slower appearance of glycerol in the systemic circulation in subjects with LD. The observation that overall lipolysis after 60 min of epinephrine infusion was not different in both groups is compatible with such a mechanism in both our HAART patients and obese patients in general.
The lipolytic response to epinephrine was delayed, which indicates impairment in the stimulation of hormone-sensitive lipase by epinephrine in HIV-1-infected patients with LD. Hormone-sensitive lipase is activated by binding of catecholamines to adrenergic receptors (AR) and is inhibited by insulin (22, 23). A differential expression of AR on the cell surface of adipocytes results in different rates of lipolysis. Binding of catecholamines to ß1-, ß2-, or ß3-AR stimulates lipolysis, whereas simultaneous activation of 
2-AR can partly impair this stimulating effect (24, 25). Increased 2-AR activity in the visceral compartment in HAART-treated subjects with LD could therefore account for the delayed lipolytic response. However, this is unlikely because in both patients with obesity and patients with the metabolic syndrome the lipolytic response of omental adipocytes seems to be enhanced rather than decreased (26, 27).
One other interesting finding is the fact that plasma norepinephrine concentrations in patients with LD were almost 2-fold higher than those in controls, as has also been described by Renard et al. (28), suggesting increased sympathetic nervous system activity in patients with LD. The plasma concentration of norepinephrine reflects the resultant of production in the adrenal glands (increased production seems an unlikely explanation for the increase in plasma concentration, because plasma epinephrine concentrations did not differ at baseline) and the balance between release and reuptake in sympathetic nerve endings. Another possible explanation could be a decreased clearance of norepinephrine due to competition with antiretroviral drugs, which to our knowledge has never been described. Therefore, we believe that the most likely explanation for the increased plasma norepinephrine concentration in the subjects with LD is increased sympathetic activity. Assuming this is correct, this could imply that the mechanism underlying the increased lipolysis in HIV infection differs in different circumstances. In patients without LD receiving no or inadequate treatment the mechanism is unknown, but is unrelated to insulin or catecholamines. TNF is a good candidate, as it is a stimulator of lipolysis (29), and TNF production is increased in the natural course of HIV (30, 31). In our study lipolysis was similarly elevated in both groups, indicating that although the factor stimulating lipolysis in uncontrolled HIV infection had disappeared, it had been replaced by another mechanism, possibly a HAART-associated increase in sympathetic activity.
To our surprise REE normalized in the HIV+LD group despite the increase in plasma norepinephrine concentrations. These data are in contrast with those previously reported by Shevitz et al. (32). In a large longitudinal study they found a strong positive correlation between the use of HAART and REE, independent of HIV-1 RNA levels. However, there is no documentation that these subjects were suffering from LD (32). We measured fat-free mass in both groups using BIA. This could lead to an overestimation of FFM, as BIA mainly measures resistance and reactance in the extremities, which is likely to be increased more in the LD group because these patients had severe lipoatrophy of their limbs. In the LD group we also measured FFM using whole-body, dual-energy x-ray absorptiometry (DEXA) as a tool to monitor any improvement in body appearance after the protease inhibitor withdrawal. FFM measured by DEXA was indeed 4.2 kg lower compared with that determined by BIA (P = 0.009) in this group, with a 97% correlation between the two measurements. Therefore, we calculated the REE adjusted for FFM using the DEXA data in the LD group and the BIA data in the control group. The linear regression model used showed that REE adjusted for FFM was significantly lower in the LD group compared with the control value (P = 0.002).
Taken together these data suggest that HIV LD counteracts the HAART-associated hypermetabolic state, which is already known to be elevated by HIV infection. The absence of an increase in REE despite an increased sympathetic activity could be explained by selective sympathetic stimulation of the adipose tissue, as this organ contributes little to basal REE.
In summary, basal lipolysis in patients with LD was not different compared with that in patients with no (adequate) antiretroviral therapy in the presence of increased plasma insulin concentrations, indicating lipolysis to be resistant to the suppression by insulin. The lipolytic response to epinephrine infusion in patients with LD was normal, albeit delayed. Plasma norepinephrine concentrations were increased in patients with LD, indicating increased sympathetic activity. The fasting REE was lower and remained lower during epinephrine infusion in the patients with LD. This suggests that HAART-associated LD as a result of concomitant sympathetic stimulation of adipose tissue has only minor effects on changes in lipolysis induced by HIV infection itself, but normalizes REE.
Acknowledgments
We acknowledge Rob Simonse, research nurse of the Reverse study, for his enthusiastic attitude and for all his logistic assistance during the entire study protocol. We thank An Ruiter and Mignonne Fakkel from the Department of Clinical Chemistry and the Department of Endocrinology for excellent analytical assistance, Gideon Allick from the Metabolic Unit for his help during the day, and Prof. Dr. M. Pfaffendorf, from the Department of Pharmacology for his input in the discussion regarding the regulation of visceral blood flow. We also thank S. N. Blank, F. J. B. Nellen, J. K. M. Schattenkerk, M. H. Godfried, J. T. M. van der Meer, T. van der Poll, J. M. Prins, A. Verbon, and W. E. M. Schouten (Department of Infectious Diseases, Tropical Medicine, and AIDS, Academic Medical Center, Amsterdam, The Netherlands); F. W. N. M. Wit, E. H. Gisolf, T. A. Ruys, and M. H. E. Reijers (International Antiviral Therapy Evaluation Center, Academic Medical Center, Amsterdam, The Netherlands); A. van Eeden (Jan van Goyen Clinic, Amsterdam, The Netherlands); and F. P. Kroon (Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands) for their efforts to recruit patients for the Reverse study.
Footnotes
The Reverse study was supported by a grant by Glaxo-Wellcome Netherlands.
Abbreviations: AR, Adrenergic receptor; BIA, body impedance analysis; BMI, body mass index; DEXA, dual-energy x-ray absorptiometry; FFA, free fatty acids; HAART, highly active antiretroviral therapy; HIV-1, human immunodeficiency virus type 1; LD, lipodystrophy syndrome; Ra, rate of appearance; REE, resting energy expenditure.
Received June 7, 2002.
Accepted August 8, 2002.
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, LD group; 
, HIV group. Data are the least square mean ± SE.
, HIV group.