Among antiretroviral drug-associated toxicity, which causes body fat redistribution, the so-called HIV/highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome (HALS) is the most feared by patients and caregivers.1 HALS is feared because of the devastating consequences in terms of self-image and psychological health, and because there is no intervention to successfully reverse this complication.1
Antiretroviral drugs with a known deleterious profile on adipocyte biology and function are the thymidine analogs and to a lesser extent didanosine, whereas nonnucleoside reverse transcriptase (RT) inhibitors have been considered benign drugs with respect to their effects on adipose depots.2 First-generation protease inhibitors also exhibited significant fat toxicity, and their negative effects were synergistic with those of nucleoside RT inhibitors (NRTIs).3 In the ACTG 5142 trial, efavirenz (EFV)-based regimes were found to be associated with a higher risk of developing lipoatrophy, defined as a limb fat loss >20% from the baseline, across all NRTI groups compared with that in the case of lopinavir/ritonavir (LPV/r).4
This intriguing finding prompted us to perform a molecular and clinical study to assess the effects of LPV/r and EFV, both combined with tenofovir/emtricitabine (TDF/FTC) on gene markers of adipocyte biology and function in subcutaneous adipose tissue (SAT), in HIV-infected, antiretroviral-naive patients, who started their first HAART regime. Because adipocyte function is already disturbed in naive patients,1 our working hypothesis was that molecular markers of adipocyte biology and function would improve in both arms.
PATIENTS AND METHODS
Patients were selected from the same HIV-1 infection clinic cohort, with the baseline visit between July 2008 and January 2010, at the Hospital de la Santa Creu i Sant Pau, which serves a population of 1778 HIV-1–infected patients on active follow-up. Inclusion criteria were as follows: an established diagnosis of HIV-1 infection and antiretroviral-naive patients who started antiretroviral therapy with a TDF/FTC combined fixed dose formulation (Truvada; Gilead Sciences, Foster City, CA) plus LPV/r or with TDF/FTC/EFV compact pill (Atripla; BristolMyersSquibb, NY and Gilead Sciences, Foster City, CA). The primary endpoint of the study was the change in molecular markers of adipocyte biology and function at the SAT level. The secondary endpoint was the change in limb fat mass measured by dual x-ray absorptiometry from baseline to 48 weeks.
Baseline and follow-up measurements and treatments were done according to standard clinical practice at the HIV infection clinic. At study entry, each patient had an HIV infection history and demographic data recorded and anthropometric, blood pressure, viroimmunological, and metabolic parameters measured. These measurements were repeated every 3 months. Dual energy x-ray absorptiometry scans and abdominal SAT biopsies were performed at baseline and at week 48. Gene expression in SAT was assessed with baseline and 48-week samples (see below).
At the time of study entry, no patient used any other drug known to influence glucose or lipid metabolism or fat distribution, which included anabolic hormones or systemic corticosteroids, recombinant human growth hormone, or appetite stimulants. All patients provided a written informed consent before enrollment. The study was approved by the institutional review board and complied with the provisions of the Good Clinical Practice guidelines and the Declaration of Helsinki. Additional exclusion criteria included the following: fasting glucose >6.1 mmol/L, daily alcohol intake ≥40 g, hypothyroidism, serum creatinine >150 mmol/L, and alanine aminotransferase, aspartate aminotransferase >5× upper limit of normal, anemia, >10% loss in body weight in the preceding 6 months, and any active AIDS-defining disease. The patients were instructed not to modify their diet, exercise, or other habits during the study. The diagnosis of AIDS was based on the 1993-revised case definition of the Centers for Disease Control and Prevention CDC.5 Written informed consent was obtained from the patients at study entry. The study was approved by the Ethics Committee of the Hospital de la Santa Creu i Sant Pau.
Body Composition Measurements
The subjects were weighed on calibrated scales after removing their shoes, outdoor clothing, and other heavy items. Body mass index (BMI) was calculated by dividing the weight in kilograms by the square of the height in meters. Waist circumference was measured to the nearest millimeter using anatomical landmarks as defined for the Third National Health and Nutrition Evaluation Survey.6
Whole-body dual x-ray absorptiometry scans (Hologic QDR-4500A; Hologic, Inc, 590 Lincoln St, Waltham, MA) were conducted by a single operator on all the patients. The operator was blinded to the antiretroviral treatment. The percentage of fat in the arms, legs, and central abdomen (calculated from the mass of fat versus lean and bone mass), and total lean body mass in kilograms, was recorded. To assess fat symmetry distribution, the following fat ratios were analyzed: trunk/limb fat ratio by dividing trunk fat by appendicular fat,7 fat mass ratio by dividing the percentage of trunk fat by the percentage of lower limb fat,8 fat mass index by dividing whole-body fat by squared height,9 and leg fat percentage normalized to BMI by dividing the percentage of leg fat mass by BMI.10
Definition of Lipoatrophy, Lipohypertrophy, and Metabolic Syndrome
Lipoatrophy was defined as a decrease in limb fat >20% with respect to the baseline value, whereas lipohypertrophy was defined as an increase in trunk fat >20% with respect to the baseline value.4,11 The metabolic syndrome was defined according to the US National Cholesterol Education Program Adult Treatment Panel III Guidelines12 and modified as recommended in the latest American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement.13
Biochemistry Laboratory Measurements
All laboratory investigations were performed after 12 hours of overnight fast and at least 15 minutes after the placement of a peripheral intravenous catheter as previously described.14 Insulin resistance was estimated by the homeostasis model assessment method (HOMA-R) as the product of the fasting concentrations of plasma insulin (microunits per milliliter) and plasma glucose (millimoles per liter) divided by 22.5.15
Fat Tissue Samples
Fat tissue samples were obtained from SAT abdominal depot through a small surgical biopsy performed by an 8-mm punch under local anesthesia with mepivacaine, at baseline, and at week 48. One half of the SAT obtained was immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction (see below).
Genes Profiled in Subcutaneous Fat
The genes profiled in subcutaneous fat together with a brief description of its function are summarized in Table 1. After homogenization in RLT buffer (Qiagen, Hilden, Germany), DNA was isolated using standard phenol/chloroform procedures, and RNA was isolated using a column affinity–based methodology that included on-column DNA digestion (RNeasy; Qiagen). One microgram of RNA was transcribed into cDNA using MultiScribe RT and random-hexamer primers (TaqMan Reverse Transcription Reagents; Applied Biosystems, Foster City, CA). For quantitative mRNA expression analysis, TaqMan RT-polymerase chain reaction (PCR) was performed on the ABI PRISM 7700HT sequence detection system (Applied Biosystems). The TaqMan RT-PCR reactions were performed in a final volume of 25 μL using TaqMan Universal PCR Master Mix, No AmpErase UNG reagent, and primer pair probes specific for transcripts encoding peroxisome proliferator–activated receptor-gamma (PPAR-γ) (Hs00234592_m1) and CCAAT enhancer–binding protein-α (C/EBPα) (Hs00269972_s1), glucose transporter-4 (GLUT4) (Hs00168966_m1), adipocyte protein-2/fatty acid–binding protein-4 (aP2/FABP4) (Hs00609791_m1), the proinflammatory cytokines tumor necrosis factor-α (TNF-α, Hs00174128_m1), interleukin 18 (IL-18) (Hs99999040_m1), monocyte chemotactic protein-1 (MCP-1/CCL-2) (Hs00234140_m1), and 78-kDa glucose-regulated protein (GRP78). The transcript levels for the mitochondrial DNA (mtDNA)-encoded genes cytochrome b (Cyt b) (Hs02596867_s1) and cytochrome oxidase subunit II (Cox II) (Hs02596865_g1) were determined using the same Taqman RT-PCR reaction method. mtDNA levels were assessed using a cyt b probe (MT-CYB) and referred to nuclear DNA, as determined by the amplification of the intronless gene CEBPα (CEBPA).16 Controls with no RNA, primers, or RT were included in each set of experiments. Each sample was run in duplicate, and the mean value of the duplicate was used to calculate the mRNA levels for the genes of interest. Values were normalized to that of the reference control RPLO (Hs99999902_m1) using the comparative 2-[INCREMENT]CT method, following the manufacturer's instructions. Parallel calculations performed using the reference gene PPIA (Hs99999904_m1) essentially yielded the same results.
Data are expressed as frequencies and percentages or median with interquartile range (IQR, percentile 25–percentile 75). HIV-1 RNA copies per milliliter were analyzed after a log10 transformation. We used the Fishers exact test to compare categorical variables and the Mann–Whitney test and the Wilcoxon test for independent and dependent continuous data. Within-group and between-group analyses were the primary and secondary endpoints, respectively. All analyses were performed with the Statistical Package for Social Sciences version 17.0 (SPSS, Chicago, IL), and the level of significance for all tests was set at the 2-sided 0.05 level.
The study group was made up of 44 patients, 23 treated with TDF/FTC plus LPV/r and 21 with TDF/FTC/EFV. Demographics of patients at baseline are shown in Supplemental Digital Content 1 (http://links.lww.com/QAI/A529).
Both groups were well balanced with respect to virologic and immunological parameters at baseline, including the percentage of patients with <200 CD4 cells and those with viral load ≥5 log10 copies per milliliter (see Supplemental Digital Content 1, http://links.lww.com/QAI/A529). Seventy percent of the patients had an undetectable viral load at week 48, and among those who had it detectable, the median viral load was 42 (IQR, 25–51) copies per milliliter (see Supplemental Digital Content 1, http://links.lww.com/QAI/A529).
Metabolic Changes Over Time
Metabolic, anthropometric, and fat parameters, including fat distribution symmetry indexes, were well balanced without statistically significant differences between groups at baseline (see Supplemental Digital Content 2, http://links.lww.com/QAI/A529). Eight patients (18.2%) had metabolic syndrome at baseline, a percentage that did not increase significantly during the study period (11, 25%). Over the 48 weeks of the study, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very low-density lipoprotein (VLDL) cholesterol, and triglycerides increased significantly in patients exposed to LPV/r, whereas in the EFV arm, only total cholesterol and LDL cholesterol had significant increases (see Supplemental Digital Content 3, http://links.lww.com/QAI/A529). There were no significant differences in the total cholesterol/HDL ratio between groups either at baseline or at week 48. There were also no significant changes over time in fasting glucose, fasting insulin, and HOMA, except for fasting glucose in the EFV group (see Supplemental Digital Content 3, http://links.lww.com/QAI/A529). There were no statistically significant differences in the metabolic parameters between groups at week 48, except for a higher fasting glucose in the EFV-treated group (see Supplemental Digital Content 3, http://links.lww.com/QAI/A529). No patient was taking lipid-lowering drugs at baseline, whereas 5 (12.7%) in the LPV/r and 1 (4.7%) in the EFV arm were taking these drugs at week 48 (P = 0.2304).
Body Composition Changes Over Time
Whole-body fat, lower limb fat, and limb fat mass increased significantly from baseline in the LPV/r-treated group, whereas a trend for a significant increase was observed in the same parameters in the EFV-treated group (Table 2). The mean increase in the limb fat mass at week 48 was 994 ± 732 g in the LPV/r group and 1181 ± 628 g in the EFV group, respectively (P = 0.9832). Two (9.5%) patients in the LPV/r group and 2 (8.7%) in the EFV group had a limb fat loss ≥20% with respect to baseline at week 48 (P = 1.0). Percentages of limb fat loss >10% of the baseline at week 48 were 21.7% and 14.3%, respectively (P = 0.7010). Two patients (8.7%) in the LPV/r and none in the EFV group had an increase in trunk fat mass ≥20% from baseline (P = 0.5101). Fat distribution symmetry indexes did not change significantly over time (see Supplemental Digital Content 3, http://links.lww.com/QAI/A529). At week 48, there were no significant differences between both groups in terms of fat parameters and fat distribution symmetry indexes. Bone mineral content (BMC) and bone mineral density (BMD) significantly decreased over the study period, and at week 48, the BMC was significantly lower in the LPV/r group compared with that in the EFV group (Table 2).
Mitochondrial DNA and Gene Expression in SAT Over Time
Paired fat biopsies were available for 18 patients (78.3%) in the LPV/r group and for 17 patients (80.9%) in the EFV group. There were no statistically significant differences with the whole group between patients who had paired biopsies in each arm in terms of viroimmunological, metabolic, and fat parameters at baseline.
Mitochondrial DNA and Mitochondrially Related Genes
MtDNA did not increase significantly, and Cox II did not change significantly in either group (Fig. 1). Cyt b mRNA significantly increased in the EFV-treated group but not in the LPV/r-treated group (Fig. 1). The mtDNA abundance and Cox II and Cyt b gene expression levels in the LPV/r and EFV groups were comparable (P = 0.8819, P = 0.1372, and P = 0.1979, respectively) at baseline and were not different at week 48 (P = 0.3871, P = 0.8880, and P = 0.8430, respectively).
Adipocyte Differentiation and Lipid Metabolism
The expression of PPAR-γ and C/EBPα did not change significantly during the study (Fig. 2). The expression of marker genes of the terminal differentiation of adipose cells, such as GLUT4 and aP2/FABP, did not change significantly (Fig. 2). Transcript levels for adipogenesis-related genes at baseline were not significantly different, with the exception of GLUT4 that was higher in the LPV/r-treated group (P = 0.0014). Between-group comparison at week 48 did not show differences for any of those transcripts (P = 0.8950, P = 0.2601, and P = 0.4678, respectively).
Genes Involved in Inflammation
MCP-1 and TNF-α transcripts did not change significantly over 48 weeks in the LPV/r-treated group, whereas they showed a significant increase in the EFV-treated group (Fig. 3). GRP78 gene expression, an indicator of the endoplasmic reticulum stress process closely related to inflammation, did not change significantly in both groups (Fig. 3). MCP-1 increased significantly only in the EFV group, whereas the IL-18 transcript level significantly decreased in the LPV/r group but increased significantly in the EFV group (Fig. 3).
MCP-1, IL-18, GRP78, and TNF-α transcript levels at baseline were comparable between groups (P = 0.6677, P = 0.1372, P = 0.9736, and P = 0.9473, respectively), and at week 48, there were no significant differences for MCP-1, GRP78, and TNF-α transcript levels (P = 0.2219, P = 0.5743, and 0.7917, respectively), but there were significant differences for IL-18 gene expression (P = 0.0050).
Our study shows that LPV/r and EFV both combined with TDF/FTC for 48 weeks are associated with different changes in molecular markers of adipocyte biology and function. None of them was associated with relevant changes in mtDNA. LPV/r had a neutral effect on the markers studied, whereas EFV was associated with an increase in Cyt b and in markers of inflammation. From a clinical point of view, both groups were associated with increases in subcutaneous fat without concomitant changes in the fat distribution symmetry indexes. From a metabolic point of view, LPV/r was associated with significant increases in total, HDL, LDL, and VLDL cholesterol; triglycerides, whereas EFV was associated with increases in total, LDL, and VLDL cholesterol. The total cholesterol/HDL ratio did not change significantly in either arm. Most probably, lipid changes were tempered by the known lipid-lowering effect of TDF.17 On the other hand, TDF has a higher impact on bone mineralization than nucleoside backbones that include abacavir and lamivudine.18
A number of clinical trials have examined the effect of LPV/r and EFV on fat depots; in 2 of them,4,11 EFV was associated with a higher incidence of lipoatrophy defined as >20% limb fat loss from the baseline value. However, in these 2 studies, the NRTI backbone in the EFV arm contained zidovudine11 or was composed of stavudine and zidovudine in 66% of the patients in the second one.4 Notwithstanding that, whichever NRTI backbone was used, EFV was associated with higher degrees of lipoatrophy than was LPV/r.4 These results are at odds with fat substudies in other clinical trials that show increases in subcutaneous fat in antiretroviral-naive patients who start an EFV-based regime in combination with TDF/FTC.19,20 Moreover, no differences were detected in these studies regarding the percentage of patients who experienced fat loss either with the 10% or the 20% threshold.19,20 Therefore, EFV from a clinical point of view seems safe in terms of SAT toxicity. In our study, both groups showed an increase in subcutaneous fat, although this increase only reached statistical significance for patients in the LPV/r arm. In addition, fat distribution symmetry indexes remained unchanged throughout the study, which suggests that the fat gained was symmetrically distributed. As expected, BMC and BMD significantly decreased during the study, without differences between groups at 48 weeks. It is well known that bone mineralization parameters decrease when HAART is started, and that this decrease is greater if TDF is part of the HAART regime and even greater when TDF is combined with a boosted protease inhibitor.18,21
LPV/r has been associated with a range of disturbances in adipocyte biology and function in in vitro studies, including impairment of adipocyte differentiation22,23 and upregulation of inflammatory-related genes.22 Moreover, LPV/r has been associated with adverse effects on mitochondrial function measured by an increased reactive oxygen species production and has also adversely affected insulin sensitivity.23 These findings are at odds with our ex vivo findings of a neutral action of TDF/FTC plus LPV/r on SAT expression of genes related to adipocyte differentiation, inflammation, and mitochondrial function. EFV has been linked in in vitro studies to the impairment of adipocyte differentiation, decreased lipogenesis, promotion of inflammation,22,24,25 and mitochondrial toxicity.26 EFV has also been associated with changes in mitochondrial bioenergetics reducing cell respiration by inhibition of complex I of the mitochondrial respiratory chain, reducing intracellular ATP levels, and increasing ROS production.27 In an ex vivo study, a decrease in the Cyt b gene expression and an increase in the UCP-2 gene expression were found, suggesting a shift to anaerobic metabolism within SAT.28 We did not find any sign of mitochondrial toxicity, measured by mtDNA content, among patients exposed to EFV, whereas Cyt b gene expression even increased significantly during the study. This discrepancy may be explained by the fact that in the case of toxicity mitochondrial mass may increase.29 In addition, in the aforementioned study, half of the patients were exposed to AZT, a known mitochondrial toxic.28 Therefore, when Cyt b expression was analyzed in the light of AZT or TDF exposure, significant differences between arms were found.
Our study did not reveal relevant changes in adipogenic genes caused by LPV/r- and EFV-based HAART for 48 weeks. The main difference in terms of gene expression at the SAT level between LPV/r- and EFV-based HAART was that the latter was associated with the upregulation of the genes encoding for inflammatory cytokines (ie, TNF-α, IL-18, and MCP-1). This finding is in line with those from in vitro adipocyte models in which EFV is associated with increases in TNF-α and MCP-1 transcript levels.22,25 In these models, LPV/r was also associated with such an increase,22,25 but to a lesser extent. This was not reproduced in our ex vivo samples for LPV/r-treated patients. On the other hand, protease inhibitors, including lopinavir, have been reported to cause endoplasmic reticulum stress in multiple tissues and cells including adipocytes,30,31 and this effect is known to be coupled to a proinflammatory induction.32 Our present data on unaltered GRP78, a marker of endoplasmic reticulum stress, indicate that this process does not seem to be involved in the preferential proinflammatory induction observed in the LPV/r-exposed SAT. The establishment of an inflammatory environment at the SAT and systemic levels is thought to play an important part in the development of HALS.33 The meaning of the increase of SAT inflammatory markers in the EFV group is unknown at present, although their long-term consequences may merit further study.
Our study has limitations; first, this is a nonrandomized study, and therefore, no causal relationship must be established. Notwithstanding that, both groups were comparable with regard to clinical, virologic, fat, and even SAT molecular levels at baseline. Second, the limited number of patients and samples may have prevented us from finding relevant differences in both molecular and clinical parameters. Third, our study lasted only 48 weeks; most probably changes at 96 weeks would have been more pronounced as seen in the fat substudy of ACTG 5142.4 Fourth, we have not explored the mitochondrial bioenergetics in samples from our patients, and this has been described both in vitro and ex vivo as a possible mechanism of SAT toxicity by EFV.34 Fifth, we have not explored the expression of genes involved in glucocorticoid generation, which may play a role in adipocyte differentiation.34 We have not performed a correlation between systemic and SAT inflammatory markers. However, the inflammatory process in SAT may not necessarily correlate with inflammatory events that take place at the systemic level.35 Finally, 13.6% of patients received statins after starting HAART, which may potentially have altered gene expression.
In summary, the expression of genes involved in adipocyte differentiation and mitochondria-related genes did not change significantly in SAT from LPV/r-exposed patients, whereas Cyt b and especially inflammation-related genes were significantly upregulated in SAT from TDF/FTC/EFV-exposed patients after 48 weeks of therapy. TDF/FTC plus LPV/r or EFV increased limb fat, although the statistical significance was reached only in the LPV/r-treated group.
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