Highly active antiretroviral therapy (HAART) has become the standard of care for HIV-infected patients and has induced a dramatic decline in the incidence of opportunistic infections, malnutrition and thus mortality of these patients . This therapeutic efficiency is based on the association of drugs that can simultaneously impair viral replication and the maturation of virions. Indeed, HAART regimens combine protease inhibitors (PI) with reverse transcriptase inhibitors which may be either nucleoside analogues (NRTI) or non-nucleoside analogues (NNRTI). Unfortunately, it quickly became apparent that these treatment regimens also induced major abnormalities of lipid metabolism generally referred to as the lipodystrophic syndrome. Although there is still an ongoing discussion about the case definition 3 years after its first description, it is generally admitted that the main clinical signs of this syndrome are region-specific disturbances of fat distribution so that in treated patients one can observe a severe loss of peripheral adipose tissue and/or the accumulation of fat in the abdomen, breast or neck [2–6]. This syndrome is frequently associated with dyslipaemia [5,7,8], insulin resistance [7,9] and less often with diabetes mellitus [7,10]. These abnormalities together have a prevalence of up to 60% in treated patients  and result in a real public health concern because the social life of treated patients is severely perturbed and the risk of atherosclerosis is greatly increased.
From an aetiological point of view, this syndrome was initially regarded as a perverse effect of PI as it arose with the generalized use of this class of antiviral agent [7,11,12]. However, recent reports have also shown evidence of peripheral fat wasting in NRTI-treated, PI-naive patients [13,14], although it has been stated that this NRTI-associated lipodystrophy can present some distinguishable clinical and metabolic features such as acidaemia and neuropathy . In fact, because most patients have been given various combinations of PI and NRTI epidemiological studies have failed to attribute unambiguously the cause of this syndrome to one drug or even a class of therapeutic agent.
Whatever its aetiology, lipodystrophy can be considered as a consequence of region-specific disturbances in adipocyte number, which is controlled by a balance between two opposite processes, apoptosis and differentiation . Therefore, several studies have focused on a possible direct effect of antiviral drugs on these processes, which has been studied experimentally on primary cultures of preadipocytes  or murine preadipocyte cell lines [18,19]. Indeed, in vitro differentiation of these cells is considered to be an efficient model that has been shown to closely resemble fat cell development in vivo . In particular, this cell model has been used extensively to unravel the transcriptional cascade underlying the differentiation process that can be characterized by the sequential expression of specific genes [21,22]. During an early stage, while expression of genes specifically present in preadipocytes decreases, the lipoprotein lipase (LPL) gene is transcriptionally activated  and its expression thus precedes those of the CAAT/element binding protein family [24,25] and the peroxisome proliferator-activated receptor-γ2 genes . During a second stage, cells synthesize triglycerides, store them as lipid droplets and express genes encoding proteins involved in this function. Fatty acid synthases (FAS) that generate fatty acids and malic enzyme (that provides reduced coenzyme for this synthesis) are typical members of this group of proteins. Therefore, peak expression of LPL (on day 7) and FAS or malic enzyme (on day 11) may be considered to reflect the compliance of both stages of the differentiation process.
Murine cell lines have been used to study the effects of drugs belonging to the PI group, but until now, no study has been made of the effects of reverse transcriptase inhibitors. We present here a first study of the effects of therapeutic doses of NRTI on the differentiation process of 3T3-F442A preadipocyte cells. In addition, as most patients suffering from lipodystrophy have been treated with combination therapy, we decided to study effects of these compounds when they were added concomitantly to cell cultures. Here we clearly demonstrate that the effects produced by specific combinations of drugs were different from those elicited by each drug separately.
Materials and methods
Cell culture sera were from Calbiochem (Meudon, France). Trizol, phenol–chloroform, cell culture medium and all enzymes were from Life Technologies (Cergy-Pontoise, France). Saquinavir and nelfinavir were gifts of Roche Laboratories (Meylan, France). Stavudine and didanosine were gifts of Bristol-Myers-Squibb Laboratories (Paris, France). Indinavir, ritonavir and lamivudine were given by Merck (Darmstadt, Germany), Abbott (Rungis, France) and GlaxoWellcome Laboratories (Marly le Roi, France), respectively. Zidovudine was from Sigma (Saint-Quentin Fallavier, France).
Cell culture and treatment
The 3T3-F442A cell line was used at passage number 11–16 in all studies. During the growth phase, the monolayer fibroblasts were maintained in Dulbecco's modified Eagle's medium with 100 mg/l penicillin/streptomycin, 800 mg/l biotin-pantothenate (medium 1) and 10% newborn calf serum at 37 °C in 5% CO2. When cell confluence reached 70% media were replaced by medium 1 supplemented with 5% newborn and 5% foetal calf serum. At confluence, media were replaced by medium 1 plus 10% foetal calf serum and insulin (100 μg/l). From this time (day 0), cells were incubated for 7 or 11 days in the absence or presence of test compounds at the concentrations indicated (Table 1) within the range achieved in human serum. Incubation media were changed every 2 days. Control assays were performed to evaluate the drug concentrations remaining in media after 48 h incubation. Compared to drug concentrations after changing the medium, the percentages of the drugs remaining were 39% (indinavir), 50% (saquinavir), 55% (nelfinavir) and 72% (ritonavir). Because the compounds used in the study were solubilized in ethanol, treated and control cells were cultured in the presence of 0.1% ethanol.
On day 7 or 11, cells were washed twice rapidly with phosphate-buffered saline, lysed by adding 1 ml Trizol reagent to the plates, scraped from the plates and transferred to Eppendorf tubes. Chloroform (500 μl; Merck) was added. Mixes were centrifuged for 15 min at 10 000 g and superior layers carefully removed then mixed with 200 μl isopropanol (Merck). RNA was collected by centrifugation for 10 min at 9000 g, washed twice with 75% ethanol and treated with DNase (10 U per assay) for 30 min. RNA was extracted once more by using a phenol : chloroform : isoamyl alcohol mixture (25 : 24 : 1, v : v) then rinsed twice with 75% ethanol. RNA was recovered in diethylpyrocarbonate-treated water and quantified by spectrophotometry at 260 nm (any aliquot with an A260 :A280 ratio < 1.8 was discarded).
Multistandard quantitative RT–PCR
Expression of LPL and malic enzyme messenger RNA (mRNA) was assayed by a quantitative multistandard reverse transcriptase (RT)–PCR method that makes use of malic enzyme and LPL sequence conservation between mouse and rat . Moreover, this protocol allowed normalization of the amounts of malic enzyme and LPL mRNA relative to that of EF1α, a gene showing invariant expression. Total RNA purified from 3T3-F442A cells was mixed with a constant amount of total RNA prepared from Sprague Dawley rat visceral adipocytes that contains rat EF1α, malic enzyme and LPL mRNA and thus acted as a multistandard source. The mixture was then submitted to a non-preferential reverse transcription (200 U RT per assay) in the presence of random hexanucleotides and separate amplifications of malic enzyme, LPL and EF1α sequences were performed. Within each amplification reaction, primers were designed to hybridize with mouse and rat sequences with the same efficiency. Amplified rat and mouse sequences could, however, be distinguished after digestion with specific restriction enzymes (see Table 2) and electrophoretic resolution on a 2% (w : v) agarose gel. Gels were stained by Gelstar base intercalant (FMC Bioproducts, Rockland, USA) and photographed under UV light by a computer-assisted camera (Kodak, Rochester, New York, USA). Quantification of each band was performed by densitometry with Kodak 1D Image Analysis 3.0 software.
Real-time quantitative RT–PCR
Reverse transcribed FAS and vitamin D receptor (VDR) mRNA were amplified on ABI PRISM 7700 (Applied Biosystems, Courtaboeuf, France) using the SYBR green fluorescence method. Primers and sizes of amplification products are indicated in Table 2. Each FAS and VDR amplification was checked for an efficiency equal to that of the 18S ribosomal RNA (rRNA) gene that was used as a reference. Real-time amplifications were then analysed using the Sequence Detector software (Applied Biosystems) and quantification of the target mRNA was carried out by comparison of the number of cycles required to reach reference and target threshold values (ΔΔCT method).
Each molecule was tested on three or four cell culture plates. Control and vehicle (ethanol) cultures were incubated under the same conditions and their number was equal to that of treated plates. The values given by the treated plates were compared to the values given by the standard plates (ANOVA with Fisher's analysis using the Statview F software). Differences were considered significant when P < 0.01 and highly significant when P < 0.001.
To evaluate the interference of antiviral drugs in the differentiation process of preadipocytes into adipocytes, 3T3-F442A cells were allowed to grow to confluence and were maintained in medium supplemented with 10% foetal calf serum and insulin in the presence of antiviral drugs at concentrations similar to those observed in plasma of treated patients. In addition, as differentiation is a time-dependent process, expression of LPL and malic enzyme genes were measured on day 7 and day 11 after the induction of the process. Comparison with control cultures of the same age indicated that the differentiation process was delayed. Measurements of specific mRNA were performed by quantitative multistandard RT–PCR [36,37]. To take into account inherent variations due to the amplification process, the amounts of LPL or malic enzyme mRNA were normalizedd according to the expression of EF1α, a gene reported to have invariant expression. This point was checked in preliminary experiments, and we did not observe any variation of the expression of this gene in variously aged and differently treated cultures (data not shown).
Effects of PI
We first examined the effects of PI on the expression of the LPL (Fig. 1a) and malic enzyme (Fig. 1b) genes. We observed that PI, at therapeutic concentrations (Table 1), interfere in the differentiation process; even though they all belong to the PI family, these drugs produced various responses. In saquinavir-treated cells, the expression of the early LPL gene, which was weaker at day 7 in treated cells than in control cells, reached plateau values equivalent to those of the control at day 11. However, the expression of the late malic enzyme gene was lower than that observed in control cells on both day 7 and day 11. It can thus be deduced from these observations that 2 μM saquinavir delayed the differentiation process. Unlike saquinavir, indinavir did not seem to have any differentiation-related effects as indinavir-treated cells showed levels of malic enzyme RNA similar to those observed in control cells both at day 7 and day 11. Interestingly, whereas on day 7 the expression of the LPL gene could be weaker in indinavir-treated cells than in control cells, it must be noted that on day 11 indinavir induced a stronger LPL gene expression than that observed in control cells. A third example of response to PI was provided when cells were treated with nelfinavir. Indeed, in cells cultured in the presence of 10 μM nelfinavir, we observed a highly significant decrease in mRNA for both LPL and malic enzyme genes on day 7 and then a severe loss of cell viability that rendered analyses impractical on day 11.
Effect of NRTI
Fig. 2 shows that, when measured on day 7 as well as on day 11, a highly significant decrease in expression of the LPL gene was observed in zidovudine-treated cells. Lamivudine-treated cells also presented a decrease in LPL gene expression on day 7, which was not observed anymore on day 11. Other drugs did not induce any significant modifications in the amounts of LPL mRNA (Fig. 2a). When the mRNA encoding malic enzyme was quantified in NRTI-treated adipocytes, didanosine did not significantly modify the pattern of expression of this gene, whereas stavudine and lamivudine induced an increase in malic enzyme gene expression only at day 11. It is noteworthy that of the four drugs tested, only zidovudine significantly modified both LPL and malic enzyme gene expression and could thus be regarded as inhibiting the differentiation process.
Effect of combinations of PI and NRTI
As shown in Fig. 3, expression of the LPL and malic enzyme genes was modified greatly when preadipocytes were treated with certain specific combinations of drugs. In most cases, the expression of both genes on day 7 was weaker in cells treated with drugs than in control cells. Nevertheless, when mRNA was measured on day 11, a restoration was observed so that expression of LPL or malic enzyme genes in treated cells was stronger than that in control cultures. In addition, several combinations of drugs modified the response of each drug tested separately. For example, whereas LPL and malic enzyme expression were clearly diminished in zidovudine-treated cells, on day 11 in cells treated with zidovudine plus lamivudine there was restoration of expression up to values superior than those of the control. Another example was provided by cultures supplemented with indinavir. This drug, that by itself did not modify the expression of the malic enzyme gene (see Fig. 1b) led to a clear (× 2.5) and highly significant increase of malic enzyme gene expression when it was added to cultures together with zidovudine and lamivudine. Because in these cells LPL expression was similar to that in control cells (Fig. 3a) it can be suggested that this combination promotes the onset of the lipogenic pathway earlier in treated adipocytes than in control cells. This would induce a higher ability for these cells to esterify free fatty acids and synthesize endogenous lipids. A similar assertion might be deduced from the expression patterns observed when cultures were treated with stavudine and lamivudine. Indeed, in these cells at day 11, LPL expression strongly increased while each drug used separately did not alter expression of this gene (Fig. 2a). The association of indinavir with both stavudine and lamivudine also induced significant increases in LPL and malic enzyme gene expression at day 11. Interestingly, nelfinavir, even in association with stavudine and lamivudine, led to cell death as observed previously.
In order to confirm the potentiating effect of a combination of several antiviral drugs on adipocyte metabolism, we studied the expression of the FAS gene in cells aged for 11 days. We performed a real-time quantification and replaced the internal reference EF1α by 18S rRNA. In addition, we also measured VDR mRNA. Unlike the FAS gene, the VDR gene is not differentiation-dependent and its expression along the differentiation process remains approximately constant . Results of these experiments are presented in Fig. 3c. Whereas 10 μM indinavir induced a clear increase in FAS gene expression as compared with the control, NRTI had no affect. Interestingly, the combination of stavudine and lamivudine led to a clear increase in FAS gene expression. In addition, combination of zidovudine and lamivudine with indinavir increased the amount of FAS mRNA up to a value significantly higher than that elicited by indinavir alone. It is noteworthy that, with similar treatment, there was no increase in the expression of the VDR gene.
Since the first description of lipodystrophy in HAART-treated patients, several epidemiological studies have pointed towards a role of PI in inducing deleterious effects on adipose tissue [7,11,12]. This has been supposed to result from the competitive inhibition of proteins involved in lipid metabolism and the differentiation process , although to date experimental data have failed to support such a hypothesis [17,19]. PI have also been proposed to impair the insulin-dependent docking to cell membrane of the adipocyte-specific glucose transporter GLUT4  or the proteolytic activity of proteasomes . On the other hand, NRTI, as they are closely related to nucleotides, have been presumed to impair the replication of mitochondrial DNA and induce severe dysfunction of these organelles [41–44]. Nevertheless most patients affected with lipodystrophy have been given both PI and NRTI and it has been shown recently that inclusion of NRTI in the treatment of PI-treated patients increased the risk of lipodystrophy . It is thus surprising that neither the effects of NRTI, nor a possible interaction between NRTI and PI have ever been investigated in experimental studies. We decided to evaluate the expression of adipocyte-specific genes in 3T3-F442A cells treated with therapeutic concentrations of various PI and NRTI. We decided to undertake this study on the 3T3-F442A cell line because, unlike the mesenchymal stem cell line C3H10T1/2, it is strictly committed in the adipocyte lineage ; in addition, unlike the 3T3-L1 cell line, 3T3-F442A does not require the addition of iso-butyl-methyl xanthine to the culture medium, a drug shown to strongly attenuate the effects of indinavir on adipocytes .
In accordance with previous results obtained with 3T3-L1 or C3H10-T1/2 cells, our results show that different PI produce opposite effects on the differentiation process, which excluded a class effect of these drugs. PI produced three different patterns: an anti-adipogenic effect (saquinavir), an increase in LPL and FAS expression (indinavir) and a loss of cell viability (nelfinavir). The anti-adipogenic response that we observed in cells treated with saquinavir was similar to that observed by Zhang et al. in 3T3-L1 cells . The decrease in the expression of the malic enzyme gene seen on day 7 and day 11 was similar to that of the differentiation-dependent aP2 gene observed by these authors. Nevertheless, concerning the effect of indinavir our data differed from theirs as the decrease in the expression of the LPL gene that we observed on day 7 (as did Zhang et al.) was reversed to an increase on day 11, when the adipocytes were fully mature. Interestingly, our results are in agreement with those presented by Gagnon et al. who observed an increase in the accumulation of triglycerides when 3T3-L1 cells were cultured in the presence of indinavir . This observation was reinforced by our demonstration that FAS expression also was increased in indinavir-treated cells (Fig. 3c). The effect of nelfinavir leading to loss of cell viability was unexpected but, during the completion of this work, Dowell et al. published similar data .
Concerning the effects of NRTI, we present here a first set of experimental data that demonstrates that these drugs can also interfere in the differentiation process, although less clearly than did PI. For example, the presence of 1 μM zidovudine during the overall process strongly depressed the expression of LPL and malic enzyme, which can be ascribed to an anti-adipogenic effect of this drug. Nevertheless a similar effect was not clearly observed when adipocytes were treated with other NRTI, as the absence of parallel decrease of LPL and malic enzyme gene expression seems to exclude a true adipogenic effect of lamivudine, stavudine and didanosine. Furthermore NRTI did not modify significantly the expression of the FAS gene.
Our main goal was to evaluate the effects of combination of antiviral drugs on cell differentiation and, in fact, our results clearly demonstrate that the combination of therapeutic doses of antiviral drugs resulted in effects that cannot be regarded as simply additive. Several striking examples are provided in the current study (see Results). For example, the depressing effects of zidovudine on both LPL and malic enzyme expression were reversed into a clear activating effect when this drug was associated with indinavir – which by itself induced only a weak activation of these genes. The combination of indinavir with stavudine and lamivudine, which induced significant increases in LPL and malic enzyme gene expression provides another example as this increase was not observed when cells were treated by each drug separately. Even when indinavir induced a clear increase in the expression of a gene, which is true of the FAS gene, the addition of nucleoside analogues that by themselves did not produce any effect increased the response of cells further. Interestingly the expression of the VDR gene, which is not involved in the terminal differentiation process, was not altered by NRTI and PI or a combination of these.
In conclusion, our data suggest that, like PI although to a lesser extent, NRTI interfere with the differentiation process of adipocytes. In addition, they provide evidence for intricate interactions between PI and NRTI in a cell model. Therefore the current study draws attention to the potential importance of drug interactions in the understanding of mechanisms leading to lipodystrophy.
The authors thank C. Solas and B. Lacarelle (Laboratoire de Pharmacocinétique, CHU Timone, Marseilles) for their skilful quantification of antiviral drugs in culture media and M. Gastaldi for careful reading of the manuscript.
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