Effects of antiretroviral drug combinations on the differentiation of adipocytes
Roche, Régis; Poizot-Martin, Isabellea; Martin-El Yazidi, Claire; Compe, Emmanuel; Gastaut, Jean-Alberta; Torresani, Janine; Planells, Richard
From INSERM Unit 476, School of Medicine of Marseilles and aCISIH, Sainte-Marguerite Hospital, Marseilles, France.
Requests for reprints to R. Planells, INSERM Unité 476, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France.
Received: 12 April 2001;
revised: 13 July 2001; accepted: 2 August 2001.
Objective: Preadipocyte cell lines present a cell model with which to understand the physiopathological mechanisms underlying lipodystrophy syndrome, a common complication observed in patients treated with highly active antiretroviral therapy (HAART) that, in general, is associated with the use of protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors (NRTI). The aim of this study was to evaluate the effects of NRTI and of PI and NRTI combinations in this cell model.
Methods: The differentiation of 3T3-F442A cells was studied by monitoring the expression of specific genes in the presence of therapeutic concentrations of antiretroviral drugs. Messenger RNA (mRNA) was quantified by two reverse transcription–PCR-based methods.
Results: In the presence of 2 μM saquinavir, 30 μM ritonavir or 1 μM zidovudine preadipocytes delayed their differentiation, whereas the use of 10 μM nelfinavir led to cell death. Indinavir (10 μM) promoted lipoprotein lipase expression whereas 1 μM lamivudine or 1μM stavudine enhanced slightly the expression of the malic enzyme gene. However, the combination of indinavir, lamivudine and stavudine led to a large increase in both lipoprotein lipase and malic enzyme mRNA transcription whereas the combination of indinavir, lamivudine and zidovudine led to a 2.5-fold increase in the expression of the lipogenic malic enzyme gene. Similar potentiating effects of NRTI and PI were observed on the expression of the fatty acid synthase gene.
Conclusions: Our data suggest that, like PI (although to a lesser extent) NRTI interfere with the differentiation process of adipocytes. In addition, we demonstrate that the effects produced by combinations of NRTI and PI are different from those elicited by each drug separately. This point may be particularly relevant in understanding the physiopathological mechanisms underlying the lipodystrophic syndrome.
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.
1. Hammer SM, Squires KE, Hughes MD. et al
. A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. AIDS Clinical Trials Group 320 Study Team.
N Engl J Med 1997, 337: 725–733.
2. Pujol RM, Domingo P, Xavier Matias G. et al
. HIV-1 protease inhibitor-associated partial lipodystrophy: clinicopathologic review of 14 cases. J Am Acad Dermatol 2000, 42: 193–198.
3. Lo JC, Mulligan K, Tai VW, Algren H, Schambelan M. `Buffalo hump: in men with HIV-1 infection. Lancet 1998, 351: 867–870.
4. Roth VR, Kravcik S, Angel JB. Development of cervical fat pads following therapy with human immunodeficiency virus type 1 protease inhibitors. Clin Infect Dis 1998, 27: 65–67.
5. Miller KD, Jones E, Yanovski JA, Shankar R, Feuerstein I, Falloon J. Visceral abdominal-fat accumulation associated with use of indinavir. Lancet 1998, 351: 871–875.
6. Stocker DN, Meier PJ, Stoller R, Fattinger KE. `Buffalo hump ‘ in HIV-1 infection. Lancet 1998, 352: 320–321.
7. Carr A, Samaras K, Burton S. et al
. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 1998, 12: F51–F58.
8. Periard D, Telenti A, Sudre P. et al
. Atherogenic dyslipidemia in HIV-infected individuals treated with protease inhibitors. The Swiss HIV Cohort Study.
Circulation 1999, 100: 700–705.
9. Walli R, Herfort O, Michl GM. et al
. Treatment with protease inhibitors associated with peripheral insulin resistance and impaired oral glucose tolerance in HIV-1-infected patients. AIDS 1998, 12: F167–F173.
10. Carr A, Samaras K, Chisholm DJ, Cooper DA. Pathogenesis of HIV-1-protease inhibitor-associated peripheral lipodystrophy, hyperlipidaemia, and insulin resistance. Lancet 1998, 351: 1881–1883.
11. Tsiodras S, Mantzoros C, Hammer S, Samore M. Effects of protease inhibitors on hyperglycemia, hyperlipidemia, and lipodystrophy: a 5-year cohort study. Arch Intern Med 2000, 160: 2050–2056.
12. Graham NM. Metabolic disorders among HIV-infected patients treated with protease inhibitors: a review. J Acquir Immune Defic Syndr 2000, 25 (suppl 1): S4–S11.
13. Saint-Marc T, Partisani M, Poizot-Martin I. et al
. A syndrome of peripheral fat wasting (lipodystrophy) in patients receiving long-term nucleoside analogue therapy. AIDS 1999, 13: 1659–1667.
14. Madge S, Kinloch-de-Loes S, Mercey D, Johnson MA, Weller IV. Lipodystrophy in patients naive to HIV protease inhibitors. AIDS 1999, 13: 735–737.
15. Carr A, Cooper DA. Adverse effects of antiretroviral therapy. Lancet 2000, 356: 1423–1430.
16. Martin RJ, Hausman GJ, Hausman DB. Regulation of adipose cell development in utero. Proc Soc Exp Biol Med 1998, 219: 200–210.
17. Wentworth JM, Burris TP, Chatterjee VK. HIV protease inhibitors block human preadipocyte differentiation, but not via the PPAR gamma/RXR heterodimer. J Endocrinol 2000, 164: R7–R10.
18. Lenhard JM, Furfine ES, Jain RG. et al
. HIV protease inhibitors block adipogenesis and increase lipolysis in vitro
. Antiviral Res 2000, 47: 121–129.
19. Zhang B, MacNaul K, Szalkowski D, Li Z, Berger J, Moller DE. Inhibition of adipocyte differentiation by HIV protease inhibitors. J Clin Endocrinol Metab 1999, 84: 4274–4277.
20. Green H, Kehinde O. Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line. J Cell Physiol 1979, 101: 169–171.
21. Brun RP, Kim JB, Hu E, Altiok S, Spiegelman BM. Adipocyte differentiation: a transcriptional regulatory cascade. Curr Opin Cell Biol 1996, 8: 826–832.
22. Fajas L, Fruchart JC, Auwerx J. Transcriptional control of adipogenesis. Curr Opin Cell Biol 1998, 10: 165–173.
23. Amri EZ, Dani C, Doglio A, Grimaldi P, Ailhaud G. Coupling of growth arrest and expression of early markers during adipose conversion of preadipocyte cell lines. Biochem Biophys Res Commun 1986, 137: 903–910.
24. Lin FT, Lane MD. Antisense CCAAT/enhancer-binding protein RNA suppresses coordinate gene expression and triglyceride accumulation during differentiation of 3T3-L1 preadipocytes. Genes Dev 1992, 6: 533–544.
25. Tang QQ, Lane MD. Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation. Genes Dev 1999, 13: 2231–2241.
26. Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 1994, 8: 1224–1234.
27. Dieleman JP, Gyssens IC, van der Ende ME, de Marie S, Burger DM. Urological complaints in relation to indinavir plasma concentrations in HIV-infected patients. AIDS 1999, 13: 473–478.
28. Regazzi MB, Villani P, Maserati R. et al
. Clinical pharmacokinetics of nelfinavir combined with efavirenz and stavudine during rescue treatment of heavily pretreated HIV-infected patients. J Antimicrob Chemother 2000, 45: 343–347.
29. Merry C, Barry MG, Mulcahy F. et al
. The pharmacokinetics of combination therapy with nelfinavir plus nevirapine. AIDS 1998, 12: 1163–1167.
30. Gatti G, Di Biagio A, Casazza R. et al
. The relationship between ritonavir plasma levels and side-effects: implications for therapeutic drug monitoring. AIDS 1999, 13: 2083–2089.
31. Merry C, Barry MG, Mulcahy F, Halifax KL, Back DJ. Saquinavir pharmacokinetics alone and in combination with nelfinavir in HIV-infected patients. AIDS 1997, 11: F117–F120.
32. Somadossi J-P. Cellular nucleoside pharmacokinetics and pharmocology: a potentially important determinant of antiretroviral efficacy. AIDS 1998, 12: S1–S8.
33. Seifert RD, Stewart MB, Sramek JJ, Conrad J, Kaul S, Cutler NR. Pharmacokinetics of co-administered didanosine and stavudine in HIV-seropositive male patients. Br J Clin Pharmacol 1994, 38: 405–410.
34. Taylor S, van Heeswijk RP, Hoetelmans RM. et al
. Concentrations of nevirapine, lamivudine and stavudine in semen of HIV-1-infected men. AIDS 2000, 14: 1979–1984.
35. Kewn S, Veal GJ, Hoggard PG, Barry MG, Back DJ. Lamivudine (3TC) phosphorylation and drug interactions in vitro. Biochem Pharmacol 1997, 54: 589–595.
36. Khiri H, Reynier P, Peyrol N, Lerique B, Torresani J, Planells R. Quantitative multistandard RT-PCR assay using interspecies polymorphism. Mol Cell Probes 1996, 10: 201–211.
37. Marchand-Pinatel S, Planells R, Merten MD, Kammouni W, Figarella C. A quantitative multistandard reverse transcriptase-polymerase chain reaction assay of the cystic fibrosis transmembrane conductance regulator: its usefulness in studying efficiency of gene transfer. Analyt Biochem 2000, 283: 200–206.
38. Dace A, Martin-el Yazidi C, Bonne J, Planells R, Torresani J. Calcitriol is a positive effector of adipose differentiation in the OB 17 cell line: relationship with the adipogenic action of triiodothyronine. Biochem Biophys Res Commun 1997, 232: 771–776.
39. Murata H, Hruz PW, Mueckler M. The mechanism of insulin resistance caused by HIV protease inhibitor therapy. J Biol Chem 2000, 275: 20251–20254.
40. Schmidtke G, Holzhutter HG, Bogyo M. et al
. How an inhibitor of the HIV-I protease modulates proteasome activity. J Biol Chem 1999, 274: 35734–35740.
41. Brinkman K, ter Hofstede HJ, Burger DM, Smeitink JA, Koopmans PP. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway. AIDS 1998, 12: 1735–1744.
42. Brinkman K, Smeitink JA, Romijn JA, Reiss P. Mitochondrial toxicity induced by nucleoside-analogue reverse-transcriptase inhibitors is a key factor in the pathogenesis of antiretroviral-therapy-related lipodystrophy. Lancet 1999, 354: 1112–1115.
43. Miro O, Gomez M, Pedrol E, Cardellach F, Nunes V, Casademont J. Respiratory chain dysfunction associated with multiple mitochondrial DNA deletions in antiretroviral therapy-related lipodystrophy. AIDS 2000, 14: 1855–1857.
44. Walker UA, Setzer B, Volksbeck SI. Toxicity of nucleoside-analogue reverse-transcriptase inhibitors [letter; comment]. Lancet 2000, 355: 1096.1096.
45. van der Valk M, Gisolf EH, Reiss P. et al
. Increased risk of lipodystrophy when nucleoside analogue reverse transcriptase inhibitors are included with protease inhibitors in the treatment of HIV-1 infection. AIDS 2001, 15: 847–855.
46. Dowell P, Flexner C, Kwiterovich PO, Lane MD. Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV protease inhibitors. J Biol Chem 2000, 275: 41325–41332.
47. Gagnon A, Angel JB, Sorisky A. Protease inhibitors and adipocyte differentiation in cell culture. Lancet 1998, 352: 1032.1032.
This article has been cited 45 time(s).
Metabolism-Clinical and ExperimentalAdipokines in the HIV/HAART-associated lipodystrophy syndromeMetabolism-Clinical and Experimental
The HIV-1 nucleoside reverse transcriptase inhibitors stavudine and zidovudine alter adipocyte functions in vitro
Journal of InfectionRisk of premature atherosclerosis and ischemic heart disease associated with HIV infection and antiretroviral therapyJournal of Infection
An objective case definition of lipodystrophy in HIV-infected adults: a case-control study
Comparative Biochemistry and Physiology C-Toxicology & PharmacologySite-specific differences in the action of NRTI drugs on adipose tissue incubated in vitro with lymphoid cells, and their interaction with dietary lipidsComparative Biochemistry and Physiology C-Toxicology & Pharmacology
Clinical Infectious Diseases
Turnover of adipose components and mitochondrial DNA in humans: Kinetic biomarkers for human immunodeficiency virus-associated lipodystrophy and mitochondrial toxicity?
Clinical Infectious Diseases, 37():
Antiretroviral drugs with adverse effects on adipocyte lipid metabolism and survival alter the expression and secretion of proinflammatory cytokines and adiponectin in vitro
Antiviral Therapy, 9(6):
BiochimieHIV antiretroviral treatment alters adipokine expression and insulin sensitivity of adipose tissue in vitro and in vivoBiochimie
New England Journal of Medicine
Medical progress - Cardiovascular risk and body-fat abnormalities in HIV-infected adults
New England Journal of Medicine, 352(1):
Reverse transcriptase inhibitors alter uncoupling protein-1 and mitochondrial biogenesis in brown adipocytes
Antiviral Therapy, 10(4):
Bmc Infectious DiseasesPersistence of lipoatrophy after a four-year long interruption of antiretroviral therapy for HIV1 infection: case reportBmc Infectious Diseases
MitochondrionIn vitro evidence of inhibition of mitochondrial protease processing by HIV-1 protease inhibitors in yeast: a possible contribution to lipodystrophy syndromeMitochondrion
Adipocytes targets and actors in the pathogenesis of HIV-associated lipodystrophy and metabolic alterations
Antiviral Therapy, 9(2):
Altered adipokine response in murine 3T3-F442A adipocytes treated with protease inhibitors and nucleoside reverse transcriptase inhibitors
Antiviral Therapy, 10(2):
Some HIV protease inhibitors alter lamin A/C maturation and stability, SREBP-1 nuclear localization and adipocyte differentiation
AtherosclerosisHIV-related lipodystrophy and related factorsAtherosclerosis
Journal of Pharmacology and Experimental TherapeuticsProtease inhibitor treatments reveal specific involvement of matrix metalloproteinase-9 in human adipocyte differentiationJournal of Pharmacology and Experimental Therapeutics
Altered fat differentiation and adipocytokine expression are inter-related and linked to morphological changes and insulin resistance in HIV-1-infected lipodystrophic patients
Antiviral Therapy, 9(4):
In combination, nucleoside reverse transcriptase inhibitors have significant effects on 3T3-L1 adipocyte lipid accumulation and survival
Antiviral Therapy, 11(2):
Sexually Transmitted Infections
A qualitative study of the psychosocial implications of lipodystrophy syndrome on HIV positive individuals
Sexually Transmitted Infections, 79(2):
AIDSExpression of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma co-activator 1, IL-6 and CD45 in subcutaneous adipose tissue in lipodystrophy associated with highly active antiretroviral therapyAIDS
Some HIV antiretrovirals increase oxidative stress and alter chemokine, cytokine or adiponectin production in human adipocytes and macrophages
Antiviral Therapy, 12(4):
AIDSDifferential effect of HIV protease inhibitors on adipogenesis: intracellular ritonavir is not sufficient to inhibit differentiationAIDS
Mitochondrial toxicity of indinavir, stavudine and zidovudine involves multiple cellular targets in white and brown adipocytes
Antiviral Therapy, 12(6):
Hormone and Metabolic ResearchThe effect of the HIV protease inhibitor ritonavir on proliferation, differentiation, lipogenesis, gene expression and apoptosis of human preadipocytes and adipocytesHormone and Metabolic Research
Insulin resistance, glucose intolerance and diabetes mellitus in HIV-infected patients
Antiviral Therapy, 12(2):
Antiviral ResearchEffect of ritonavir and atazanavir on human subcutaneous preadipocyte proliferation and differentiationAntiviral Research
Antimicrobial Agents and ChemotherapyMitochondrial and metabolic effects of nucleoside reverse transcriptase inhibitors (NRTIs) in mice receiving one of five single- and three dual-NRTI treatmentsAntimicrobial Agents and Chemotherapy
Clinical and Experimental ImmunologyLow plasma level of adiponectin is associated with stavudine treatment and lipodystrophy in HIV-infected patientsClinical and Experimental Immunology
Expert Opinion on Therapeutic Patents
Cardiovascular risk associated with antiretroviral therapy in IV-infected patients
Expert Opinion on Therapeutic Patents, 16():
Toxicologic PathologyThe Role of Protease Inhibitors in the Pathogenesis of HIV-Associated Lipodystrophy: Cellular Mechanisms and Clinical ImplicationsToxicologic Pathology
Hiv Clinical TrialsA Meta-Analysis of Six Placebo-Controlled Trials of Thiazolidinedione Therapy for HIV LipoatrophyHiv Clinical Trials
Rosiglitazone in the treatment of HAART-associated lipodystrophy - a randomized double-blind placebo-controlled study
Antiviral Therapy, 8(3):
British Journal of PharmacologyLipid lowering effects of Momordica charantia (Bitter Melon) in HIV-1-protease inhibitor-treated human hepatoma cells, HepG2British Journal of Pharmacology
Pathogenesis of lipodystrophy and lipid abnormalities in patients taking antiretroviral therapy
AIDS Reviews, 9(1):
Antimicrobial Agents and ChemotherapyLong-term treatment with lopinavir-ritonavir induces a reduction in peripheral adipose depots in miceAntimicrobial Agents and Chemotherapy
Environmental and Molecular MutagenesisMorphological and molecular course of mitochondrial pathology in cultured human cells exposed long-term to zidovudineEnvironmental and Molecular Mutagenesis
InternistAdverse events of antiretroviral therapy: aspects of pathogenesisInternist
Intracellular disposition and metabolic effects of zidovudine, stavudine and four protease inhibitors in cultured adipocytes
Antiviral Therapy, 8(5):
AIDSSerum adipocytokines are related to lipodystrophy and metabolic disorders in HIV-infected men under antiretroviral therapyAIDS
Journal of Biological ChemistryHuman immunodeficiency virus protease inhibitors accumulate into cultured human adipocytes and alter expression of adipocytokinesJournal of Biological Chemistry
JAIDS Journal of Acquired Immune Deficiency SyndromesDrug-Specific Effect of Nelfinavir and Stavudine on Primary Culture of Human PreadipocytesJAIDS Journal of Acquired Immune Deficiency Syndromes
JAIDS Journal of Acquired Immune Deficiency SyndromesMitochondrial Abnormalities in HIV-Infected Lipoatrophic Patients Treated With Antiretroviral AgentsJAIDS Journal of Acquired Immune Deficiency Syndromes
JAIDS Journal of Acquired Immune Deficiency SyndromesNelfinavir Induces Necrosis of 3T3F44-2A Adipocytes by Oxidative StressJAIDS Journal of Acquired Immune Deficiency Syndromes
Adipocytes; cell model; lipodystrophy; protease inhibitors; reverse transcriptase inhibitors
© 2002 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.