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Antimicrobial Agents and Chemotherapy, August 2008, p. 2882-2889, Vol. 52, No. 8
0066-4804/08/$08.00+0     doi:10.1128/AAC.01505-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Zidovudine Impairs Adipogenic Differentiation through Inhibition of Clonal Expansion

Metodi V. Stankov, Reinhold E. Schmidt, Georg M. N. Behrens,^* for the German Competence Network HIV/AIDS

Clinic for Clinical Immunology and Rheumatology, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover 30625, Germany

Received 20 November 2007/ Returned for modification 4 February 2008/ Accepted 2 May 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Lipoatrophy is a prevalent side effect of treatment with thymidine analogues. We wished to confine the time point of the antiadipogenic effect of zidovudine (AZT) during adipogenesis and to evaluate the antiproliferative effect of AZT on adipocyte homeostasis. We investigated the effects of AZT on adipogenesis in 3T3-F442A cells and studied their proliferation, differentiation, viability, and adiponectin expression. Cells were exposed to AZT (1 µM, 3 µM, 6 µM, and 180 µM), stavudine (d4T; 3 µM), or dideoxycytosine (ddC; 0.1 µM) for up to 15 days. Differentiation was assessed by real-time PCR and quantification of triglyceride accumulation. Proliferation and clonal expansion were determined by a [³H]thymidine incorporation assay. When they were induced to differentiate in the presence of AZT at the maximum concentration in plasma (C_max) and lower concentrations, 3T3-F442A preadipocytes failed to accumulate cytoplasmic triacylglycerol and failed to express normal levels of the later adipogenic transcription factors, CCAAT/enhancer-binding protein and peroxisome proliferator-activated receptor . AZT exerted an inhibitory effect on the completion of the mitotic clonal expansion, which resulted in incomplete 3T3-F442A differentiation and, finally, a reduction in the level of adiponectin expression. In addition, AZT impaired the constitutive proliferation in murine and primary human subcutaneous preadipocytes. In contrast, incubation with d4T and ddC at the C_max did not affect either preadipocyte proliferation or clonal expansion and differentiation. We conclude that the antiproliferative and antiadipogenetic effects of AZT on murine and primary human preadipocytes reveal the impact of the drug on fat tissue regeneration. These effects of the drug are expected to contribute to disturbed adipose tissue homeostasis and to be influenced by differential drug concentration and penetration in individual patients.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nucleoside reverse transcriptase inhibitors (NRTIs) have been shown to cause mitochondrial toxicity both in vitro and in vivo (24). According to the "DNA polymerase gamma (pol-) hypothesis," NRTIs may (i) affect mitochondrial DNA (mtDNA) chain elongation (by acting as chain terminators), (ii) compete with natural nucleotides (by acting as competitive inhibitors), (iii) inhibit the exonucleolytic proofreading activity of DNA pol- (by acting as inductors of fidelity errors), and (iv) decrease mtDNA repair (by impairing the removal by DNA pol- because of a lack of a 3'-OH group in NRTIs) (7, 25, 32). This hypothesis has been used to explain a variety of phenotypic findings observed in human immunodeficiency virus (HIV)-infected patients receiving NRTIs, including hepatic steatosis/lactic acidosis, myopathy, and peripheral polyneuropathy (24), and has been proposed as a major mechanism contributing to the development of lipoatrophy seen in HIV-infected patients while they are receiving antiretroviral combination therapy (6). We and others, however, have observed that zidovudine (AZT) leads to only limited mtDNA depletion (22, 36, 41) but, nevertheless, has been associated with lipoatrophy (29, 30). Thus, it is very likely that AZT affects the biology of adipocytes through different mechanisms. So far, several non-pol--related toxic effects of AZT have been proposed, including interference with adenylate kinase, ADP/ATP translocase, and physical interference with the mitochondrial membrane structure (14, 23).

White adipose tissue expansion takes place after birth, and the potential to generate new fat cells through precursor differentiation persists even in adults (3). After stimulation of differentiation, growth-arrested preadipocytes undergo at least one round of DNA replication and cell division, the so-called clonal expansion (31). Complex changes in the pattern of gene expression accompany clonal expansion, which has been shown to be critical for the differentiation process (39). Specifically, DNA synthesis precedes the expression of late differentiation markers (5, 21). Transcription factors such as peroxisome proliferator-activated receptor (PPAR) and CCAAT/enhancer-binding protein (C/EBP) initiate the full adipocyte differentiation program. During this program, preadipocytes acquire a spherical shape and change their cytoskeleton components and morphology. Subcutaneous abdominal adipose tissue from lipoatrophic patients treated with highly active antiretroviral therapy contained a higher percentage of small adipocytes in comparison to the percentage in the subcutaneous abdominal adipose tissue of control subjects and demonstrated decreased levels of expression of adipogenic transcription factors, such as C/EBP and PPAR (16) as well as the adipocyte-specific marker adiponectin (37). Given the known antiproliferative effects attributed to AZT (19, 34, 41) and the essential role of mitotic clonal expansion for adequate adipogenesis (44), we hypothesized that AZT leads to both impaired preadipocyte proliferation and impaired clonal expansion.

We aimed to address the role of AZT on adipocyte differentiation using 3T3-F442A cells. We conducted a detailed analysis of the effects of the drug on preadipocyte proliferation, adipocyte differentiation, and adipocyte survival. The results from our investigation indicate that AZT has primary effects on the differentiation of 3T3-F442A preadipocytes during mitotic clonal expansion and has a subsequent impact on PPAR and C/EBP expression, finally leading to impaired differentiation and the decreased expression of adiponectin. AZT may promote adipose tissue atrophy by affecting early proliferation-dependent differentiation and by disturbing the fat tissue regeneration capacity through the inhibition of precursor proliferation.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell culture. 3T3-F442A preadipocytes (17) were kindly provided by Jacqueline Capeau (INSERM, France) and were cultured as described previously (11). Preadipocytes were maintained in Dulbecco modified Eagle medium (DMEM) containing 5% newborn calf serum with 100 U penicillin, 100 µg/ml streptomycin, 4 µg/ml pantothenic acid, and 8 µg/ml biotin (preadipocyte medium). Subconfluent preadipocytes were cultured for 2 days in preadipocyte medium supplemented with 5% newborn calf serum and 5% fetal calf serum (FCS). Committed preadipocytes must withdraw from the cell cycle before they undergo adipose conversion. For preadipose cell lines as well as for primary preadipocytes, growth arrest is required for adipocyte differentiation and is normally achieved through contact inhibition when a confluence of cells is reached (18). For differentiation, cells were incubated 2 days after confluence (designated day 0) in preadipocyte medium containing 10% FCS and 1 µM insulin (Sigma-Aldrich, St. Louis, MO), and incubation was continued until day 9. NRTIs were dissolved in dimethyl sulfoxide. The therapeutic maximum concentrations in plasma (C_max) of AZT (6 µM), stavudine (d4T; 3 µM), and dideoxycytosine (ddC; 0.1 µM) were used (9, 42, 43). For AZT, several additional concentrations were used and ranged from about the C_max to 30 times the C_max (1 µM, 3 µM, 6 µM, and 180 µM). The highest concentration of the solvent used in the incubation experiments (0.1% dimethyl sulfoxide) did not affect cellular viability or preadipocyte differentiation. The degree of preadipocyte differentiation was evaluated every second day by Oil red O staining and microscopic evaluation for the acquisition of a spherical shape and lipid droplet accumulation. Cell numbers and viability were determined microscopically by trypan blue staining after trypsinization of the cells.

Isolation of primary human subcutaneous preadipocytes was conducted essentially as described previously (45, 46), with some modifications. Fat tissue samples were kindly provided by Kerstin Reimers, Department of Plastic Surgery, Hannover Medical School. Fat tissue was dissected and minced. After removal of the fibrous tissue and blood vessels, the adipose tissue was digested in Hank's balanced salt solution containing 2 mg/ml collagenase in a 37°C shaking water bath at 90 cycles/min and was then incubated for 1 h (instead of 30 min) until the digests had a milky appearance. An additional step was added to the original protocol and consisted of homogenization of the solution, dilution with Hank's balanced salt solution, and centrifugation at 800 rpm and 4°C for 5 min. The upper fat phase was then transferred to another vial, the middle phase was removed, and the pellet was added to the upper phase in the vial. Continuing with the original protocol, the vial was diluted with Hank's balanced salt solution supplemented with 0.5% bovine serum albumin (instead of DMEM and 10% fetal bovine serum) and was centrifuged at 1,500 rpm and 4°C for 10 min. After centrifugation, the supernatant and the floating fat were removed, and the vial with the pellet was diluted with Hank's balanced salt solution supplemented with 0.5% bovine serum albumin and centrifuged at 1,500 rpm and 4°C for 10 min. The supernatant was removed, and the pellet was subjected to additional filtrations to obtain the preadipocyte fraction. The primary human subcutaneous preadipocytes were maintained in DMEM-Ham's F-12 medium (1:1) (instead of DMEM only) containing 10% fetal bovine serum and 172 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate. The cell culture media were supplemented with 100 U penicillin and 100 µg/ml streptomycin.

RNA preparation and real-time quantitative PCR. Total RNA extraction was conducted with an RNeasy lipid tissue mini kit (Qiagen, Hilden, Germany). cDNA was synthesized from total RNA in 20 µl by using oligo(dT)-specific primers and an Omniscript RT kit (Qiagen). The primers were designed with Primer3 software (available at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Real-time quantitative PCR analyses for the genes encoding C/EBPβ, C/EBP, PPAR, and adiponectin were performed in a final volume of 25 µl with a QuantiTect SYBR green PCR kit (Qiagen). The following primers were used: transferrin receptor gene (housekeeping gene)-specific forward primer 5'-TCCGCTCGTGGAGACTACTT-3' and reverse primer 5'-ACATAGGGCGACAGGAAGTG-3', C/EBPβ gene-specific forward primer 5'-ACTTCAGCCCCTACCTGGAG-3' and reverse primer 5'-GGCTCACGTAACCGTAGTCG-3', C/EBP gene-specific forward primer 5'-TGGACAAGAACAGCAACGAG-3' and reverse primer 5'-TCACTGGTCAACTCCAGCAC-3', PPAR gene-specific forward primer 5'-CGAGTCTGTGGGGATAAAGC-3' and reverse primer 5'-GGATCCGGCAGTTAAGATCA-3', and adiponectin gene-specific forward primer 5'-GGAACTTGTGCAGGTTGGAT-3' and reverse primer 5'-CCAAGAAGACCTGCATCTCC-3'. Amplifications were performed in specifically designed optical 96-well plates with a spectrofluorometric thermal cycler (iCycler; Bio-Rad, Munich, Germany). The gene of interest and the housekeeping gene products were amplified separately under identical cycling conditions. An initial cycle at 95°C for 15 min of incubation was performed for activation of the HotStar Taq DNA polymerase, and then 42 cycles of one step at 94°C (15 s) for denaturation, one step at 55°C (30 s) for annealing, and one step at 72°C (30 s) for extension were performed. The products were also analyzed by melting curve analysis and on an ethidium bromide-stained agarose gel to ensure that a single amplicon of the expected size was indeed obtained. To measure the efficiency of the PCR, serial dilutions of reverse-transcribed RNA (1, 1/5, and 1/25) were amplified, and a standard curve was obtained by plotting the cycle threshold values as a function of the amount of starting reverse-transcribed RNA, the slope of which was used for calculation of the efficiency of the PCR by using the iCycler software. The relative quantification for any given gene was calculated after the standard curve value for a given gene (gene A) was divided by that for calibrator gene B (the transferrin receptor gene) for treated and control cells. The transferrin receptor gene was chosen because it has been shown to be superior to several other commonly used housekeeping genes for the analysis of adipocyte and preadipocyte differentiation (15). The expression of some standard housekeeping genes, such as actin, has been shown to be highly influenced during early differentiation (35). The effect of AZT was calculated by comparing the mean values obtained from three independent experiments. In each of three experiments, RNA from three independent culture plates was isolated and pooled. The pooled RNA was reverse transcribed, and cDNA was obtained by three separate reactions. This cDNA was again pooled and measured in a real-time PCR in triplicate. The triplicate real-time PCR results from three independent experiments were used for statistical evaluation. These were compared to the mean value of the control cells subjected to the same procedure.

Proliferation assay. For the preadipocyte proliferation assay, cells were incubated in 96-well plates for 24 h in preadipocyte medium and the indicated AZT concentration or vehicle. After that, [³H]thymidine was added for an additional 24 h and the cells were then harvested and washed through a filter. For clonal expansion, the cells were initiated to differentiate in 96-well plates 2 days after confluence (designated day 0) by using preadipocyte medium supplemented with 10% FCS, 1 µM insulin, and the indicated AZT concentration or vehicle for 24 h. After that, [³H]thymidine was added for an additional 24 h and the cells were then harvested and washed through a filter. The filters were placed into a beta counter in which the radioactivity and, therefore, the amount of [³H]thymidine incorporated was measured. The data represent mean values (counts per minute) obtained from three experiments with 8 to 16 separate wells per group under the same experimental conditions.

Adipocyte staining with Oil red O. For Oil red O (Sigma-Aldrich, Munich, Germany) staining, adherent cells were fixed in 10% formalin, washed, and stained with a 0.0021% (wt/vol) Oil red O solution (60% isopropanol, 40% water). At this level, Oil red O staining was examined by conventional fluorescence microscopy. For objective quantification of the triacylglyceride content, the cells were dried, Oil red O was extracted with 100% isopropanol, and the fluorescence was measured at 495 nm.

Statistics. Statistical analyses were done by unpaired Student's t test or by the Mann-Whitney test, where appropriate. Comparisons of more than two groups were performed by analysis of variance with Bonferroni's post hoc analysis. The level of significance was set at a P value of <0.05. All data are means ± standard errors of the means (SEMs). All calculations were performed with SPPS software (version 15.0) for Windows.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
AZT affects precursor proliferation. Given the known antiproliferative effects attributed to AZT (19, 34, 41), we decided to analyze the effects of this and other NRTIs on our model 3T3-F442A cell line. To this end, proliferating 3T3-F442A preadipocytes were cultured in the presence of AZT (6 µM), d4T (3 µM), or ddC (0.1 µM), concentrations that have been shown to correspond to the C_max (9, 42, 43), or in the presence of vehicle for 2 days. As shown in Fig. 1A, a 24-h [³H]thymidine incorporation assay confirmed the existence of a unique AZT-mediated antiproliferative effect on murine 3T3-F442A cells. When AZT was analyzed at different concentrations (1 µM, 6 µM, and 180 µM), it demonstrated dose-dependent inhibition of proliferation, starting already at doses as low as 1 µM (Fig. 1B). In addition, we were able to confirm that the antiproliferative effect of AZT is not restricted to 3T3-F442A preadipocytes but can also be demonstrated in primary human subcutaneous preadipocytes (Fig. 1C). We conclude that AZT has the ability to significantly impair the constitutive proliferation of murine and human preadipocytes.

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FIG. 1. Effects of AZT and other NRTIs on precursor proliferation. Nondifferentiated proliferating 3T3-F442A cells were cultured in the presence of AZT, d4T, or ddC at concentrations near the Cmax (6 µM, 3 µM, or 0.1 µM, respectively) (A) and in the presence of different AZT concentrations (1 µM, 6 µM, and 180 µM) (B) or vehicle (black columns) for 2 days. Primary human subcutaneous preadipocytes were cultured in the presence of AZT at a concentration near the Cmax or vehicle (black column) for 2 days (C). The [3H]thymidine incorporation assay was used to determine cell proliferation. Different cpm in different panels with similar conditions result from different cell numbers at the start of culture. The graphs show representative data of one of at least three independent experiments with 4 to 16 replicates per experiment. Values are the means ± SEMs, and analysis of variance with Bonferroni post hoc analysis (A and B) or Student's t test (C) was performed for the comparisons. *, P < 0.05 versus the results for the control; **, P < 0.01 versus the results for the control; ***, P < 0.001 versus the results for the control.

AZT perturbs differentiation-dependent triacylglyceride accumulation and adipogenic marker expression. Extending our earlier observations (36), we aimed to determine whether and specifically when AZT affects adipogenesis. 3T3-F442A preadipocytes were induced to differentiate in the presence or absence of AZT (1 µM and 6 µM), d4T (3 µM), or ddC (0.1 µM); the drugs were added from day 0 to day 9 (from the time after start of differentiation). Adipocytes exposed to AZT presented a significant decrease in cell numbers and decreased amounts of triacylglycerol droplets compared to the number and amounts for vehicle-treated cells at day 9, similar to earlier observations (10, 36, 40) and as confirmed by objective quantification by Oil red O staining (Fig. 2A to E). In contrast, 3T3-F442A adipocytes exposed to vehicle proceeded through the differentiation process unaffected and acquired a typical mature phenotype with a round shape and multiple lipid droplets. Under these experimental conditions, cultures exposed to ddC and d4T did not seem to differ from the control cultures.

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FIG. 2. Impaired adipocyte differentiation after exposure to AZT. The 3T3-F442A adipocyte phenotype (determined by Oil red O staining of the cells) was examined by conventional fluorescence microscopy at day 9 after the initiation of differentiation in the absence of drug (A) or in the presence of AZT (6 µM) (B), d4T (3 µM) (C), or ddC (0.1 µM) (D). Only the AZT-exposed culture presented a decrease in cell numbers and a decreased amount of triacylglyceride droplets compared to the numbers and the amount for the control. (E) 3T3-F442A adipocytes were stained with Oil red O; and staining was quantified at 495 nm on day 9 after the initiation of differentiation in the presence or the absence of AZT, d4T, or ddC. (F to I) Effects of AZT (1 µM and 6 µM) on the expression of the differentiation factors, including C/EBPβ (F), PPAR (G), and C/EBP (H), in differentiating 3T3-F442A cells. Preadipocytes were cultured in the presence of vehicle (black columns) or the indicated drug concentrations from day –7, induced to differentiate on day 0, and differentiated until day +9. Total RNA was isolated on days +1 (for C/EBPβ), +4 (for PPAR), and +6 (for C/EBP). On day +9, the cells were assessed for their adiponectin contents by measurement of the level of mRNA expression (I). (E) Data from three independent experiments; (F to I) data from three independent experiments with triplicate samples per experiment. Values are the means ± SEMs. The Mann-Whitney U test (E), Student's t test (F), and analysis of variance with Bonferroni post hoc analysis (G to I) were performed for the comparisons. *, P < 0.05 versus the results for the control; **, P < 0.01 versus the results for the control; ***, P < 0.001 versus the results for the control.

Using the well-defined adipogenic transcription factors involved in the differentiation of preadipocytes, we (36) and others (10, 40) have already confirmed that AZT can lead to incomplete adipogenesis in both white and brown adipocytes. However, only either drug concentrations above the C_max were studied or the decrease in the amounts of the end-stage factors of differentiation was analyzed, leaving questions regarding the time point and the mechanisms of impaired adipogenesis unresolved. If, indeed, mitotic clonal expansion is essential for adequate adipogenesis, as has been proposed in studies with rapamycin (44), we proposed that AZT should lead to separate effects on the expression of early and late (after clonal expansion) transcription factors in adipocyte differentiation. Preadipocytes were cultured for 7 days in the absence or the presence of different concentrations of AZT (1 µM and 6 µM) or vehicle and were then induced to differentiate. In contrast to the experimental conditions of our earlier studies with 3T3-L1 cells (36), mRNA was already isolated at days +1, +4, and +6 of the differentiation protocol and was reverse transcribed. Real-time PCR analysis was then performed to assess the levels of expression of several transcription factors involved in a cascade during preadipocyte differentiation. We measured the levels of C/EBPβ mRNA expression at day +1 after the addition of AZT to the culture on day –7 to determine whether AZT had any potential effect on C/EBPβ induction before the drug had an effect on clonal expansion. As shown in Fig. 2F, cultures with 6 µM AZT showed a slight increase but no significant alteration in the level of expression of this factor before clonal expansion. Further analysis of the levels of expression of other adipogenic transcription factors following clonal expansion, including PPAR and C/EBP, on days +4 and +6, which are the times of the peak expression of these factors during adipogenesis, respectively, demonstrated a marked and dose-dependent reduction even at an AZT concentration of 1 µM compared to the level of expression by vehicle-treated cells (Fig. 2G and H). The impaired differentiation of 3T3-F442A cells was confirmed by a significant decrease in the level of adiponectin expression (Fig. 2I), as has previously been shown for 3T3-L1 cells (36). When they are considered together, these results indicate that AZT treatment of differentiating preadipocytes, starting from day –7, induces the disruption of the patterns of expression of the two main adipogenic factors, C/EBP and PPAR, whose expression normally peaks after the successful completion of clonal expansion. Thus, we concluded that under these conditions, AZT exerted an inhibitory effect on 3T3-F442A adipocyte differentiation.

Impact of AZT on mitotic clonal expansion in differentiating preadipocytes. Given the antiproliferative effects of AZT that we observed in proliferating 3T3-F442A cells (Fig. 1) and the impaired adipogenesis (Fig. 2) (10, 40), we hypothesized that AZT treatment affects mitotic clonal expansion. 3T3-F442A cells were induced to differentiate in the absence or the presence of AZT (1 µM, 3 µM, 6 µM, and 180 µM), d4T (3 µM), or ddC (0.1 µM); and clonal expansion was quantitated by a 24-h [³H]thymidine incorporation assay. The data revealed that AZT had a statistically highly significant (P < 0.001) inhibitory effect at concentrations near the C_max and higher (Fig. 3), leading to an at least 30% reduced level of thymidine incorporation. Control cultures exposed to d4T or ddC during differentiation did not show reduced proliferation. In order to exclude the possibility that the reduced level of thymidine incorporation and the reduction in the numbers of cells stained with Oil red O (Fig. 2B and E) are a result of increased cell death, we examined the effects of AZT on the viability of the cell line (detached and attached cells together) using trypan blue staining. In cultures of differentiating 3T3-F442A cells incubated for 8 days with 1 µM or 6 µM AZT, there was only a minor decrease in the number of viable cells from about 95% (untreated) to 93% and 91%, respectively (Fig. 3C). Taken together, we conclude that increased cell death rates are unlikely to sufficiently explain the reduced Oil red O staining in our models.

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FIG. 3. Effect of AZT on cell viability and on mitotic clonal expansion in 3T3-F442A cells. Mitotic clonal expansion, which takes place within the first 3 days of the differentiation program, was measured 24 h after the initiation of differentiation by a 24-h [3H]thymidine incorporation assay. (A) 3T3-F442A preadipocytes were induced to differentiate in the presence of AZT, d4T, or ddC at concentrations near the Cmax (6 µM, 3 µM, and 0.1 µM, respectively) and (B) in the presence of different AZT concentrations (1 µM, 3 µM, and 180 µM) or vehicle (black columns). The different cpm in different panels with similar conditions result from different cell numbers at the start of culture. (C) Viability was measured on day +8 of the differentiation protocol by using trypan blue dye exclusion. (A and B) Representative data from one of at least three independent experiments with 4 to 16 replicates per experiment; (C) data from five independent experiments. Values are the means ± SEMs, and analysis of variance with Bonferroni post hoc analysis (A to C) was performed for the comparisons.*, P < 0.05 versus the results for the control; ***, P < 0.001 versus the results for the control.

Addition of AZT after the successful completion of clonal expansion does not affect either triacylglyceride accumulation or adipogenic marker expression. To further prove our hypothesis that clonal expansion is, indeed, the critical step of the antiadipogenic AZT effect, we treated differentiating 3T3-F442A cells with increasing doses of AZT immediately after clonal expansion and monitored their phenotypic differentiation. As shown in Fig. 4A to D, the accumulation of cytoplasmic triacylglycerides on day 9 was unaffected by AZT compared to the effect of vehicle treatment of the cells, as confirmed by objective quantification by Oil red O staining. Furthermore, the addition of 6 µM AZT after the successful completion of clonal expansion did not result in decreases in the levels of PPAR (Fig. 4E) and C/EBP expression (Fig. 4F), in accordance with the lack of reduced triaglyceride accumulation (Fig. 4A to D). When the results are considered together, they indicate that the presence of AZT during the process of clonal expansion is necessary for the disruption of the patterns of expression of the two main adipogenic factors, C/EBP and PPAR, and for the drug to have an effect on triacylglyceride accumulation. Taken together, we conclude that AZT significantly affects the constitutive proliferation and mitotic clonal expansion of murine and human preadipocytes after the induction of differentiation but does not cause a major difference in cell viability in comparison to that for the controls.

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FIG. 4. Adipocyte differentiation with AZT exposure after the completion of clonal expansion. The 3T3-F442A adipocyte phenotype (determined by Oil red O staining of the cells) was examined as described in the legend to Fig. 2 by conventional fluorescence microscopy for untreated control cells (A) and cells treated with 1 µM AZT (B) or 6 µM AZT (C). (D) 3T3-F442A adipocytes were stained with Oil red O, and the dye was quantified at 495 nm on day 9. (E and F) Effects of AZT (6 µM) on the expression of the differentiation factors PPAR (E) and C/EBP (F), determined on days +4 and +6, respectively, in differentiating 3T3-F442A cells when AZT was added after the completion of clonal expansion. (D) Results of four independent experiments; (E and F) results of three independent experiments with triplicate samples per experiment. Values are the means ± SEMs, and analysis of variance with Bonferroni post hoc analysis (D) or Student's t test (E and F) was performed for the comparisons. Values are the means ± SEMs. *, P < 0.05 versus the results for the control.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The 3T3-F442A cell line is one of the most widely used and well-characterized models for the study of adipocyte differentiation and function (8, 26, 27). After stimulation, 3T3-F442A preadipocytes change the pattern of gene expression and acquire adipocyte characteristics, like a spherical shape and the accumulation of triacylglyceride-rich lipid droplets, as signs of differentiation. Using this well-characterized model and NRTI concentrations corresponding to the C_max (9, 42, 43), the present study provides evidence that AZT leads to impaired adipogenesis by the inhibition of clonal expansion and to disturbed adipose homeostasis through the reduction of the preadipocyte proliferation rate. This seems to be a specific feature of AZT that we confirmed in 3T3-L1 cells (data not shown), whereas other nucleoside analogues, including ddC and d4T, cause a substantial depletion of mtDNA or have been implicated in lipoatrophy (d4T), had no antiproliferative effect (Fig. 1), and did not impair adipogenesis (Fig. 2). Preadipose cell lines undergo one or two rounds of DNA replication and cell division after growth arrest (31). This so-called mitotic clonal expansion takes place within the first 3 days of differentiation and is followed by the expression of an adipogenic cascade of transcription factors involved in the acquisition of adipocyte characteristics (39). It has been shown that the inhibition of DNA synthesis at this early stage inhibits the differentiation into fat cells (4, 5, 21). The drug rapamycin is able to inhibit the process of clonal expansion (44), and inhibition of this expansion interferes with the subsequent adipocyte differentiation, confirming the critical role of clonal expansion in cell line models of differentiation.

Two families of transcription factors, C/EBPs and PPARs, involved in terminal differentiation by transactivation of adipocyte-specific genes, are induced early during adipocyte differentiation. After the hormonal induction of differentiation, the expression of PPAR becomes detectable on about the second day and its levels peak in mature adipocytes (8, 26, 27). PPAR expression is preceded by a transient increase in the levels of expression of the C/EBPβ and C/EBP isoforms (8, 27). C/EBP is another key player in adipocyte differentiation (8, 26, 27), and its constitutive expression alone induces 3T3-L1 cell differentiation (27). In contrast to the findings of previous studies, which have demonstrated that AZT-treated adipocytes have decreased levels of C/EBP and PPAR expression at the end of the differentiation process (10, 36), we analyzed the expression of these factors sequentially during the differentiation process at the time points at which their expression is supposed to commence or peak in order to evaluate the particular time and cell process in which the differentiation cascade is affected. Our time data confine the dose-dependent effects of AZT on adipogenesis to the period of clonal expansion, as addition of the drug to the cultures after the successful completion of clonal expansion did not lead to any morphological changes or to reduced levels of expression of the differentiation markers. We propose that the antiproliferative effect of AZT is the effect that exerts the major impact on adipocyte homeostasis in both murine and human cells. In this regard, AZT appears to be able to inhibit preadipocyte differentiation at the point of mitotic clonal expansion, most likely through the inhibition of the early S phase, with subsequent effects on the cascade of expression of the differentiation markers C/EBP and PPAR. C/EBPβ activates the expression of both the C/EBP and the PPAR genes during preadipocyte differentiation through the C/EBP regulatory elements in their promoter regions (13, 27). We found that the early C/EBPβ expression was not affected or was even slightly increased, which would even favor increased adipogenesis (27). However, we wish to emphasize the fact that C/EBPβ expression starts within 4 h after the induction of adipocyte differentiation, but at this point this transcription factor is unable to bind to the C/EBP regulatory element in the C/EBP promoter. Only when preadipocytes enter S phase at the beginning of mitotic clonal expansion, C/EBPβ begins to acquire the capacity to bind to the C/EBP regulatory element and concomitantly become associated with the centromere (38). We propose that effects on these processes could account for the observed decrease in the levels of expression of C/EBP and PPAR without a detectable decrease in the level of C/EBPβ expression. Therefore, most likely, a mechanism in which AZT prevents the normal differentiation-dependent expression of C/EBP and PPAR by antagonizing the clonal expansion in connection with or without C/EBPβ could be suggested. Downregulation of the expression of C/EBP was accompanied by a subsequent decrease in the level of expression of adiponectin, one of its downstream genes. The overall decrease in the level of expression of the adipogenic factor in combination with reduced levels of triacylglyceride accumulation suggest disturbed differentiation. The only minimal increase in cell death was insufficient to explain the impaired Oil red O staining. However, although differentiation was impaired as determined microscopically (in which the acquisition of the adipocyte morphology was detected) and as estimated by determination of the level of triacylglyceride accumulation, a mechanism in which AZT may perturb additional molecular pathways independent of C/EBPβ, C/EBP, and PPAR expression cannot be excluded.

The molecular mechanism responsible for the antiadipogenic effects of AZT is not known. In vitro experiments demonstrated that incubation in the presence of AZT results in intracellular drug accumulation and the formation of mono-, di-, and triphosphate anabolites (20). mtDNA depletion has been suggested, but as we have recently described in 3T3-L1 cells, significant depletion has not been observed even at the highest drug concentrations used (36), and we were unable to detect mtDNA depletion in 3T3-F442A cells upon treatment with AZT (data not shown). Mechanistically, the antiproliferative effect of AZT has been hypothesized to be mediated by its inhibitory effect on telomerase activity (19, 33). However, it seems unlikely that such a mechanism would account for the antiproliferative activity observed in our experimental system, as the observed effects occurred in a relatively short period of time. Recently, speculation about the physical interference of AZT with the mitochondrial membrane structure was prompted by reports showing that AZT induces in vitro a reduction in the mitochondrial membrane potential even without mtDNA depletion (12). Another recent paper suggested the dissociation between the mtDNA content, transcription, and mitochondrial activity in AZT-treated 3T3-F442A cells (40). On the other hand, elegant experiments have demonstrated that AZT interferes with cellular DNA and RNA (1, 2, 28). Some of these mechanisms might account for the observed short-term antiproliferative effect.

We conclude from the results of our studies that AZT has a strong influence on the proliferation capacity of 3T3-F442A and primary human subcutaneous preadipocytes, which may lead to a decrease in the renewal potential of fat tissue. In 3T3-F442A cells, it affects the differentiation process through the inhibition of clonal expansion. The antiproliferative and antiadipogenic effects of AZT might induce disturbances in the regeneration capacity of adipose tissue in vivo. This effect of the drug is expected to be influenced by the differential drug concentrations and levels of penetration in individual patients.

 
We thank Sabine Buyny for excellent technical assistance and Jacqueline Capeau for providing the cell line.

This work was supported by the Competence Network HIV/AIDS (C15) by BMBF.

 
* Corresponding author. Mailing address: Clinic for Clinical Immunology and Rheumatology, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover 30625, Germany. Phone: 49 (0) 511 532 5713. Fax: 49 (0) 511 532 5324. E-mail: behrens.georg{at}mh-hannover.de

Published ahead of print on 12 May 2008.

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Antimicrobial Agents and Chemotherapy, August 2008, p. 2882-2889, Vol. 52, No. 8
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