Although highly active antiretroviral treatment (HAART) including protease inhibitors (PIs) has drastically lowered morbidity and immediate mortality in HIV-1-infected patients [1,2], it frequently induces hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia and lipodystrophy [3,4]; this, consequently, increases the risk of cardiovascular complications [5,6]. Recent studies revealed that HAART-related adverse effects are common and persist in patients remaining on treatment [3,6]. A single-nucleotide polymorphism in the gene encoding the sterol-regulatory element-binding protein (SREBP)-1c, also called adipocyte determination and differentiation factor (ADD)-1, has been recently found to be associated with this HAART-related syndrome . Furthermore, PI drugs inhibit SREBP-1c/ADD-1 in vitro [8,9]. To determine the effect of the PI indinavir on SREBP-1c/ADD-1 and the consequences of a possible inhibition, effector genes specifically activated by SREBP-1c/ADD-1 were studied in cell culture in the presence of indinavir.
Materials and methods
Indinavir sulfate and simvastatin were provided by Merck & Co. Inc., Rahway, New Jersey, USA. The inactive lactone prodrug simvastatin was converted into its active dihydroxy-open form (L-644128) by acidic hydrolysis according to the manufacturer's protocol. Aqueous solutions of indinavir sulfate and the active dihydroxy-open form of simvastatin were prepared.
Reporter gene constructs
Lipoprotein lipase gene
The pGL2–lipoprotein lipase (LPL) gene construct contained nucleotides −1910 to −9 (a of the a tg translation start site was assigned +1 in all the constructs) of the LPL gene promoter (GenBank X68111; nucleotides 1 to 1902 according to GenBank numbering), comprising three putative sterol-regulatory elements. The promoter was amplified by polymerase chain reaction (PCR) using the oligonucleotide sequences 5′-GG GGTACC TGCAGGAGTATTCTAT ATAAGATAG-3′ and 5′-CCC AAGCTT CGCGTC CCTCTGGAGGAGCTGCAAG-3′ (restriction sites are shown in bold), digested, purified, and ligated into the pGL2–Basic vector (Promega, Madison, Wisconsin, USA).
Fatty acid synthase gene
The pGL2–fatty acid synthase (FAS) gene construct contained nucleotides −1485 to −1246 of the FAS gene promoter (GenBank X54671; nucleotides 1381 to 1620 according to GenBank numbering), comprising the key regulatory elements such as the sterol-regulatory element. The promoter was cloned into pGL2 following PCR (5′-GGA GGTACC GCGTTCCTT GTGCTCCAGCGCGC-3′; 5′-CAG AAGCTT CTG GACGGGACGCTGCTGCCGTCTCTC-3′).
Low-density lipoprotein receptor gene
The pGL2–low-density lipoprotein receptor (LDLR) gene construct contained nucleotides −328 to −61 of the LDLR gene promoter (GenBank L29401; nucleotides 380 to 627 according to GenBank numbering), comprising one sterol-regulatory element. The promoter was cloned into pGL2 following PCR (5′-AGCT G GTACC CGGAGACCCAAATACAACA-3′; 5′-TGT CC AAGCTT GAAACCCTGGCTTCCCGCGA-3′).
Confirmation of constructs
All constructs were confirmed by sequencing and contained sequences identical to those previously published and demonstrated to be functional [10–13]. In each experiment, the regulatory sequences of the inserts have been demonstrated to be inhibited by sterols as a negative and activated by simvastatin as a positive control.
Monolayers of human embryonic kidney (HEK)293 and hepatoma (Hep)G2 cells were set up (day 0, 5 × 106 cells/100 mm poly-d-lysine-coated Petri-dish) and cultured (37°C, 5% CO2) in Dulbecco's Modified Eagle Medium (DMEM) (Gibco BRL, Paisley, UK) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma, St Louis, Missouri, USA), and 5% (v/v) fetal calf serum (FCS) (Gibco) for 15 h.
HEK293 and HepG2 cells were independently cotransfected with 8.4 μg/dish of the pGL2–LPL, pGL2–FAS or the pGL2–LDLR luciferase reporter gene constructs, with 0.2 μg pRL–cytomegalovirus (CMV) (Promega), a plasmid encoding the Renilla luciferase, as an internal control for transfection efficiency, and with the vectors without inserts. The cells were incubated (7 h), treated with trypsin and transferred to medium A [DMEM supplemented with 5% (v/v) FCS, 100 U/ml penicillin, and 100 μg/m streptomycin] or medium B [DMEM supplemented with 5% (v/v) calf lipoprotein-deficient serum (LPDS) (Sigma), 50 μmol/l sodium mevalonate, 100 U/ml penicillin, and 100 μg/m streptomycin]. The cells were distributed over 96-well plates and incubated for 17 h. Each well contained 2 × 104 viable cells.
Twenty-four hours after transfection, indinavir was added at final concentrations of 0, 10–3, 10–2, 10–1, 1, 101, 5 × 101, 102, 2 × 102, 7.5 × 102, 103, 2 × 103, 5 × 103, 2 × 104, and 105 nmol/l. As a control of the inhibition of the SREBP-regulated reporter genes, the cells were incubated with 1 μg/ml 25-hydroxycholesterol and 10 μg/ml cholesterol (Sigma); as a control of the activation of the SREBP-regulated reporter genes, the cells were incubated with the dihydroxy-open form of simvastatin at final concentrations of 3 × 104 or 4 × 104 nmol/l.
Following incubation for 24 h, the media were discarded, the cells were washed with 1 × phosphate-buffered saline (PBS), and passive lysis buffer (25 μl/well; Promega) was added. The 96-well plates were shaken for 20 min. The luciferase and Renilla activities were determined by the Dual-LuciferaseTM Reporter Assay System (Promega). Luciferase activities were normalized according to the Renilla activities.
Reversibility of indinavir effects
To determine whether indinavir-induced inhibition of the activation of the SREBP-dependent genes was reversible or not, two series of HEK293 cells were cotransfected with pGL2–FAS luciferase reporter gene constructs and with pRL–CMV. These experiments were identical to those described above except that two sets of cells were used instead of one. Both sets of cells were first incubated with indinavir for 24 h at the concentrations specified above. Thereafter, indinavir was completely washed out with 1 × PBS. The first set of cells was incubated for another 24 h with fresh medium not containing indinavir. The second set of cells was incubated for another 24 h with fresh medium again containing indinavir at various concentrations. After incubation, the cells were harvested to determine the activation of the SREBP-dependent genes as described above.
Antagonization of indinavir effects by simvastatin
To determine whether indinavir-induced inhibition of the SREBP-dependent genes could be partially or entirely antagonized by statins, simvastatin in combination with indinavir was added to the cell culture medium. The net effects of the combination of simvastatin (constant final concentration of 3 × 104 nmol/l) and indinavir (final concentrations of 10, 5 × 101, 102, 2 × 102 nmol/l) were determined following 24 h of incubation with the respective combinations. The cells were harvested and the activation of the SREBP-dependent FAS gene was determined as described above.
RepGene, a spreadsheet template for the management of reporter gene assays was used for planning and evaluation of the experiments . The significance level of the effect of indinavir on Renilla-normalized luciferase activities was determined by analysis of variance (ANOVA) using StatView 4.5 (Abacus Concepts Inc., Berkeley, California, USA).
Indinavir inhibited the LPL and FAS reporter gene activities, measured as normalized relative light units, in a dose-dependent fashion (Fig. 1a,b). ANOVA confirmed the significance of the interaction between the different indinavir concentrations and the inhibition of the LPL gene activity (P = 0.0158) as well as the interaction between the different indinavir concentrations and the inhibition of the FAS gene activity (P = 0.0358). Indinavir did not inhibit the LDLR reporter gene activity significantly.
In the LPL reporter gene experiments, inhibition of the gene activity was detectable starting from an indinavir concentration of 1 nmol/l. At a concentration of 103 nmol/l, indinavir inhibited the LPL gene activity from baseline by 12.4% but did not reach a plateau at this concentration. At the highest concentration tested (105 nmol/l) indinavir inhibited the LPL gene activity from baseline by 57.1% (difference baseline versus highest concentration:P = 0.041) (Fig. 1a).
In the FAS reporter gene experiments, inhibition of the gene activity was detectable starting from an indinavir concentration of 10–2 nmol/l. At a concentration of 103 nmol/l, indinavir inhibited the FAS gene activity from baseline by 30.3%. The effect reached a plateau at this concentration (Fig. 1b).
Two sets of HEK293 cells were identically prepared except that, following incubation with indinavir and washing of the cells, the first series was incubated with medium containing no indinavir, the second series with medium containing indinavir. After a second incubation period, no statistically significant differences regarding the indinavir-induced inhibition of the SREBP-dependent genes were detectable between the two series. It was, therefore, concluded that indinavir-induced effects were not reversible.
Toxicity of indinavir causing a decrease in the viability of the cells with increasing concentrations of indinavir was first excluded on the morphological level. Cell toxicity was not detected microscopically, even at high indinavir concentrations (≥ 103 nmol/l). Cell toxicity as an explanation for the decrease of the LPL and FAS gene activities attributed to indinavir was further excluded by incubating the cells with the active dihydroxy-open form of simvastatin, a strong activator of the SREBP pathways. When simvastatin was combined with various concentrations of indinavir, the cells were still able to upregulate the SREBP-1c/ADD-1-dependent genes, as shown for FAS (Fig. 2): increasing concentrations of indinavir (0, 10, 5 × 101, 102, and 2 × 102 nmol/l) decreased the rate of the gene activation induced by a constant simvastatin concentration (3 × 104 nmol/l) again in a dose-dependent fashion. At an indinavir concentration of 2 × 102 nmol/l combined with a simvastatin concentration of 3 × 104 nmol/l, the FAS gene activity decreased by 36.4% compared with the activation achieved with simvastatin alone. However, the combination of 3 × 104 nmol/l simvastatin with 2 × 102 nmol/l indinavir still resulted in a net activation of the gene, demonstrating that the cells maintained their ability to upregulate their SREBP-1c/ADD-1-dependent genes despite the presence of indinavir (Fig. 2).
The effects of indinavir on the LPL and FAS reporter genes were similarly detectable when liver cells (HepG2 cells) instead of HEK293 cells were transfected (data not shown). The effects of indinavir on the LPL and FAS reporter genes were also detectable when the cells were incubated in FCS or LPDS media. Indinavir, sterols or simvastatin did not influence the activity of the empty vectors (pGL, pRL) (data not shown).
In the present study, the influence of indinavir on the activation of effector genes involved in the triglyceride, cholesterol, and insulin metabolism was investigated.
The major findings were that indinavir (i) decreased the activities of the SREBP-1c/ADD-1-dependent LPL and FAS reporter genes in a dose-dependent manner and (ii) did not decrease the activity of the SREBP-1c/ADD-1-independent LDLR reporter gene.
Inhibition of the LPL and FAS reporter gene activities was detectable starting from an indinavir concentration of 10–1 nmol/l (LPL) or 10–2 nmol/l (FAS). For comparison: concentrations of 5 × 101 to 102 nmol/l indinavir inhibit viral spread by 95% in cell culture , concentrations of 102–103 nmol/l indinavir correspond to physiological mean plasma concentrations in patients on treatment, and concentrations of 103–105 nmol/l correspond to mean plasma concentrations usually not achieved in the steady state . Since we observed clear decreases (12.4% for LPL and 30.3% for FAS) at an indinavir concentration of 103 nmol/l, we expect that administration of indinavir in recommended doses (2.4 g daily) will result in changes in the expressions of the LPL and FAS genes in vivo as well. In contrast, a significant inhibition of the LDLR reporter gene activity was not detectable, even at an indinavir concentration of 2 × 104 nmol/l. This result was not unexpected. We previously hypothesized that the pathophysiological mechanism to induce the hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia, and lipodystrophy syndrome observed in HIV-infected patients is mediated by the SREBP-1c/ADD-1 pathway rather than by the SREBP-2 pathway . Since the gene encoding the LDLR is activated by SREBP-2 but much less by SREBP-1c/ADD-1 , the lack of a significant inhibition of the LDLR reporter gene activity by indinavir is in line with our hypothesis.
Using other experimental approaches, PI have been shown to inhibit SREBP-1c/ADD-1 , insulin-stimulated glucose uptake , insulin signalling [9,18] and adipocyte determination and differentiation [9,19,20], and to induce adipocyte apoptosis  and modulate proteosome activity . In particular, the results of Caron et al. demonstrate a clear indinavir-induced impairment of SREBP-1 . Caron et al. focused on the indinavir-induced inhibition of insulin effects and adipocyte determination and differentiation, which are both mediated by SREBP-1c/ADD-1. Our results demonstrate the indinavir-induced inhibition of the lipoprotein and fatty acid metabolism, both also mediated by SREBP-1c/ADD-1, and are, therefore, in agreement with the observations of these authors. Thus, the indinavir-induced inhibition of effector genes regulated by SREBP-1c/ADD-1 leads to hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia and lipodystrophy: the syndrome observed in HIV-infected patients on HAART.
SREBP-1c/ADD-1 plays a central role in the regulation of triglycerides, cholesterol, insulin, and adipose tissue formation. Administration of PIs, associated with hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and peripheral lipodystrophy, affects SREBP-1c/ADD-1 and, therefore SREBP-1c/ADD-1-dependent effector genes. SREBP-1c/ADD-1 specifically activates the genes encoding the LPL and FAS [10,11,23–25] as well as the adipocyte determination and differentiation .
SREBP-1c/ADD-1 controls the peripheral clearance of triglyceride- and cholesterol-rich lipoproteins via regulation of LPL [25,27–29]. Inhibition of LPL results in hypertriglyceridaemia and hypercholesterolaemia (Fig. 3), as observed in inherited LPL deficiency syndromes (Fig. 3) [30–33]. SREBP-1c/ADD-1 controls fatty acid synthesis via regulation of FAS [11,24] and the determination and differentiation of adipocytes [26,27]. SREBP-1c/ADD-1 controls the insulin effect via regulation of a series of genes such as those encoding LPL , FAS [11,24], and glucokinase (Fig. 3). Inhibition of these genes results in a decreased insulin effect and, thus, insulin resistance [34,35]. Plasma insulin then increases in compensation . Consequently, inhibition of LPL, FAS and the gene encoding the glucokinase results in hyperinsulinaemia, as observed in inherited defects affecting, for example, the genes encoding LPL [30–32] and glucokinase  (Fig. 3). In line with these observations are our previous findings of a significant, parallel increase in plasma cholesterol and plasma insulin levels in HIV-1-infected subjects treated with PIs .
Inhibition of fatty acid synthesis and adipocyte determination and differentiation results in decreased fatty acid synthesis and adipose tissue formation [9,19,20] and might, therefore, explain particular aspects of peripheral lipodystrophy in humans (Fig. 3).
Proteolytic cleavage of SREBP-2 activates the gene encoding the LDLR strongly whereas proteolytic cleavage of SREBP-1c/ADD-1 activates LDLR much less . Similarly, simvastatin activates the SREBP-2-dependent LDLR strongly (approximately 500%, Fig. 1c) but activates the SREBP-1c/ADD-1-dependent LPL and FAS genes much less (approximately 100%, Fig. 1a,b). Inhibition of the SREBP-2-dependent LDLR gene (as in familial hypercholesterolaemia caused by LDLR gene defects) results in hypercholesterolaemia but usually not in hypertriglyceridaemia . In HAART-associated hyperlipidaemia, however, hypertriglyceridaemia is the predominant phenotype. In line with this latter observation, in our cell culture experiments, indinavir inhibited LDLR much less, indicating that the SREBP-1c/ADD-1-mediated mechanisms play the predominant role in the development of HAART-associated adverse effects.
We previously cloned and characterized the promoters of the differentially spliced genes of SREBP-1, SREBP-1a and SREBP-1c/ADD-1 and characterized the SREBP-2 gene . Very recently, we identified in the gene encoding SREBP-1c/ADD-1 a marker predictive of the hypertriglyceridaemia, hypercholesterolaemia, hyperinsulinaemia and lipodystrophy syndrome . Although, in our experience, other cell lines such as HepG2 cells are more difficult to transfect than HEK293 cells, analogous experiments carried out using HepG2 cells instead of HEK293 cells resulted in similar inhibitory effects on FAS activation. Hence, there is strong evidence from others [8,9] as well as from our own previous  and present results that inhibition of SREBP-1c/ADD-1 is involved in the development of the HAART-related hyperlipidaemia, hyperinsulinaemia, and lipodystrophy syndrome. Nevertheless, it remains to be clarified how PIs mediate the inhibition of SREBP-1c/ADD-1. The HIV-1 protease (GenBank 230883) and the site-1 protease (GenBank 4506775), a sterol-regulated protease that cleaves the SREBP molecules , share a sequence homology at their catalytic sites. Despite this sequence homology, the HIV-1 protease and the site-1 protease belong to different classes of proteases. Therefore, because of considerable differences in the three-dimensional structures of the HIV-1 protease and the site-1 protease and based on steric models, it is unlikely that PI influence the SREBP pathway by direct inhibition of the site-1 protease. Similarly, these considerations apply to the hypothesis that PIs interact with the adipocyte-enhancer binding protein . Furthermore, if the site-1 protease was inhibited, we would expect quantitatively similar effects regarding the impairment of the activation of SREBP-1c/ADD-1- and SREBP-2-dependent genes. However, this was clearly not the case in our experiments.
A possible explanation for the differential inhibition of SREBP-1c/ADD-1- and SREBP-2-dependent genes is based on recent experiments suggesting differences in the regulation of the amount of mature SREBP-1c/ADD-1 and SREBP-2. While SREBP-1c/ADD-1 is controlled by two independent mechanisms, by cleavage activation and by the rate of mRNA degradation , there is no evidence that SREBP-2 is regulated by the latter .
In summary, our demonstration of the inhibition of SREBP-1c/ADD-1-dependent genes explains the major metabolic effects described in association with HAART.
We thank Dr Daniel Bur (Allschwil), Prof. Manuel Battegay and Prof. Christoph Moroni (Basel), Prof. Bernard Hirschel (Geneva), Prof. Walter Wahli and Prof. Beatrice Desvergne (Lausanne), and Prof. Charles Rice (Los Angeles) for valuable discussions. Indinavir sulfate and simvastatin were kindly provided by Merck & Co. Inc., Rahway, New Jersey, USA.
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