Effects of nevirapine and efavirenz on human adipocyte differentiation, gene expression, and release of adipokines and cytokines

Abstract

The non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz are drugs of choice for initial antiretroviral treatment for HIV-1 infection. Although NNRTIs have not traditionally been associated with the appearance of adipose alterations, recent data suggest that efavirenz may contribute to adipose tissue alterations in antiretroviral-treated patients, consistent with its ability to impair differentiation of adipocytes in cell cultures. No such effects have been reported for nevirapine, the other most commonly used NNRTI. In this study, we determined the effects of nevirapine on differentiation, gene expression and release of regulatory proteins (adipokines and cytokines) in differentiating human adipocytes, and compared them with those of efavirenz. Efavirenz caused a dose-dependent repression of adipocyte differentiation that was associated with down-regulation of the master adipogenesis regulator genes SREBP-1, PPARγ and C/EBPα, and their target genes encoding lipoprotein lipase, leptin and adiponectin, which are key proteins in adipocyte function. In contrast, nevirapine does not affect adipogenesis and causes a modest but significant coordinate increase in the expression of SREBP-1, PPARγ and C/EBPα and their target genes only at a concentration of 20 μM. Whereas efavirenz caused a significant increase in the release of pro-inflammatory cytokines (interleukin [IL]-8, IL-6, monocyte chemoattractant protein-1), plasminogen activator inhibitor type-1 and hepatocyte growth factor (HGF), nevirapine either had no effect on these factors or decreased their release (IL-6 and HGF). Nevirapine significantly increased adiponectin release, whereas efavirenz strongly repressed it. Moreover, nevirapine inhibited preadipocyte endogenous reverse transcriptase activity, whereas efavirenz did not alter it. It is concluded that, in contrast with the profound anti-adipogenic and pro-inflammatory response elicited by efavirenz, nevirapine does not impair adipogenesis.

Introduction

Alterations in adipose tissue distribution (lipodystrophy syndrome) and systemic metabolic disturbances (dyslipidemia and insulin resistance) appear frequently in HIV-1-infected patients under highly active antiretroviral treatment (HAART). HAART employs a drug regimen that typically includes nucleoside-analog reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs) and/or non-nucleoside analog inhibitors of reverse transcriptase (NNRTIs). The complex metabolic alterations that arise with these treatment regimens reflect the action of HAART drug combinations on the susceptible status of HIV-1-infected patients. Although individual drug treatments cannot account for the development of the lipodystrophy syndrome, NRTIs, such as stavudine and zidovudine, are thought to predispose toward peripheral lipoatrophy, whereas PIs are considered to favor insulin resistance and dyslipidemia (Villarroya et al., 2005). NNRTIs are drugs of choice for initial antiretroviral treatment of HIV-1-infected patients, in combination with drugs from other families. Unlike NRTIs, NNRTIs do not inhibit DNA polymerase-γ, and therefore are not expected to elicit mitochondrial toxicity, a major suspected cause of adipose tissue alterations in HAART-treated HIV-1-infected patients.

Efavirenz, an NNRTI, is the preferred third agent to include in antiretroviral regimes according to most international antiretroviral treatment guidelines (Hammer et al., 2008, Gazzard et al., 2008). This is because efavirenz has never been surpassed in clinical trials, and, in fact, has shown better antiretroviral efficacy than PIs in pivotal clinical trials (Staszewski et al., 1999, Riddler et al., 2008). Nevirapine, though more restricted in use than efavirenz, is the other NNRTI commonly used in several European countries, especially in developing countries where a compact pill with stavudine and lamivudine is often used for initial antiretroviral therapy (Zhou et al., 2007, Colebunders et al., 2005). The 2NN study was originally intended to directly compare efavirenz and nevirapine (van Leth et al., 2004a); unfortunately, however, no conclusion could be drawn from this study in terms of non inferiority between both agents.

NNRTIs have not traditionally been associated with the appearance of lipodystrophy and are often perceived as benign in terms of adipose alterations. However, recent clinical trials have shown that efavirenz could favor lipoatrophy when used as a component of the HAART cocktail (Riddler et al., 2008, Haubrich et al., 2009). In contrast, several reports have indicated that shifting PI- or NRTI-based regimes to one containing nevirapine modestly ameliorated disturbances in the lipid profile of adults (Negredo et al., 1999) or pediatric (Gonzalez-Tome et al., 2008) patients. Switching to nevirapine also caused minor improvements in lipodystrophy (Negredo et al., 1999), but did not attenuate specific events associated with lipoatrophy, such as adipose tissue apoptosis (Domingo et al., 2001). Nevirapine appears to cause a better improvement in the lipid profile of treated patients respect to efavirenz (Fisac et al., 2005, van Leth et al., 2004b), basically by increasing HDL-cholesterol and the total cholesterol/HDL-cholesterol ratio.

There are few studies on the effects of NNRTIs on adipocytes. Using murine cell models, we reported (Rodríguez de la Concepción et al., 2005) that nevirapine favors differentiation and expression of marker genes of adipocyte function, such as peroxisome proliferator-activated receptor gamma (PPARγ), in primary brown adipocytes. This was in contrast to the anti-adipogenic effect of efavirenz in these cells. Efavirenz has been reported to decrease the capacity of mouse 3T3-L1 preadipocytes to accumulate triglycerides due to impaired lipogenesis (El Hadri et al., 2004), whereas no effects were observed for nevirapine in the murine adipogenic cell line 3T3-F422A (Caron et al., 2004). Similarly, studies of differentiating human adipocytes, which have been limited to reports on morphological changes, have shown reduced adipogenesis with efavirenz treatment (El Hadri et al., 2004) and no adipogenic effects of a single concentration (10 μg/ml) of nevirapine (Vernochet et al., 2005).

One of the main physiopathogenic components of the interplay between adipose tissue disturbances and systemic metabolic derangements in response to viral and pharmacological insults is interference with the secretory functions of adipose tissue. Adipose tissue is not only a site of fat storage, but is also responsible for releasing regulatory factors such as adipokines and pro-inflammatory cytokines that act both locally and on distant organs (e.g., liver, muscle, heart, pancreas) to influence overall metabolism. For instance, adipose tissue releases the insulin-sensitizing hormone adiponectin and other factors that affect insulin resistance, such as resistin, as well as inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin (IL)-6 (Hauner, 2005). Thus, the potential action of drugs on adipose tissue may not only affect adipose tissue development, it can also alter adipose tissue secretory functions, and thereby alter systemic metabolism.

The effects of the NNRTIs nevirapine and efavirenz on the secretory functions of adipocytes have not been investigated to date. In the present study, we conducted a comparative analysis of nevirapine and efavirenz action on adipocyte differentiation, gene expression and release of adipokines and cytokines by human adipocyte cells in culture.