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COMPARISON OF THE EFFECTS OF NEVIRAPINE RESPECT TO EFAVIRENZ ON DIFFERENTIATING AND MATURE HUMAN
ADIPOCYTES IN CULTURE
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From Jules: are these studies conducted properly and do they have clinical impact?
Reported by Jules Levin
10th Intl Workshop on Adverse Drug Reactions and Lipodystrophy in HIV, London Nov 6-8 2008
J Diaz-Delfin1, JM Gallego-Escuredo1, M Milanski1, JCDomingo1, MM Gutierrez 2, MG Mateo 2, P Domingo 2, M Giralt1, F Villarroya1
1Department of Biochemistry and Molecular Biology and Institut de Biomedicina, University of Barcelona, and 2 Department of Internal Medicina, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
INTRODUCTION
Nevirapine and efavirenz are nonnucleoside analog inhibitors of reverse trascriptase, drugs of choice for initial antiretroviral therapy in HIV-1-infected
patients. Recent data has suggested potentially deleterious effects of
efavirenz on adipose tissue in patients(1). Efavirenz has also been reported to
have negative effects in differentiation of white and brown adipocytes (2,3) whereas no effect or slight induction of adipogenesis has been reported for
nevirapine in other rodent models, including brown adipocytes (3). A common parallel study of the effects of both type of non-nucleoside analog reverse transcriptase inhibitors on human primary white adipocyets was not available.
AIM
Our aim was to perform a parallel assessment of the effects of nevirapine
respect to the action of efavirenz on human adipocytes in culture, and to
compare their actions on differentiation and on the expression of marker genes
of adipogenesis, inflammation and mitochondrial toxicity, as well as on the
release of regulatory adipokines by cultured adipocytes.
METHODS
Human adipocytes were differentiated in primary culture from precursor cells
obtained from liposuction material from healthy individuals, following already
reported methodology (4). We determined the effects of exposure to distinct concentrations of nevirapine and efavirenz of pre-adipocytes during their 10-day differentiation process into adipocytes. The same comparison was performed by studying the effects of 24h exposure of already differentiated human adipocytes to nevirapine and efavirenz. Morphology of adipocyte differentiation (number of cells accumulating lipid droplets), lactate release to the medium (Roche Accutrend) as well as changes in mRNA expression for marker genes (TaqMan
real-time PCR, Applied Biosystems) were determined. Quantification of the abundance of specific proteins released by adipocytes to the culture medium was performed using the Multiplex technology (Millipore).
Effects of efavirenz and nevirapine on human adipocyte differentiation
Human pre-adipocytes were differentiated in culture in the presence of the indicated concentrations of drugs (10 days since confluence). A, micrographs of cell morphology. B, a quantitative assessment of the extent of differentiation respect to controls (set up arbitrarily as 100%). Quantification was performed through image analysis of percent of surface occupancy by adipocytes using 4 random micrographs for each individual culture condition. Expression of data is shown as means + SEM and p values for StudentŐt t test comparison
are shown when statistically significant (P < 0.05). If not significant. NS is marked.
Mitochondrial DNA levels and mRNA expression of marker genes of mitochondriogenesis and adipogenesis in human adipocytes treated with efavirenz or nevirapine throughout differentiation
Human pre-adipocytes were differentiated in culture in the presence of the indicated concentrations of drugs (10 days since confluence). DNA and RNA were obtained. Mitochondrial DNA and specific transcripts were measured by quantitative real-time PCR using TaqMan technology and the corresponding TaqMan Assay-on-Demand probes (Applied Biosystems), previous retro-transcription with random primers. Data of mitochondrial DNA was referred to nuclear DNA (C/EBPa single-copy gene). Data of transcripts was referred to 18S rRNA and main changes were re-checked using cyclophilin mRNA as alternative housekeeping transcript. Data are shown as means ± SEM from 3 independent
experiments and are expressed as relative to values from untreated control cells which was set to 1 (dotted line, mean; shaded area, ± SEM). Statistical significance of differences respect to controls are shown as * P<0.05, **P<0.01, ***P< 0.001. Those in the comparison of Efavirenz versus Nevirapine at the same concentration of treatment of cells are shown as #.
Levels of adiponectin, MCP-1 and lactate realeased to the culture medium by human adipocytes treated with efavirenz or nevirapine throughout differentiation
Human pre-adipocytes were differentiated in culture in the presence of the indicated concentrations of drugs (10 days since confluence). 25 μl of serum-free culture medium (unchanged during the last 5 days before cell harvest) were used for quantification. Milliplex technology (Millipore) with the corresponding specific antibodies and standard curves were used for adiponectin and MCP-1 release. Lactate was measured using standarized
technology from Roche Inc. Data are shown as means ± SEM from 4 independent experiments and are expressed as relative to values from untreated control cells which was set to 1 (dotted line, mean; shaded area, ± SEM). Range levels of specific protein contents in medium (mean of controls) were: adiponectin, 0.24 μg/ml; MCP-1, 3.0 ng/ml.
mRNA expression of marker genes of mitochondriogenesis and
adipogenesis in differentiated human adipocytes treated with
efavirenz or nevirapine during 24 h
Human pre-adipocytes were differentiated in culture. When already differentiated (10 days after confluence), the cultures were treated with the indicated concentrations of drugs during 24h. RNA was obtained and specific transcripts were measured by quantitative real-time PCR using TaqMan technology and the corresponding TaqMan Assay-on-Demand probes (Applied Biosystems), previous retro-transcription with random primers. Data of transcripts was referred to 18S rRNA and main changes were re-checked using cyclophilin A mRNA as alternative housekeeping transcript. Data are shown as means ± SEM from 3 independent experiments and are expressed as relative to values from untreated control cells which was set to 1 (dotted line, mean; shaded area, ± SEM). Statistical significance of differences respect to controls are shown as * P<0.05, **P<0.01, ***P< 0.001. Those in the comparison of Efavirenz versus Nevirapine at the same concentration of treatment of cells are shown as #.
Effects of efavirenz and nevirapine on human
adipocytes already differentiated
Micrographs of cultures of differentiated human adipocytes (10 days after confluence) treated with the indicated concentrations of drugs during 24h, show no alterations in cell morphology
AUTHOR CONCLUSIONS
Efavirenz impairs morphological adipogenic differentiation and adipogenic gene expression without causing mitochondrial toxicity. In contrast, nevirapine did not impair but even induced slightly adipogenesis. Considering the evidence that alterations in the adipogenic processes take place in lipoatrophic adipose tissue from patients, inclusion of nonnucleoside reverse transcriptase inhibitors in antiretroviral regimes design should take into account the anti-adipogenic properties of efavirenz respect to nevirapine.
REFERENCES
1. Haubrich R, Riddler S, DiRienzo D, et al. 14th Conference on Retrovirus and Opportunistic Infections, Los Angeles, 25-28 February 2007.
2.-El Hadri K, Glorian M, Monsempes C, Dieudonne MN, et al. J Biol Chem. 2004, 279:15130-41.
3.-Rodr’guez de la Concepci—n ML, Yubero P, Domingo JC, et al. Antivir Ther. 2005;10:515-26.
4. Schlulter A, Yubero P, Iglesias R, Giralt M, Villarroya F. Int J Obes Relat Metab Disord. 2002 26:1277-80.
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