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NMR Metabonomics in an in vitro Model of HIV-1 latency
Background: Metabolic disorders have been identified in patients infected with the
human immunodeficiency virus (HIV). These disorders include lipodystrophy,
wasting syndrome, cardiovascular disease and glucose intolerance. Highly active
antiretroviral treatment (HAART) administered to patients can successfully
suppress the virus and decrease the prevalence of opportunistic infections
associated with AIDS but increase incidence of metabolic disorders. Glucose
tolerance test, dual-energy x-ray absorptiometry and CD4 counts are some of the
conventional tests that are used to detect and monitor disease progression and
metabolic disorders. These are single result tests that are time-consuming and
provide limited information about the metabolic disorder. A metabonomic approach
allows for the measurement of multiple metabolites simultaneously; which could
lead to the identification of markers of disease progression. Most HIVmetabonomics
studies to date used nuclear magnetic resonance (NMR)
spectroscopy and mass spectrometry (MS) spectrometry to detect multiple
metabolites from blood and urine samples simultaneously, fewer studies utilized cell
culture supernatants as metabolite source.
In the body HIV is able to seize control of cellular networks and exists as an active
or latent virus. HIV latency is mainly responsible for survival of the virus through the
production of reservoirs throughout the body. It is then of great interest to
investigate the effects of active and latent virus on cell metabolic networks. In vitro
cell models such as U1 cells, a promonocyte latently infected with HIV-1 are
suitable for such investigations because the models allow for control over activation
of the virus with stimulants. To date only one study has compared the metabolic
profiles of the active virus against non-infected cells but no study has comparatively
investigated actively and latently infected immune system U1 cells, which is what is
presented here. In this study a metabonomic approach was used to investigate
active and latently infected U1 promonocytic cells and the metabolites were
detected by NMR spectroscopy. Methods: U1 and U937 cells were cultured and lysed by the freeze-thaw method to
extract the supernatant, the extent of cell lysis was determined through flow
cytometry. NMR spectroscopy was used to detect metabolic profiles of uninfected
U937 cells (parent cell line from which U1 cells were derived) as well as actively
and latently infected U1 cells. Phorbol myristate acetate (PMA) was used as a
stimulant to activate the virus. NMR data was preprocessed for statistical analysis
with Mestrenova 10.0 software, and the metabolites were putatively identified with
the use of Chenomx software, literature searches, human metabolome database
(HMDB) and Kyoto encyclopedia of genes and genomes (KEGG). For statistical
data analysis SPSS 20.0 software was used to determine group separation and
metabolic profile differences.
Results: Glucose, lactate, glutamine/glutamate, leucine, alanine, choline,
phosphocreatine and lipid are some of the metabolites that were detected by NMR
spectroscopy and through ANOVA analysis the metabolites were determined to be
significantly different (P-value<0.05) between the three groups. A multiple
comparison table presented the group significant differences and LDA correctly
classified the experimental groups with 100% accuracy. U937 and actively infected
cells produced similar results to what was seen in other investigations where sera
and plasma were used as metabolite source. Latently infected cells produced the
more distinguishable separation among the experimental groups and the
metabolites responsible for this separation were those mainly involved in glycolysis
and lipid biosynthesis pathways. All the cell lines were treated with lactate to
evaluate the influence of one prominent metabolite on the virus and cell
metabolism. Lactate was selected because the metabolite was found to be
significantly present in the initial experiments and for its role in glycolysis (indicates
anaerobic respiration). Cysteine, an indicator of oxidative stress was produced and
some of the metabolites such as alanine and taurine were no longer detectable. Conclusion: NMR spectroscopy successfully elucidated metabolic profiles of U937
cells, U1 cells (latent virus) and PMA induced U1 cells (active virus). The technique
was highly reproducible with minimal sample preparation. Most metabolites that
were detected are those primarily associated with metabolic disorders involving
glycolytic energy metabolism. Through multiple comparisons it was determined that
latent HIV-1 infection had a profound effect on cell glycolysis as seen by the
significant alteration of lactate and the occurrence of aerobic glycolysis and
mitochondrial disruption. In this study, it was observed that the virus supports
biosynthetic pathways more than the production of energy through oxidative
phosphorylation. Cells that were exposed to lactate produced a different metabolic
profile from those that were not treated, this indicated that an increase or decrease
in concentration of a particular metabolite can affect cell metabolism in HIV infected
cells.