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. 2016 Jul 20;11(7):e0159747.
doi: 10.1371/journal.pone.0159747. eCollection 2016.

SILAC-MS Based Characterization of LPS and Resveratrol Induced Changes in Adipocyte Proteomics - Resveratrol as Ameliorating Factor on LPS Induced Changes

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SILAC-MS Based Characterization of LPS and Resveratrol Induced Changes in Adipocyte Proteomics - Resveratrol as Ameliorating Factor on LPS Induced Changes

Mark K Nøhr et al. PLoS One. .
Free PMC article

Abstract

Adipose tissue inflammation is believed to play a pivotal role in the development obesity-related morbidities such as insulin resistance. However, it is not known how this (low-grade) inflammatory state develops. It has been proposed that the leakage of lipopolysaccharides (LPS), originating from the gut microbiota, through the gut epithelium could drive initiation of inflammation. To get a better understanding of which proteins and intracellular pathways are affected by LPS in adipocytes, we performed SILAC proteomic analysis and identified proteins that were altered in expression. Furthermore, we tested the anti-inflammatory compound resveratrol. A total of 927 proteins were quantified by the SILAC method and of these 57- and 64 were significantly up- and downregulated by LPS, respectively. Bioinformatic analysis (GO analysis) revealed that the upregulated proteins were especially involved in the pathways of respiratory electron transport chain and inflammation. The downregulated proteins were especially involved in protein glycosylation. One of the latter proteins, GALNT2, has previously been described to regulate the expression of liver lipases such as ANGPTL3 and apoC-III affecting lipid metabolism. Furthermore, LPS treatment reduced the protein levels of the insulin sensitizing adipokine, adiponectin, and proteins participating in the final steps of triglyceride- and cholesterol synthesis. Generally, resveratrol opposed the effect induced by LPS and, as such, functioning as an ameliorating factor in disease state. Using an unbiased proteomic approach, we present novel insight of how the proteome is altered in adipocytes in response to LPS as seen in obesity. We suggest that LPS partly exerts its detrimental effects by altering glycosylation processes of the cell, which is starting to emerge as important posttranscriptional regulators of protein expression. Furthermore, resveratrol could be a prime candidate in ameliorating dysfunctioning adipose tissue induced by inflammatory stimulation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Experimental setup.
3T3-L1 cells were incubated with either heavy (Arg-10), medium (Arg-6) or light labeled arginine for 6 passages. Cells were differentiated and subjected to the treatment regimens according to labeling: Control (Arg-10 cells), LPS (Arg-6 cells) and LPS + resveratrol (Arg-0 cells). Whole cell lysates were divided by SDS-PAGE and 12 bands were cut and trypsinized before mass spectrometry analysis. Abbreviations: IBMX: 3-isobutyl-1-methylxanthine, FCS: fetal calf serum, FAFBSA: fatty acid free bovine serum albumin.
Fig 2
Fig 2. Differentiation of 3T3-L1 cells.
Lipoprotein lipase (lpl) expression during the differentiation process of 3T3-L1 cells.
Fig 3
Fig 3. GO analysis of regulated proteins by LPS.
(A) Upregulated proteins by LPS treatment belonged to the GO classes: respiratory electron transport chain and generation of precursor metabolites and energy processes. (B) Downregulated proteins especially belonged to the GO class protein glycosylation and to a smaller degree lipid metabolic processes. (C and D) Schematic overview of the distribution of upregulated (C) and downregulated (D) proteins in different GO classes represented here by gene name. Abbreviations: please see S1 Table.
Fig 4
Fig 4. Resveratrol as ameliorating factor on LPS-induced alterations in protein expression.
(A) Upregulated proteins by LPS were generally partially rescued and returned towards control expression levels. (B) Resveratrol partially reversed the downregulation of proteins induced by LPS. Abbreviations: please see S1 Table.
Fig 5
Fig 5. IFIT1 expression in whole adipose tissue measured by qPCR and in 3T3-L1 cells measured by and SILAC-MS.
(A) Mice treated with LPS for 28 days showed increased gene expression of IFIT1, which was ameliorated by resveratrol delivered through the diet. (B) IFIT1 protein expression measured by SILAC-MS in 3T3-L1 cells incubated with LPS and resveratrol.
Fig 6
Fig 6. STAT1 expression measured by qPCR and SILAC-MS.
(A) STAT1 gene expression measured by qPCR in 3T3-L1 cells incubated with LPS and resveratrol. (B) IFIT1 protein expression measured by SILAC-MS in 3T3-L1 cells incubated with LPS and resveratrol.

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Grant support

MKN was supported by a full PhD scholarship provided by the Graduate School of Health, Aarhus University. The study was supported by the Rasmus Riisfort Foundation, Ejnar Danielsens Foundation and the AP Møller Maersk Foundation. The study is part of the research program LIRMOI Research Center (www.LIRMOI.com), which is supported by the Danish Council for Strategic Research (Grant 10-093499). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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