Citrus aurantium L. Dry Extracts Ameliorate Adipocyte Differentiation of 3T3-L1 Cells Exposed to TNFα by Down-Regulating miR-155 Expression

Michele Campitelli,Pietro Formisano,Gregory A Raciti,Pietro Campiglia,Claudia Miele,Alessia Leone,Giuseppe Cacace,Cecilia Nigro,Antonella Desiderio,Sonia De Simone,Francesco Beguinot,Immacolata Prevenzano

doi:10.3390/nu12061587

Abstract

Citrus aurantium L. dry extracts (CAde) improve adipogenesis in vitro. These effects are dependent from an early modulation of CCAAT/enhancer-binding protein beta (C/Ebpβ) expression and cyclic Adenosine Monophosphate (cAMP) response element-binding protein (CREB) activation. C/Ebpβ and Creb are also targets of miR-155. This study investigated whether CAde regulates miR-155 expression in the early stages of adipogenesis and whether it ameliorates adipocyte differentiation of cells exposed to tumor necrosis factor-alpha (TNFα). Adipogenic stimuli (AS) were performed in 3T3-L1 pre-adipocytes treated with CAde, TNFα, or both. Gene and miRNA expression were determined by quantitative real-time PCR. Adipogenesis was evaluated by Oil-Red O staining. CAde treatment enhanced AS effects during the early adipogenesis phases by further down-regulating miR-155 expression and increasing both C/Ebpβ and Creb mRNA and protein levels. At variance, TNFα inhibited 3T3-L1 adipogenesis and abolished AS effects on miR-155, C/Ebpβ, and Creb expression. However, in cells exposed to TNFα, CAde improved adipocyte differentiation and restored the AS effects on miRNA and gene expression at early time points. In conclusion, this study identified miR-155 down-regulation as part of the mechanism through which CAde enhances adipogenesis of pre-adipocytes in vitro. Furthermore, it provides evidence of CAde efficacy against TNFα negative effects on adipogenesis.

Highlights

Adipocyte differentiation is a highly orchestrated physiological process, which involves a large number of molecular events and whose dysregulation is relevant to human disease by contributing metabolic dysfunction in obesity [1,2]. microRNAs represent a class of small non-codingRNAs (≈22 nt in length) involved in a variety of cellular processes, whose role is to repress targetNutrients 2020, 12, 1587; doi:10.3390/nu12061587 www.mdpi.com/journal/nutrientsNutrients 2020, 12, 1587 translation and to induce target mRNA degradation [3]
We have recently demonstrated the nutraceutical properties of Citrus aurantium L. dry extracts (CAde), a dry extract preparation obtained from Citrus aurantium L. (CAde) fruit juice, on the regulation of 3T3-L1 cell adipocyte differentiation and function [28]
In Adipogenic stimuli (AS) + CAde-treated cells, the expression of miR-155 resulted in levels being decreased by 31% already by 15 min, and its levels further declined by 58% at 30 min, 51% at 1 h, 54% at 2 h, and 54% at 4 h upon adipogenic induction compared to control cells at time 0 (Figure 1a)

Summary

Introduction

Adipocyte differentiation is a highly orchestrated physiological process, which involves a large number of molecular events and whose dysregulation is relevant to human disease by contributing metabolic dysfunction in obesity [1,2]. microRNAs (miRNAs) represent a class of small non-codingRNAs (≈22 nt in length) involved in a variety of cellular processes, whose role is to repress targetNutrients 2020, 12, 1587; doi:10.3390/nu12061587 www.mdpi.com/journal/nutrientsNutrients 2020, 12, 1587 translation and to induce target mRNA degradation [3]. MiR-27a and -27b, belonging to the miR-27 gene family, were identified as negative regulators of adipogenesis [7,10,11]. The expression of both is down-regulated during adipogenic differentiation of 3T3-L1 cells, and their over-expression is inhibited in vitro adipocyte formation by reducing the levels of master adipogenic regulators such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) [7]. MiR-155, initially described as the B-cell integration (bic) cluster gene in chickens [19], is a multifunctional miRNA involved in numerous physiological and pathological processes such as haematopoietic lineage differentiation, immunity, inflammation, cancer, and cardiovascular diseases [20,21]. Its role in the regulation of adipocyte differentiation has been described [22]

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Conclusion