A triterpene-enriched natural extract from Eucalyptus tereticornis modulates the expression of genes involved in adipogenesis, lipolysis, and extracellular matrix remodeling in a primary human and mouse cell line adipocyte

Abstract

Context: Obesity induces alterations in adipocyte size, tissue inflammation, vascularization, and extracellular matrix composition. Previous studies have shown that a leaf extract of Eucalyptus tereticornis Sm. (Myrtaceae), with ursolic acid, oleanolic acid, and ursolic acid lactone mixed with minor metabolites, provided a superior antiobesity effect than reconstituted triterpenoid mixtures in adipocyte cell lines and a pre-diabetic mouse model. Further identification of the molecular mechanisms of action of this mixture of triterpenes is required.

Objective: This study analyzes the effect of the natural extract and its components on early RNA expression profiles in human primary cultured adipocytes and a mouse cell line.

Materials and methods: RNA was sequenced using the DNBseq platform and the EnrichR software to perform gene enrichment analysis using the Gene Ontology database, Kyoto Encyclopedia of Genes and Genomes, and Reactome. To conduct clustering analysis, the normalized counts of each gene and applied k-means clustering were standardized.

Results: The combination of molecules in the natural extract has an additive or synergic effect that increases the number of genes regulated associated with the biological functionality of differentiating adipocytes, with UAL playing a central role. The natural extract modulates PPAR, Wnt, and Extracellular Matrix organization pathways significantly in both cellular models. Remarkably, the extract downregulates the expression of genes involved in lipid metabolism, adipogenesis, and adipocyte fat load, such as PRKAR2B, LPIN1, FABP4, Scd1, MC5R, CD36, PEG10, and HMGCS1.

Discussion and conclusions: Our study shows that Eucalyptus tereticornis extract is a promising option for treating adipocyte tissue dysfunction derived from obesity.

Keywords: Natural extract; adipocyte; adipose tissue; transcriptome; triterpenes.

Figure 1.

Correspondence in transcriptomic profiles in differentiating human and mouse adipocytes. (a) Bar chart illustrating the combined count of genes exhibiting upregulation and downregulation in differentiating adipocytes from human and mouse models. (b) Pearson correlation plot depicting the expression correlation of orthologous genes identified as differentially expressed in at least one species under logical or and (c) under logical and In the correlation plots, every data point represents an orthologous gene, with its expression quantified using log2 fold change. The blue line denotes the linear regression, while the shaded grey region indicates the 95% confidence interval. The red line signifies perfect identity. (d) Heat map of Z-score normalized orthologue DEGs by unsupervised hierarchical clustering analysis. (e) Principal component analysis (PCA) on orthologous DEGS utilizing FPKM values. The variance percentages for each principal component (PC1 and PC2) are presented. Cumulative distribution plots depict the log2 fold change for (f) upregulated genes in both mice (indicated by the blue line) and humans (shown by the purple line), as well as (g) downregulated genes. (h) Gene ontology enrichment analysis on shared orthologous DEGs in human and mouse differentiating adipocytes. (i) Bar plots depict orthologous DEGs in differentiating adipocytes from humans and mice. C: Control group, DC: Differentiating control.

Figure 2.

Gene expression profile of differentiating adipocytes treated with triterpenes. (a) PCA for human and mouse models across five treatments (DC: differentiating control, UA: Ursolic acid, UAL: Ursolic acid lactone, OA: Oleanolic acid, M1: triterpenes mix, and OBE100). (b) Bar plot of the total count of upregulated and downregulated genes for each treatment condition. (c,d) Cumulative distribution plots depict the log2 fold change for OBE100 (light blue line) and M1 or UAL (green line) treatments in comparison to differentiating control (red line) for both upregulated (c) and downregulated (d) genes.

Figure 3.

OBE100 Treatment alters the gene expression of genes related to adipogenesis, thermogenesis, and extracellular matrix organization in differentiating adipocytes. (a) Gene ontology enrichment for DEGs regulated by OBE100 in human and mouse macrophages. Down-regulated terms are depicted in blue, while up-regulated terms are shown in red. (b) Bar plots illustrating normalized expression levels of differentially expressed genes. *p ≤ 0.05 compared to DC: Differentiating control.

Figure 4.

OBE100 Extract regulates gene expression in differentiating adipocytes via triterpenes mix and the minor fraction. (a) Venn diagrams depict the modulation of DEGs by OBE and M1 treatment in human adipocytes. (b) Bar graph illustrating five expression profiles, categorized using k-means, according to the average expression of the genes in the four conditions of human adipocytes. (c) The table presents the genes belonging to each profile and the gene ontology enrichment analysis.

Figure 5.

Ursolic acid lactone regulates ECM and adipogenesis genes. (a) Venn diagrams depict the modulation of DEGs by M1 and UAL treatment in human adipocytes. (b) Bar graph illustrating five expression profiles, categorized using k-means, according to the average expression of the genes in the four conditions of human adipocytes. (c) The table presents the genes that belong to each profile, along with the gene ontology enrichment analysis.

Figure 6.

The mode of action of OBE100 involves multiple pathways and regulatory mechanisms. OBE100 treatment impacts various cellular processes, including regulating gene expression of genes related to cell cycle progression and extracellular matrix (ECM) remodeling. Central to its action is the modulation of gene expression profiles, including genes such as HMGCS1, which plays a role in lipid metabolism. OBE100 may exert its effects on HMGCS1 through transcriptional regulators like PPARα, which is known to control genes involved in lipid metabolism. Furthermore, OBE100 may influence ECM remodeling, crucial for maintaining tissue integrity and function. By modulating genes involved in ECM dynamics, such as those encoding matrix metalloproteinases and collagens, OBE100 can affect tissue architecture and cell behavior. Overall, the mode of action of OBE100 involves a complex interplay between gene expression regulation, cell cycle control, EMT modulation, and ECM remodeling, with potential implications for various physiological and pathological processes.