Obesity induced by high-fat diet is associated with critical changes in biological and molecular functions of mesenchymal stromal cells present in visceral adipose tissue

Figure 1. Experimental design. Mice were fed with a normal diet (ND) and a high-fat diet (HFD) for 20 weeks. One week before animal euthanasia, GTT and ITT tests were conducted. The MSCs isolated from visceral adipose tissue were characterized by flow cytometry, senescence-associated β-galactosidase assay, annexin V staining, and cell analysis performed at passage 3. At passage 3, for each experimental group, we collected total cellular protein content and secretomes as well as performed LC-MS/MS analysis.

Figure 2. High fat diet outcomes. (A) Average body weight of animals through 20 weeks. The histogram shows the weight of the five mice fed with HFD and the five mice with ND for 20 weeks. Data are shown with standard deviation (SD) n=5 *p<0.05, **p<0.01. (B) Insulin tolerance tests (ITT) and glucose tolerance tests (GTT). The graphs show blood glucose levels (mg/dl) in HFD- and ND-treated mice. (C) Flow cytometry results on visceral adipose-derived MSCs. The picture shows a representative example of flow cytometry analysis carried out on P0 MSCs obtained from an ND-treated mouse. Cells were positive for MSC markers CD29 and CD90 and negative for MSC marker CD45. The same results were obtained with cells isolated from HFD-treated mice (data not shown).

Figure 3. Biological assays in HFD- and ND-MSCs. (A) The histogram shows the mean percentage value of senescent cells, determined by beta-galactosidase assay. Data are expressed with SD (n = 5) ****p<0.001. Representative images of senescent cells that are stained blue are shown. (B) The histogram shows the mean percentage of annexin V-positive cells. Data are expressed with standard deviation (n = 5) **p<0.01. (C) Cell proliferation was evaluated by MTT colorimetric assay. The graph shows data coming from HFD and ND samples. (D) Representative cell cycle analysis of MSCs harvested from HFD- and ND-treated mice. Data are expressed with SD (n = 5) *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Identified pathways in ND- and HFD-MSCs. (A, B) The pictures show the signaling pathways that govern the specific core biological activities (see Table 1) of ND-MSCs and HFD-MSCs, respectively.

Figure 5. Quantitative changes in the protein expression of MSCs following HFD treatment. The volcano plot displays the results of up- and down-regulated proteins in HFD samples compared to ND samples. Panels data from cellular proteome and secretome, respectively.

Figure 6. Overexpressed proteins in HFD-MSCs associated with senescence. The picture shows the overexpressed (in red) and downregulated (in black) proteins in the proteomes and secretomes of HFD-MSCs compared to ND-MSCs. The differences in expression are expressed as LogFC. The column “Upregulated in senescence” indicates the proteins for which expression is upregulated in senescent cells, according to SeneQuest (https://senequest.net) and PubMed (https://pubmed.ncbi.nlm.nih.gov).