## 3 Results
### Differentiated 3T3-L1 Adipocytes Subjected to Metabolic Stress Produce TGF\(\beta\)  
After finding an effective approach to induce metabolic stress, we decided to verify the production of TGF\(\beta\) in our dysmetabolic cellular model. We treated differentiated adipocytes with lipid mix for 48 h; moreover, we also tested the effect of hypoxia, a well-known promoter of mitochondrial dysfunction, to compare the effect exerted by these two stressors on TGF\(\beta\) production. The secreted cytokine was measured by ELISA assay in the supernatant of treated adipocytes (henceforth referred to as conditioned medium) and found to be highly increased compared to the undetectable levels of control cells. In fact, Figure 2A shows the 8-fold increase of the levels of TGF\(\beta\) secreted in the adipocyte medium under hypoxia conditions (from 0.053 ng/mL to 0.402 ng/mL) and the 19-fold increase of TGF\(\beta\) in the medium of cells treated with the concentrated lipid mix (from 0.053 ng/mL to 1.010 ng/mL). Next, to verify whether TGF\(\beta\) was secreted by adipocytes in its active form, we investigated its efficacy in inducing vitamin D receptor (VDR) expression in pancreatic cancer cells. This property was previously demonstrated in two pancreatic cancer cell lines [29] and in this model was exploited as a readout of TGF\(\beta\) activity. As shown in Figure 2B, the conditioned medium obtained from adipocytes exposed to treatments did not increase VDR expression in human pancreatic cancer cells, when it was used unprocessed. The increased adipocyte secretion of TGF\(\beta\) not accompanied by biological activity led us to suspect the secretion of a latent form of the cytokine, as it is well known that the extracellular activation of latent TGF\(\beta\) must be a major regulatory step controlling TGF\(\beta\) effects [57]. Whereas in vivo the mechanisms activating the pro-form are numerous and not yet fully elucidated (for instance, the acidosis-dependent upregulation of thrombospondin-1 [58] and the cell-associated plasmin protease activity [59]), in vitro it is relatively easy to induce conformational changes of latent TGF\(\beta\), leading to the release of mature and active TGF\(\beta\). For instance, purified latent TGF\(\beta\) can be activated by acidic pH [59], which is a mechanism occurring also in vivo when a similar pH is generated by osteoclasts [57]. We decided to carry out the procedure of subjecting the conditioned medium to strong acidification and subsequent neutralization, which was able to activate TGF\(\beta\), and finally we could detect the expression of VDR as the readout of TGF\(\beta\) activity. Indeed, after the activation of the conditioned medium by acidification, we detected the induced expression of VDR when pancreatic cancer cells were treated with medium from hypoxic adipocytes and most strongly with medium from adipocytes exposed to lipid mix, as shown in Figure 2C. Original blots are displayed in Figure S4 (Supplementary Materials). We could conclude that dysmetabolic adipocytes secreted a latent form of TGF\(\beta\), which was modified and activated by acidification. This in vitro procedure of TGF\(\beta\) activation was previously reported [44; 60]. Since in the 3T3-L1 model we characterized the cellular response to the lipid mix, and because this stimulus is the most powerful inducer of TGF\(\beta\) secretion, in subsequent experiments we further investigated only this metabolic stress trigger. Pancreatic Cancer Cells Exposed to TGF\(\beta\) Secreted by Dysmetabolic Adipocytes Increase Their Respiratory Activity  
The data obtained in our previous work revealed the metabolic impact of TGF\(\beta\) on human pancreatic cancer cells and in particular the dose-dependent sensitivity of mitochondrial activity to cytokine treatment [29]. In fact, we showed that long-term exposure of PANC-1 and CAPAN-2 cells to a low, non-toxic dose of TGF\(\beta\) (2 ng/mL) produced a strong increase in mitochondrial respiratory activity [33].