## 4 Discussion
In addition, many studies have reported the interdependence of HIF-1\(\alpha\) and TGF\(\beta\) signaling [70; 71], and one study demonstrated that hypoxic hepatocytes increased the ratio ofactive TGF\(\beta\) to total TGF\(\beta\) in the culture medium [72]. In this study, for the first time we demonstrated that hypoxia is a metabolic factor capable of stimulating TGF\(\beta\) production in differentiated adipocytes. As a further cause of metabolic stress, we took into consideration the factors that can alter the mitochondrial function, and we focused on the uncoupling properties of fatty acids. The transfer of fatty acids through the mitochondrial membrane can proceed by a flip-flop mechanisms or can be due to a unipotent mediated by anion carriers; in both cases, the consequent release of protons into the mitochondrial matrix (acidification) causes a decrease in the protonmotive force, driving uncoupling from ATP synthesis, and the uncoupling can be observed experimentally upon the fatty acid addition to mitochondria [55; 56]. Uncoupling, like hypoxia, can be considered a stress stimulus that lowers energy levels due to impairment of mitochondrial respiration. Accordingly, the concentrated lipid mix used in this study induced a deregulation of the mitochondrial bioenergetic system of adipocytes, reproducing a condition of metabolic failure. Interestingly, both hypoxia and the fatty acid-driven uncoupling induced TGF\(\beta\) synthesis and secretion. This evidence is a novel and exciting discovery because the influence of mitochondrial dysmetabolism on cytokine production had never been investigated before and sheds a new light on the association between obesity, dysmetabolism and inflammation. In this proof-of-concept study, we established an adipose cell model that could be stressed and forced to produce TGF\(\beta\); however, in vivo, the interaction with the microenvironment is necessary to control TGF\(\beta\) activity. For instance, the activation of TGF\(\beta\) may be regulated locally at the tumor site by proteinases such as plasmin secreted by tumor and stromal cells [59], moreover it has been reported that the interaction with integrins [73] and the extracellular tumoral acidosis [58] are necessary to TGF\(\beta\) maturation. Since our simplified cellular model missed the microenvironment responsible for TGF\(\beta\) activation, it was necessary to evaluate the activity of the secreted cytokine. We chose to analyze the VDR expression as the readout of TGF\(\beta\) activity, based on our previously published observations that synthetic TGF\(\beta\) induces VDR and modulates mitochondrial activity of human pancreatic cancer cells [29]. Interestingly, we realized that in our experimental model TGF\(\beta\) was secreted in its latent form, and a transient acidification was necessary to activate the cytokine. These results are in agreement with the evidence that the tumoral acidic environment is fundamental for the activation of latent TGF\(\beta\), possibly together with other activating mechanisms, and can lead to an enhanced response to the cytokine [60]. The novelty of this study lies in the observation that mitochondrial dysfunction and hypoxia can induce TGF\(\beta\) secretion. Some hypotheses can be formulated to explain the influence of mitochondrial failure and hypoxic stress on TGF\(\beta\) gene transcription. We believe that oxidative stress generated by ROS could be the main mediator of TGF\(\beta\) modulation. ROS are produced by the aberrant function of mitochondrial complexes during hypoxic and metabolic uncoupling stress, and can control TGF\(\beta\) expression [57; 74] and hypoxia-inducible factor HIF-1\(\alpha\) activity [75], which is known to mediate the TGF\(\beta\)-dependent pathways [76]. Indeed, the increased levels of ROS can induce the transcription of TGF-\(\beta\) and stimulate its release and activation [77]. In turn, ROS production is also enhanced by HIF-1\(\alpha\) stabilized in hypoxic condition and by TGF\(\beta\), in a feed-forward loop that supports cancer progression [78].