## 3 Results  
Lipid Mix Impairs Mitochondrial Respiration and ATP Production in 3T3-L1 Adipocytes, Without Affecting Mitochondrial Integrity  
The first aim of this work was setting an in vitro experimental model of fat tissue cells that could be stimulated to reproduce a pathological condition of mitochondrial dysfunction. 3T3-L1 cells can be differentiated into adipocytes using different hormonal stimuli [51]. After differentiation, cells transform a fibroblast phenotype into a typical adipocyte appearance, and fill their cytoplasm with lipid droplets (Figure S2 in Supplementary Materials). We selected this cell culture model as it is widely used and standardized to study the effects of molecules on adipogenesis, inflammation and metabolism [51; 52; 53]. Moreover, the amino acid sequences for mature mouse and human TGF\(\beta\) are 99-100% identical [54]; therefore, if the cytokine was secreted in 3T3-L1 growth medium, its biological effects could be further investigated in human cells. To experimentally induce mitochondrial dysmetabolism, we used a lipid mix able to generate a metabolic stress by uncoupling the respiratory chain from oxidative phosphorylation. In fact, high concentrations of long-chain free fatty acids can cause uncoupling by a mechanism of fatty acid cycling [55; 56]. We analyzed the mitochondrial metabolic alterations occurring in 3T3-L1 pre-adipocytes and differentiated adipocytes in response to lipid mix treatment. First, the flow cytometric analysis with the JC-1 dye measured the mitochondrial membrane potential, proportional to mitochondrial respiratory activity, and was carried out only in undifferentiated 3T3-L1 cells treated for 48 h, because in differentiated adipocytes the abundance of lipid deposits interfered with the measurement. We observed a decrease in mitochondrial membrane potential when 3T3-L1 cells were treated with lipid mix, compared to untreated control (Figure 1A). This result suggested a possible condition of mitochondrial stress. Next, we found a decrease in ATP levels in the pre-adipocytes and fully differentiated adipocytes treated with lipid mix, compared to the respective untreated controls (Figure 1B). In addition, we detected a slight difference in ATP production between the short 12 h treatment and the longer 48 h treatment with the lipid mix, suggesting a partial protective restoration of ATP levels exerted by cells to avoid cytotoxicity. The results of both these analyses confirmed the reduced mitochondrial activity, demonstrating that a concentrated mixture of lipids could alter mitochondrial respiration, thus proposing a new model in vitro of adipocytes exposed to metabolic stress. Notwithstanding the energy loss, the integrity of mitochondrial compartment was not compromised by the metabolic derangement, as demonstrated by the analysis of intracellular cytochrome C localization. In both models of 3T3-L1 treated with the lipid mix for 48 h, the Western blot analysis revealed that the expression of cytochrome c was unchanged both in the mitochondrial and in the cytoplasmic compartment, demonstrating that the lipid mix did not induce the export of this soluble mitochondrial protein (Figure 1C). Similarly, the total expression of cytochrome C did not change. Original blots are shown in Figure S3 (Supplementary Materials). Altogether, these data suggest that although mitochondrial metabolism was altered by uncoupling agents, mitochondrial structures were not damaged and cells were protected from apoptosis. Figure 1: Lipid mix impairs mitochondrial respiration. (**A**) 3T3-L1 pre-adipocytes were incubated for 48 h with the lipid mix and the mitochondrial membrane potential was evaluated by flow cytometric measurement of the JC-1 dye. The FL2/FL1 ratio was expressed as a percentage of the value obtained  
for untreated cells. (**B**) Pre-adipocytes and 3T3-L1 differentiated adipocytes were incubated with the lipid mix for 12 h and for 48 h and mitochondrial ATP was evaluated relative to the control.