To maintain a proper lipid composition of cellular membranes is fundamental for cell viability and intracellular signalling. Therefore we hypothesised the existence of membrane quality control homeostatic mechanisms. Previously, we have shown that Pyruvate Carboxylase (PC), an enzyme involved in de novo lipogenesis and fuel partitioning, is a target of PPARγ and that PC level is decreased in PPARγ KO and obese insulin resistant mouse models. Here, we used RNAi knock-down of PC (PC-KD) in 3T3-L1 preadipocytes to elucidate the metabolic consequences associated with the decreased levels of PC in vitro. Our results show that reduced mRNA expression and protein levels of PC (by 90 95%) does not prevent fat accumulation in 3T3-L1 adipocytes. However, using 13C-Nuclear Magnetic Resonance spectroscopy and Gas Chromatography-Mass Spectrometry we found that suppressed levels and activity of PC changes the composition of fatty acid acids being synthesised resulting in increased amount of unsaturated fatty acids (fatty acids with structure 16:1, 18:1, 20:1), and decreased levels of saturated fatty acids (fatty acids with structure 10:0, 14:0, 15:0, 16:0, 22:0, 24:0). This was associated with specific gene expression (qRT PCR) changes of elongase enzymes (Elovl). Moreover, we show that the same pattern of reduction in PC expression and preferential accumulation of unsaturated fatty acids is also observed in adipose tissue of morbidly obese individuals compared to lean individuals (local ethical committee approval had been obtained). Therefore our results support the notion that that under metabolic dysregulation such as seen in obesity and insulin resistance, when there is decreased substrate flux for de novo lipid biosynthesis, adaptive mechanisms do occur to maintain the biosynthesis of unsaturated fatty acids. We speculate that this adaptation may ensure that enough unsaturated fatty acids are available to maintain membrane lipid composition.