## 2 In Vitro Models to Study CAC
### Adipose Tissue Loss  
During CAC, the loss of skeletal muscle is, to a lesser extent, accompanied by extensive remodelling and loss of adipose tissue. In fact, it was discovered that lipolysis precedes muscle wasting in some cases, therefore loss of adipose tissue can be considered an overall survival predictor in cachectic patients [70; 71]. Adipose atrophy results in increased lipolysis and fat oxidation, reduced lipogenesis, disrupted lipid accumulation and adipogenesis, as well as browning [2; 23]. In the initial stages of CAC, the white adipose tissue (WAT) representing a major energy reserve and therefore being responsible for lipid storage undergoes a process of transformation in brown adipose tissue (BAT), an active participant in thermogenesis, in a process denominated browning. Both BAT and the skeletal muscle are enriched in mitochondria expressing high levels of uncoupling proteins (UCPs) responsible for shifting the ATP production by the mitochondria to an increase in heat production and consequent lipolysis and energy expenditure [72; 73].  
The preadipocyte cell line 3T3-L1 is originally from the fibroblast cell line 3T3, established from disaggregated 17- to 19-day-old mouse embryos [72]. Over the last few years, the well-characterised cell line has become a suitable proxy model for primary human preadipocytes commonly used to study the WAT, to identify molecular markers, transcription factors and molecular pathways involved in the differentiation of preadipocyte cells (also known as adipogenesis), lipid metabolism, the activity of hormones and assess the potentiality of nutrients and other compounds, particularly those used in the treatment of obesity [74; 75]. More recently, a characterisation of the lineage of 3T3-L1 adipocytes showed that even though the basal bioenergetic expression profile corresponds to white adipocytes, the cells present attributes from both white and brown adipocytes [76]. Under appropriate culture conditions, the fibroblast-like preadipocytes can differentiate and acquire a mature adipocyte-like phenotype [77]. The current and most refined process for differentiation consists of a cocktail of pro-differentiation agents: insulin, dexamethasone and 3-isobutyl-1-methyl-xanthine (IBMX). 3T3-L1 cell differentiation results in a process of sequential expression of specific adipogenic transcription factors, each of them regulated by pro-differentiation agents. The initiation of the adipogenic gene expression program is promoted by dexamethasone which stimulates the expression of CCAAT enhancer binding protein (C/EBP) \(\delta\) and IBMX which stimulates the expression of C/EBP\(\beta\). Both transcription factors heterodimerise activating C/EBP\(\alpha\) and peroxisome proliferatorator-activated receptor (PPAR)\(\gamma\) eventually starting adipogenesis [74; 75; 77]. 3T3-L1 preadipocyte cells present a fibroblast-like morphology, throughout their conversion into adipocytes, cells lose their primitive mesenchymal character and accumulate triglycerides into lipid droplets intracellularly bound to the membrane [78; 79; 77]. Differentiation takes up to 10 to 12 days and the culture lasts until cell culture passage 10. However, with an increasing number of cell passages, the capacity of cells to differentiate decreases [77]. For the study of adipogenesis 3D technique has been applied using a system of single cells suspended in agarose capable of promoting cell differentiation without using a cocktail of agents for induction [80; 81]. Spheroids can also be made using a method of hanging droplets capable of better reproducing the adipocyte-like properties [81].