Background

Human white adipose tissue (WAT) plays a major role in body energy homeostasis. Adipocytes, specialized cells expressing specific lipid handling metabolic activities, constitute more than 90% of the volume of WAT (Lafontan, 2012). In addition to adipocytes, other cell types are present within human WAT e.g., vascular cells, immune cells (lymphocytes and macrophages) and progenitor cells involved in WAT remodeling and renewal. The metabolic activity of adipocytes is tightly controlled by the integration of both local and systemic pathways. Neurohumoral signals modulated in anabolic or catabolic conditions impact on the net adipocyte metabolic activity, i.e., energy storage or release. In post-prandial conditions, non-esterified fatty acids (NEFAs) originating from the hydrolysis of VLDL and chylomicron particles are taken up by the adipocytes and esterified to glycerol phosphate to form triglycerides packaged into a single lipid vacuole (Large et al., 2004). This process called lipogenesis, is mainly under the control of insulin. In conditions of energy demand such as exercise or fasting, hydrolysis of triacylglycerol through a process called lipolysis results in the release of glycerol and NEFAs into the circulation thereby providing energy to other tissues and organs. Lipolysis involves the sequential hydrolysis of triacylglycerol through the successive action of lipolytic enzymes, i.e., adipose triglyceride lipase, hormone sensitive lipase and monoacylglycerol lipase. In human adipocytes, lipolysis is mainly stimulated by catecholamines and atrial natriuretic peptide (ANP). Catecholamines mediate their pro-lipolytic effects through β-adrenoceptors (β1-AR and β2-AR), while alpha2-adrenoceptors are anti-lipolytic; ANP acts via NPR-A to activate lipolysis (Lafontan, 2012). It should be noted that in human AT the beta3-adrenergic receptor is almost not expressed and is not functional. The classical approach to evaluate adipocyte lipolytic responses was developed by Robdell (Robdell, 1964) who first described mature adipocyte isolation based on flotation after collagenase digestion. Although this technique is used worldwide, it presents several limitations. Firstly, the buoyancy of isolated mature adipocytes will prevent, with increasing time in vitro, their immersion into media and promote direct toxic effects through air contact, ultimately leading to cell damage and disintegration. Secondly, the isolation process per se alters adipocyte phenotype (Ruan et al., 2003). Thirdly, the isolated mature adipocytes are disconnected from their natural microenvironment including extracellular matrix and from other cell types (vascular cells, immune cells (lymphocytes and macrophages) and progenitor cells). Moreover, cytokines such as TNF-alpha and IL6, present in the AT microenvironment, are well described to impact adipocyte lipolysis. Finally, NEFAs originating from in situ lipolysis may have different fates: 1) release into the circulation, 2) re-esterification within the mature adipocytes, 3) potentially taken up by other cell types in the near vicinity of mature adipocytes including progenitor cells. Thus studies on isolated adipocytes do not take into account these different factors.

The present technique allows the study of the lipolytic responsiveness of mature adipocytes for a longer time in their natural context 1) in a closed culture chamber avoiding direct contact with air but with adequate and modulable gas exchange, 2) without the necessity of a collagenase digestion step and 3) in a maintained viable microenvironment. This approach was recently published by our groups and clearly demonstrate that lipolytic stimulation is associated with increased fatty acid uptake by the progenitor cells leading to enhanced adipogenic capacity (Gao et al., 2016).