Measurement of lipolysis

Abstract

Lipolysis is defined as the hydrolytic cleavage of ester bonds in triglycerides (TGs), resulting in the generation of fatty acids (FAs) and glycerol. The two major TG pools in the body of vertebrates comprise intracellular TGs and plasma/nutritional TGs. Accordingly, this leads to the discrimination between intracellular and intravascular/gastrointestinal lipolysis, respectively. This chapter focuses exclusively on intracellular lipolysis, referred to as lipolysis herein. The lipolytic cleavage of TGs occurs in essentially all cells and tissues of the body. In all of them, the resulting FAs are utilized endogenously for energy production or biosynthetic pathways with one exception, white adipose tissue (WAT). WAT releases FAs and glycerol to supply nonadipose tissues at times of nutrient deprivation. The fundamental role of lipolysis in lipid and energy homeostasis requires the accurate measurement of lipase activities and lipolytic rates. The recent discovery of new enzymes and regulators that mediate the hydrolysis of TG has made these measurements more complex. Here, we describe detailed methodology for how to measure lipolysis and specific enzymes' activities in cells, organs, and their respective extracts.

Keywords: ATGL; Adipose tissue; HSL; Lipase; Lipid droplet; Lipolysis; MGL; Triglyceride hydrolase activity.

Figure 10.1. Coordinated breakdown of triglycerides in the course of lipolysis.

Figure 10.2. Intracellular localization of lipases and regulatory proteins in the basal/non-lipolytic-stimulated state and upon lipolytic stimulation.

In the basal/non-lipolytic-stimulated state, CGI-58 is bound to perilipin-1 and unavailable for ATGL binding/ activation. HSL resides in the cytosol. β-Adrenergic stimulation leads to the phosphorylation of HSL and perilipin-1, which causes HSL to translocate onto the LD. ATGL also translocates from the cytosol to the LD surface. In both lipolytic states, G0S2 is localized on LD, bound to ATGL. In addition, phosphorylated perilipin-1 releases CGI-58, which is now available for ATGL binding and activation. MGL is localized on the LD, the cytosol, and the ER independent of the metabolic state of the cell.

Figure 10.3. The fate of free fatty acids (FA) and glycerol (G) generated during intracellular lipolysis.

FAs generated by the activity of ATGL, HSL, and MGL are either released into the circulation or activated by acyl-CoA synthetases (ACS) to FA-CoA. These FA-CoAs serve as building blocks for the synthesis of MG, DG, TG, and phospholipids (PL), by the activities of MGAT (monoacylglycerol-acyltransferase), DGAT (diacylglycerol-acyltransferase), or CPT (cholinephosphotransferase). Glycerol (G) is recycled by GK (glycerol kinase), GPAT (glycerolphosphate-acyltransferase), AGPAT (acylglycerolphosphate-acyltransferase), and lipin to DGs.

Figure 10.4. pH dependence of CGI-58-coactivated ATGL hydrolysis of TG.

COS-7 cell lysates containing recombinant CGI-58 and ATGL were incubated with 0.32 mM triolein as substrate and ³H-triolein as tracer in 100 mM potassium phosphate buffer of various pH (6–8). Substrates were emulsified in phospholipids (PC/PI, 3:1, w/w) containing 5% BSA (FA-free). After incubation, FAs were extracted and radioactivity was determined by scintillation counting. Data are means ± standard deviation of three replicates.

Figure 10.5. TG hydrolase activities of HSL and ATGL using different detergents for substrate preparation.

COS-7 cell lysates containing recombinant HSL (A), ATGL (B), or ATGL and CGI-58 (C) were subjected to TG hydrolase assay using 0.32 mM triolein as substrate and ³H-triolein as tracer. As control, lysates containing recombinant β-galactosidase (LacZ) were used. Substrates were prepared in 100 mM potassium phosphate buffer, pH 7.0, containing 5% BSA (FA-free). For stabilization 45 μM PC/PI (3:1, w/w), 20 mM cholate, 2% glycerol, or 0.025% Triton X-100 were used. After incubation, FAs were extracted and radioactivity was determined by scintillation counting. Data are means ± standard deviation of three replicates.

Figure 10.6. Time course of basal and stimulated lipolysis of adipocytes.

Differentiated 3T3-L1 adipocytes were incubated in DMEM containing 2% BSA (FA-free) in the absence (basal) and presence (stimulated) of isoproterenol (10 μM). After various times, the FA content of incubation media was determined and normalized to cellular protein content. Data are means ± standard deviation of three replicates.