Lipolysis and β-oxidation

Lipid mobilization is a well-orchestrated processes that mobilizes lipids from its storage form TAG to other lipids that can be used for membrane synthesis, act as signalling molecules or are utilized for oxidative phosphorylation to generate ATP.

In mammals, catecholamines, natriuretic peptides and insulin are considered to represent the major regulators of lipolysis in humans (Lafontan and Langin, 2009).

The activation via the β-adrenergic receptor by catecholamines has been studied extensively (Granneman, 2015, Heier et al., 2016). By binding of e.g. adrenaline this G protein-coupled seven-transmembrane domain receptor (7TM GPCR) becomes activated and leads to the generation of cyclic-adenosine monophosphate (cAMP) by Adenylyl cyclase. cAMP activates Protein Kinase A (PKA) that phosphorylates and activates several enzymes involved in lipolysis (Figure 4) (Berg et al., 2007).

Perilipin1 (PLIN1) a central modulator of lipid storage is localized on lipid droplets under basal conditions and binds to α/β-hydrolase domain containing 5 (ABHD5 / CGI-58). Upon PKA activation both proteins are phosphorylated by PKA (reviewed in (Londos et al., 1999). This leads to the dissociation of ABHD5 to the cytoplasm that there binds to the adipocyte triglyceride lipase (ATGL) and the complex localizes on lipid droplets again (Granneman et al., 2009). In vitro experiments showed, that this interaction is already sufficient to perform the first step in the mobilization of TAGs, the hydrolysis to Diacylglycerides (DAGs) and non-esterified fatty acids (NEFAs) (reviewed in (Oberer et al., 2011). At the same time ABHD5 stimulates through an up to now unknown mechanism the activity of ATGL (Lass et al., 2006).

Additionally, ABHD5 binds to fatty acids binding protein (FABP) (Boeszoermenyi et al., 2015). This enhances the Triacylglyceride hydrolase (TGH) activity of ATGL even more and provides a NEFA acceptor. Moreover, it has been shown that also ATGL is a phosphorylation target of PKA (Ser⁴⁰⁶) and that phosphorylation increases ATGL activity as well (Pagnon et al., 2012). Antagonistically, phosphorylation of ATGL by AMPK lowers the activity of ATGL (Kim et al., 2016).

The next step in lipolysis is the hydrolysis of DAG to Monoacylglyceride (MAG) which is performed by hormone-sensitive lipase (HSL) (Fredrikson et al., 1981, Haemmerle et al., 2002a). Under basal conditions HSL is localized in the cytoplasm but after phosphorylation it trans-locates to lipid droplets and interacts with the activated and now free (not bound to ABHD5) PLIN1 (Tansey et al., 2003). Also HSL has been described to interact with FABP (Jenkins-Kruchten et al., 2003). In the last step of lipid mobilization MAGs are degraded to free glycerol and NEFA by Monacylglycerol-lipase (MAGL). The released glycerol is then transported to the liver and metabolised to pyruvate or used for gluconeogenesis (Berg et al., 2007). The NEFAs now can be used for re-esterification or are utilized for oxidative phosphorylation.

Figure 4 Schematic overview of mammalian lipid mobilization for ATP regeneration. β-adrenergic signalling leads to elevated cAMP levels that activate PKA. Phosphorylation of PLIN1 leads to the release of ABHD5 that can interact with ATGL and FABP to catalyse the first step in lipid mobilization the hydrolysis of TAGs. PLIN1 and phosphorylated HSL can now interact that leads to trans-location of HSL the main DAG lipase from the cytoplasm to lipid droplets. In the last step the Monoacylglycerol

(MAG) generated by HSL is hydrolysed by MAGL. The released fatty acids from storage lipid mobilization are activated by Acyl/CoA-synthetase (ACS) and subsequently broken down in peroxisomes and mitochondria. All longer NEFAs (>C8) are transported into mitochondria via the carnitine-shuttle. The rate limiting step in the transport is catalysed by Acyl(palmitoyl)-transferase I (CPTI). The end-product of β-oxidation Acetyl-CoA can be utilized in the tri-carbonic acid cycle (TCA) to produce electron donors for oxidative phosphorylation (OXPHOS) to finally generate ATP. Peroxisome proliferator-activated receptor α (PPARα) can sense NEFAs generated by ATGL and improve cellular substrate oxidation and respiration. Alternatively, this can be simulated by activated p-cAMP responsive element binding protein (CREB) or nuclear factor of activated T-cells (NFAT).

For the later NEFAs need to be activated, a process that actually needs energy in form of ATP. At the outer membrane of mitochondria, the NEFAs are bound to Coenzyme A by Acyl-CoA-synthetase (ACS) under the consumption of ATP. This two-step reaction is coupled with inorganic pyro-phosphatase cleaving the liberated pyrophosphate from the ATP into two separate phosphate ions consuming one molecule of water (Berg et al., 2007). This shifts the reaction of NEFA to Acyl-CoA towards it end-product and makes it irreversible. Subsequently, Acyl-CoA is transported into the mitochondrial matrix by utilizing carnitine-shuttle. The activated NEFAs are conjugated to the zwitterion carnitine by carnitine acyl(palmitoyl)-transferase I (CPT1) that is located on the outer mitochondrial membrane as well.

After that the acylated carnitine is shuttled to the inner mitochondrial membrane by a translocase by a simultaneous transport of one carnitine molecule to the outer side again. Arrived at the inner site the acyl carnitine is de-acylated by CPT2 (Berg et al., 2007). Especially, the acylation of carnitine by CPT1 is rate limiting and tightly regulated as CPT1 is inhibited allosterically by Malonyl-CoA that is generated by Acetyl-CoA carboxylase (ACC) from Acetyl-CoA. As Malonyl-CoA is an intermediate of fatty acid synthesis, high amounts inhibit beta-oxidation in mitochondria and boost lipogenesis. Particularly, medium chain fatty acids (MCFAs) depend on the Carnitine shuttle to be available for β-oxidation and subsequently oxidative phosphorylation (Figure 5). However, various ACSs exist with different acyl-chain length specificities and cellular localization (Faust et al., 2014). This mechanism is used in the hypothalamus in order to regulate food intake and glucose production (Lam et al., 2005) (Figure 5).

Figure 5 Schematic overview of lipid breakdown for ATP synthesis and its regulation. Chemical reactions that happen during β-oxidation in mitochondria are shown in A. Peroxisome β-oxidation only differs in the first step lipid oxidation as shown in the box in A (modified after Faust et al.2012; red (best hit) and blue are predicted Drosophila genes coding for the depicted enzyme of this reaction). An electron transport chain over NADH-coenzyme Q oxidoreductase (complex I), Succinate-Q oxidoreductase (complex II), Q-cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV) generates a proton gradient by transporting H⁺ into the interluminal space of mitochondria. The stored energy in this gradient is used by ATP-Synthase to generate ATP (B). In the hypothalamus the generation of “activated” long-chain fatty acids (LCFA-CoA) lead to signalling for decreased food intake and glucose production, shown in C. Signalling by like insulin or leptin over AMPK lead to increased Malonyl-CoA amounts that block fatty acid transport into mitochondria by blocking CPT1 that enhances LCFA-CoA accumulation in the hypothalamus (picture C from (Aguilera et al., 2008))

Most of the NEFAs (90%) are directed to mitochondria for oxidative phosphorylation (OXPHOS). However, especially very-long chain fatty acids (VLCFAs) and poly-unsaturated fatty acids (acyl residues have numerous C=C double bonds) are processed in a different cell organelle the peroxisomes. These organelles have certain characteristics. The β-oxidation in peroxisomes terminates at Octanoyl-CoA and in the first reaction of β-oxidation the dehydrogenation of Acyl-CoA the Flavoprotein-dehydrogenase transfers electrons to oxygen and thereby generates H2O2 (Figure 5).

Subsequently, hydrogenperoxide is broken down to oxygen and water by catalase. In contrast, the electrons are fixed inFlavin adenine dinucleotide hydrochinone (FADH2)

in mitochondria. The following steps in β-oxidation take place as in mitochondria but are performed by different isoforms of the proteins (Berg et al., 2007).