Lipolysis in white adipose tissue: enzymes, mechanisms, and regulation

Lipolysis is the biochemical process responsible for the hydrolysis of triacylglycerols, commonly known as triglycerides (TAG), into free fatty acids and glycerol, a reaction catalyzed by enzymes known as lipases.

Its biological significance stems from the inability of triglycerides to independently cross cell membranes, necessitating their breakdown for energy utilization or transport to various tissues.

In vertebrates, this process is vital in three distinct contexts: gastrointestinal, vascular, and intracellular lipolysis.

Gastrointestinal lipolysis, mediated by lingual, gastric, and pancreatic lipases, allows for the catabolism and absorption of dietary triglycerides.
Vascular lipolysis, facilitated by lipoprotein lipase (LPL) within the capillary bed, enables the hydrolysis of triglycerides associated with plasma lipoproteins.
Intracellular lipolysis is responsible for the mobilization of triglycerides stored within intracellular lipid droplets.

While gastrointestinal and vascular lipolysis are essential for nutrient transport between organs, intracellular lipolysis serves as the cornerstone of cellular energy metabolism, finely regulated in response to changes in nutritional and hormonal status.

Contents

• Intracellular lipases
• ATGL, HSL, MGL and lipolysis
  • Adipose triglyceride lipase
    • Catalytic activity
    • CGI-58
    • G0S2
    • PEDF
  • Hormone-sensitive lipase
    • Catalytic activity
    • Molecular architecture
  • Monoacylglycerol lipase
    • Catalytic activity
• Hormonal regulation in white adipose tissue
  • β-Adrenergic stimulation
    • ATGL and β-adrenergic stimulation
    • HSL and β-adrenergic stimulation
  • Lipolysis inhibition by insulin
• Non-hormonal regulation in white adipose tissue
• ATGL-mediated lipolysis in non-adipose tissues
  • Role of perilipins
• References

Intracellular lipases

Intracellular hydrolysis of triglycerides involves two types of lipases: acid and neutral.

The acidic lipases have a pH optimum between 4 and 5.
They are the most important acid triacylglycerol hydrolases in lysosomes, and can hydrolyze cholesterol esters as well. These enzymes are thought to act primarily on lipoprotein-associated lipids, following their receptor-mediated endocytosis and sorting to lysosomes. However, their action is also associated with macroautophagy, a lysosomal pathway that catabolizes cytoplasmic inclusions such as aggregates of misfolded proteins, as well as damaged and superfluous organelles, releasing into the cytosol the hydrolysis products.

The cytosolic neutral lipases, with a pH optimum around 7.
They include:

• adipose acylglycerol lipase (EC 3.1.1.3);
• hormone-sensitive lipase (EC 3.1.1.79);
• monoacylglycerol lipase (EC 3.1.1.23).

In the remainder of this article, we will analyze the intracellular lipolysis by the aforementioned neutral lipases and its hormonal and non-hormonal regulation, with particular attention to white adipose tissue.

ATGL, HSL, MGL and lipolysis

Fatty acids deposited in the white adipose tissue as triacylglycerols represent the largest energy store in higher eukaryotes.

When energy demand increases, such as during intense and prolonged physical activities, triacylglycerol hydrolysis occurs and fatty acids are released into the blood.

In adipose tissue the sequential action of these three enzymes leads to complete hydrolysis of triacylglycerols. In this process, both in vivo and in cultured adipocytes, ATGL and HSL account for more than 90% of the lipolytic activity.

Enzymes involved in intracellular lipolysis, their chromosomal location, molecular domains, substrates, and regulatory mechanisms.

Enzyme	Chromosomal location	Key molecular domains	Primary substrate	Main regulators
ATGL	11p15.5	Patatin domain (N-terminal) and hydrophobic C-terminal region	Triacylglycerols	Activated by CGI-58; inhibited by G0S2
HSL	19q13.2	α/β-hydrolase catalytic domain and regulatory domain (phosphorylation sites)	Diacylglycerols	Activated by PKA and translocation induced by phosphorylated perilipin-1
MGL	3q21.3	α/β-hydrolase fold and flexible hydrophobic lid	Monoacylglycerols	Constitutively active (not regulated by PKA or lipolytic hormones)

Adipose triglyceride lipase

Of the three aforementioned neutral lipases, ATGL is the most recently discovered.

Its central role in triacylglycerol catabolism became clear after studies conducted on mutant mice lacking the enzyme, and on humans with mutations in the gene encoding it. In humans, for example, a systemic triacylglycerol accumulation, and therefore of body fat, and cardiomyopathy have been observed. This clearly indicates that ATGL activity is required for efficient mobilization of triacylglycerols in adipose and non-adipose tissues.
In fasted mutant mice lacking the enzyme, the lack of non-esterified fatty acids causes a high glucose consumption for energy purposes, and hypoglycaemia, hypometabolism, and hypothermia occur after fasting for more than 6 hours.

The human enzyme, encoded by a gene on chromosome 11p15.5, belongs to the family of proteins containing a patatin domain, which includes 8 murine and 9 human members. The patatin domain is present in the N-terminal half of the protein and also contains the active site of the enzyme. Conversely, the C-terminal half has primarily a regulatory function, and also includes the hydrophobic region responsible for the binding of lipid droplets, essential for in vivo enzyme activity.

Orthologous enzymes are present in essentially all eukaryotes, including invertebrates, vertebrates, as well as fungi and plants.

Enzyme activity is subject to regulation by interacting with activating and inhibitory proteins, some of which are localized on lipid droplets.

Catalytic activity

The enzyme catalyzes the first step in triacylglycerol hydrolysis, leading to the formation of diacylglycerols (DAG) or diglycerides, and fatty acids:

Triacylglycerol + H₂O → Diacylglycerol + Fatty acid

ATGL preferentially hydrolyzes sn-2 ester bonds, but as a consequence of the interaction with CGI-58, its selectivity broadens to the sn-1 bond.

Compared to the activity towards triacylglycerols, the enzyme exhibits minor or no catalytic activity towards monoacylglycerols (MAG) or monoglycerides, diacylglycerols, cholesterol esters, and retinol esters.

CGI-58

Like pancreatic lipase and LPL, which are much more active in the presence of protein coactivators, ATGL catalytic activity is increased by the activator protein comparative gene identification-58 (CGI-58), which therefore stimulates the first step of intracellular lipolysis.

It is a highly conserved protein among species, encoded in humans by a gene on chromosome 3p21. It interacts with the patatin domain of ATGL. The maximal stimulation occurs at approximately equimolar concentrations of the two proteins.

The importance of its stimulatory action is underlined by the fact that its deficiency or malfunctioning results in a severe systemic accumulation of triacylglycerols both in men and mice.

CGI-58 is regulated primarily by its interaction with perilipin-1, a protein that covers lipid droplets. CGI-58, at least in vitro, also acts as an acyl-CoA-dependent acylglycerol-3-phosphate acyltransferase.

G0S2

It is an inhibitor of ATGL, originally identified in mononuclear blood cells, where it acts at the G0 to G1 transition of the cell cycle, and therefore called G0/G1 switch protein 2 (G0S2).

In humans, it is encoded by a gene on chromosome 1q32.2.

It is present in many tissues, with the highest levels in adipose tissue and liver, followed by ovary, muscle, and kidney. It is found in different cellular compartments, such as cytoplasm, mitochondria, endoplasmic reticulum, and lipid droplets. These different cellular locations may reflect the different functions that the protein performs, such as the regulation of:

• lipolysis;
• cell cycle;
• possibly apoptosis, through its ability to interact with Bcl2, a mitochondrial antiapoptotic factor.

G0S2, like CGI-58, interacts with the patatin domain of ATGL, and, at least in vitro, lipid droplet binding and enzyme inhibition depend on physical interaction between the N-terminal region of G0S2 and the patatin domain of the enzyme. However, it does not seem that this interaction directly competes with the binding of the activator CGI-58.

PEDF

Another protein involved in the regulation of ATGL activity seems to be pigment epithelium derived factor (PEDF), which induces the hydrolysis of triacylglycerols in adipose tissue, liver, and muscle via lipase activity.

It is a widely expressed protein, belonging to the noninhibitory Serpin family, and with a large spectrum of activities, such as anti-inflammatory, antioxidative, neuroprotective, antitumorigenic, and antiangiogenic effects. The protein binds to the enzyme and activates it. Its activity may be involved in the development of hepatic steatosis and the pathogenesis of insulin resistance.

Finally, it seems that ATGL delivery to lipid droplets requires vesicular transport. In fact, its translocation to the lipid droplets is blocked in the absence of Sar1, ARF1 or GBF1, which are protein components of the transport machinery; and the enzyme remains associated with the endoplasmic reticulum, from which lipid droplets are believed to bud off.

Hormone-sensitive lipase

In the early sixties of last century it was noted that lipolytic activity in adipose tissue was induced by hormones. In 1964, both hormone-sensitive lipase and monoacylglycerol lipase of adipose tissue were isolated and characterized.

HSL is encoded by a gene on chromosome 19q13.2. Alternative splicing leads to significant variations in the 5′ region of the transcripts, and therefore tissue-specific mRNAs and proteins of different sizes.

The enzyme expression profile is essentially similar to that of ATGL. The highest abundance of mRNA and protein is found in white and brown adipose tissue; in many other tissues and cells, including muscle, pancreatic β-cells, steroidogenic cells, and macrophages, HSL gene expression is low.

Unlike adipose triglyceride lipase, which has orthologous enzymes in all eukaryotes, hormone-sensitive lipase is less ubiquitous; for example, no orthologous proteins are known in birds, in D. melanogaster, C. elegans, and S. cerevisiae.

Finally, unlike ATGL, no mutations in HSL gene have been observed in humans.

Catalytic activity

HSL is more efficient as a diacylglycerol hydrolase than triacylglycerol hydrolase, in vitro by a factor of 10-fold. This suggested that HLS was was the rate-limiting enzyme in the catabolism of triacylglycerols in adipose and many other tissues. Many years later, in 2000, it was however observed that enzyme-deficient mice showed no signs of triacylglycerol accumulation in adipose and other tissues, whereas they accumulated large quantities of diacylglycerols in many tissues. This suggested that HSL was more important as diacylglycerol hydrolase than as triacylglycerol hydrolase, which is now commonly accepted.

Diacylglycerol + H₂O → Monoacylglycerol + Fatty acid

However, it is a multifunctional enzyme able to hydrolyze, in addition to diacylglycerols in which it has a stereo-preference for sn-3 ester bonds, and triacylglycerols, in which it preferentially hydrolyses sn-1 ester bonds, also the ester bonds of other lipids, such as monoacylglycerols, retinyl esters, and cholesteryl esters.

Molecular architecture

Three main domains have been identified in the structure of the protein: an N-terminal, a C-terminal, and a regulatory module.

Mediating enzyme dimerization, lipid binding, and interaction with FABP4, a protein that increases HSL catalytic activity, is the primary role of the N-terminal domain.

Contained within the C-terminal domain is a structural fold common to many esterases and lipases, known as the α/β hydrolase fold. This region houses the classical catalytic triad of human hydrolases, Ser⁴²⁴, Asp⁶⁹³, and His⁷², which constitutes the active site.

The third region is the regulatory module of HSL. It is located within the catalytic domain and contains at least five phosphorylation sites, on as many serine residues, two of which, Ser⁶⁵⁰ and Ser⁶⁶³, seem to be particularly important for its activity.

Monoacylglycerol lipase

Monoacylglycerol lipase is considered the rate-limiting enzyme for the catabolism of monoacylglycerols resulting from the hydrolysis of triglycerides, both lipoprotein and intracellular, and of phospholipids.

The enzyme is encoded by a gene on chromosome 3q21.3, and is ubiquitously expressed, with highest expression in adipose tissue; however, high expression levels are also found in hepatocytes and muscle cells.

The enzyme is localized on lipid droplets, cell membranes, and in cytoplasm.

Unlike ATGL and HSL, neither the mRNA concentration nor the catalytic activity of MGL appears to be regulated hormonally or by cellular energy charge. The protein shares structural homologies with esterases, lysophospholipases, and haloperoxidases.

Catalytic activity

The enzyme catalyzes the hydrolysis of monoacylglycerols into fatty acids and glycerol:

Monoacylglycerol + H₂O → Fatty acid + Glycerol

The importance of its action has been confirmed by studies conducted on mutant mice: its deficiency compromises lipolysis, leading to an accumulation of monoacylglycerols in both adipose and non-adipose tissues.

However, since other enzymes, such as HSL and ABHD6, possess monoacylglycerol hydrolase activity, it is not yet entirely clear whether in vivo the activity of MGL is the only one relevant to complete the lipolytic process.

Hormonal regulation in white adipose tissue

In white adipose tissue, triacylglycerol hydrolysis on the surface of lipid droplets is mainly regulated post-translationally by phosphorylations, protein-protein interactions, and translocations of the participating proteins.

β-Adrenergic stimulation

Adrenaline and glucagon, as a result of binding to specific G-protein coupled receptors (such as β-adrenergic receptors for adrenaline), activate ATGL and HSL, which hydrolyze triacylglycerols in a coordinated manner.

A central role in the process leading to enzyme activation is performed by perilipin-1, a lipid-droplet associated protein found only in cells which can be β-adrenergically stimulated. As a result of such stimulation, perilipin-1 is phosphorylated on six serine residues by protein kinase A (PKA).
β-Adrenergic stimulation, like fasting, has little effect on the expression of CGI-58 in adipose tissue.

ATGL and β-adrenergic stimulation

In unstimulated adipocytes, CGI-58 is localized on the surface of lipid droplets, mostly bound to perilipin-1, and does not interact with ATGL. In this state, ATGL activity, and therefore lipolysis, are low.
Phosphorylation of perilipin-1 by PKA leads to the release of CGI-58 which in turn, in a poorly understood manner, activates ATGL.
Even ATGL can be phosphorylated on at least two serine residues, Ser⁴⁰⁴ and Ser⁴²⁸, but not by PKA. However, the importance of these phosphorylations in the regulation of the enzyme activity is not yet clear.

HSL and β-adrenergic stimulation

In adipose tissue, its activity is strongly induced by the stimulation of β-adrenergic receptors.
Phosphorylation increases moderately, by about two-fold, enzyme activity. For complete activation, HSL must gain access to lipid droplets, a process mediated by phosphorylated perilipin-1: in the basal state, in fact, the protein is unphosphorylated and prevents HSL binding to lipid droplets, whereas phosphorylation allows its binding to the N-terminal region of the protein, granting access to the droplets.
Therefore, β-adrenergic stimulation, via HSL phosphorylation and translocation to the lipid droplet surface, leads to an increased activity of the enzyme in white adipose tissue of about 100-fold.
Other kinases, such as AMPK or Ca²⁺/calmodulin-dependent kinase also phosphorylate the enzyme to modulate its activity.

In summary, β-adrenergic stimulation coordinates the release of CGI-58, to activate ATGL, and the translocation of HSL onto lipid droplets, ensuring efficient and sequential mobilization of fatty acids.

Lipolysis inhibition by insulin

Insulin mediates the deactivation of lipolysis by acting at the transcriptional level, on the activity of ATGL and HSL, and via sympathetic nervous system.

• Transcriptional regulation. The hormone causes the down-regulation of ATGL and HSL gene expression and stimulates the expression of the inhibitory protein G0S2, which contributes to curbing enzymatic activity.
• Enzymatic control. Through the activation of PKB/AKT, insulin promotes cAMP hydrolysis via phosphodiesterases. The resulting inactivation of PKA blocks the phosphorylation of HSL and perilipin-1, preventing the enzymes from accessing the lipid droplets.
• Central mechanism. Insulin also acts on the central nervous system, inhibiting the release of signals from the sympathetic nervous system; this indirectly reduces the phosphorylation of HSL and perilipin-1 in peripheral tissues.

Non-hormonal regulation in white adipose tissue

Non-hormonal factors can also play a role in the regulation of triacylglycerol hydrolysis.

Recent works have shown an inhibitory effect of long chain acyl-CoAs, such as palmitoyl-CoA and oleoyl-CoA, on ATGL activity.
Note: palmitic acid and oleic acid are two highly abundant fatty acids in white adipose tissue.
Similarly to what is described for HSL, long chain acyl-CoAs act as non-competitive inhibitors. This inhibition may be an effective feedback mechanism for the control of triacylglycerol hydrolysis and the protection of the cells from lipotoxic concentrations of molecules such as diacylglycerol, acyl-CoAs, and ceramides.

Receptor-interacting protein 140 (RIP-140), as a result of an increase in cellular lipid content, induces lipolysis by binding to perilipin-1 on lipid droplets. Through this interaction, perilipin-1 more efficiently recruits HSL to lipid droplets and enhances complex formation between ATGL and its coactivator CGI-58.

ATGL-mediated lipolysis in non-adipose tissues

ATGL and HSL are also expressed in most non-adipose tissues, although at significantly lower levels. This has raised the question whether the intervention of other lipases occurs to ensure efficient lipolysis.
For example, in the liver of fasted mice, ATGL accounts for less than 50% of neutral hydrolysis of triacylglycerols, an activity that is, however, important because its absence leads to the development of hepatosteatosis. But even in the absence of ATGL, a good activity on triacylglycerols is retained in the hepatocytes. This ensures no apparent defect in VLDL production, whose assembly and secretion requires a considerable mobilization of hepatic triacylglycerol stores.

Role of perilipins

In non-adipose tissues, such as skeletal muscle, heart, and liver, ATGL-mediated lipolysis follows a different mechanism.
Perilipin-5 replaces perilipin-1.

During fasting, it recruits both CGI-58 and ATGL to lipid droplets by direct binding of the two proteins. The role of perilipin-5 in this complex is not yet clear, but it seems to be involved in the interaction of lipid droplets with mitochondria and the inhibition of ATGL-mediated triacylglycerol hydrolysis.

In skeletal muscle, hormone-sensitive lipase is activated by phosphorylation in response to muscle contraction and adrenaline. In non-adipose tissues lacking perilipin-1, the role of HSL is less well characterized and the enzyme expression is low. The interaction between HSL and lipid droplets may involve perilipin-2 and perilipin-5.

References

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Domande Frequenti

What is the function of ATGL in intracellular lipolysis?

Adipose triglyceride lipase initiates intracellular lipolysis by hydrolyzing the first ester bond of triglycerides to generate diacylglycerols and a free fatty acid. It is the rate-limiting enzyme for lipid mobilization and is strictly regulated by CGI-58.

How does insulin inhibit intracellular lipid breakdown?

Insulin stops lipolysis by promoting cAMP hydrolysis via phosphodiesterases. This deactivates protein kinase A (PKA), blocking the phosphorylation of HSL and perilipin-1, thereby preventing enzyme access to the core of lipid droplets.

What are the main differences between perilipin-1 and perilipin-5?

Perilipin-1 is mainly expressed in white adipose tissue and controls hormone-induced lipolysis. Perilipin-5 is instead expressed in highly oxidative tissues like skeletal muscle and the heart, where it mediates metabolic interactions between droplets and mitochondria.