Adipose tissue lipolysis is an important process in which TAG are mobilized, releasing fatty acids and glycerol and thus providing the body with substrates during fasting. The dysregulation of lipolysis, which can occur in obesity or lipodystrophic states, can lead to elevated levels of fatty acids in the circulation, which is associated with an increased risk for developing cardiovascular diseases and diabetes. The lipolytic machinery is complex and includes as central components perilipin, HSL, and ATGL, in addition to several factors and interacting proteins. Here, we present data suggesting PTRF as a novel physiologically regulated and critical component of the lipolytic machinery.
We found that levels of PTRF expression vary in different adipose tissue depots. Moreover, PTRF expression is under strict hormonal and nutritional control in WAT but not in brown adipose tissue. Although catecholamines and fasting increase PTRF expression, insulin and refeeding decrease its expression. This differential regional expression and regulation of PTRF might contribute to the metabolic heterogeneity observed among different adipose depots (29
). The induction of PTRF expression by fasting and catecholamines in WAT supports with a role of PTRF during lipid mobilization. Of note, PTRF also is expressed in skeletal muscle, another tissue depot that exhibits lipolytic activity. Future experiments will determine whether skeletal muscle PTRF also could be regulated in a similar manner.
Consistent with nutritional and hormonal regulation, manipulation of PTRF expression in cultured cells also changed lipid homeostasis. PTRF overexpression and knockdown resulted in enhancement and suppression of lipolysis, respectively. This is in line with PTRF subcellular location at the lipid droplets and at TAG-synthesizing caveolae subclass.
Lipolysis is an important process in which TAG are mobilized, releasing fatty acids and glycerol and thus providing the body with substrates during fasting. The lipolytic machinery is complex and includes as central components perilipin, HSL, and ATGL in addition to several factors and interacting proteins. Recently, both HSL and perilipin were localized to TAG-synthesizing caveolae subclass (31
). Other lipolytic components, such as the β-adrenergic receptors, as well as PKA also were found to associate with caveolar membranes (32
). It is therefore possible that PTRF, together with other lipolytic proteins, facilitates lipolysis of TAG in lipid droplets as well as in caveolae. This is consistent with the finding that the localization of both PTRF and HSL in caveolae is under insulin control, where insulin, which inhibits lipolysis, induced translocation of both PTRF and HSL from caveolae to the cytosol (16
During fasting, hormones including catecholamines induce lipolysis through binding to β-adrenergic receptors, activating PKA, which phosphorylates perilipin and HSL at multiple sites (5
). PKA-dependent phosphorylation of HSL is necessary for docking of HSL at the surface of lipid droplets and for activation of lipolysis (28
). However, localization of PTRF to caveolae in the plasma membrane is not influenced by β-adrenergic stimulation (16
). Insulin, on the other hand, inhibits lipolysis through activating phosphodiesterase 3B, which causes degradation of cAMP and loss of PKA activation (3
). Our data show that during lipolysis, PTRF also is phosphorylated by PKA at multiple sites and that this PKA-dependent phosphorylation of PTRF plays an essential role during lipolysis. A number of phosphorylation sites in the mouse and human PTRF sequences have been previously identified. However, the functional significance of PTRF phosphorylation has not previously been characterized. Here, we provide evidence for an adipocyte-specific functional role of PTRF phosphorylation, as mutation of PTRF at serine 42, threonine 304, or serine 368 to alanine significantly abrogated the lipolytic response in 3T3-L1 adipocytes.
In addition to phosphorylation, the action of HSL is dependent on its interaction with other proteins such as perilipin. This interaction is necessary for translocation of HSL from the cytosol to the lipid droplets during lipolysis (35
). Furthermore, HSL interacts with the protein lipotransin, which docks HSL at the surface of the lipid droplets (36
). Because PTRF has been shown to interact with HSL (16
), it is possible that PTRF also serves as a bridge between HSL and PKA to mediate phosphorylation and activation of HSL during lipolysis. Indeed, the decrease in lipolysis in cells expressing the PTRF mutants S42A, T304A, and S368A was accompanied by a reduction in serine phosphorylation of HSL. Thus, phosphorylation of PTRF is required for the subsequent phosphorylation of HSL and initiation of lipolysis. It remains to be determined whether the interaction between PTRF and HSL is phosphorylation dependent and whether PKA-phosphorylated PTRF interacts with other lipolytic proteins. It also would be of interest to determine whether PTRF itself had TAG hydrolase activity or if it serves specifically to modulate the activity of other hydrolases such as HSL.
Recent studies (11
) have demonstrated that PTRF is required for the formation of caveolae because knockdown of PTRF leads to loss of caveolae. Loss of caveolae was accompanied with rapid degradation of caveolin-1 protein (11
). It is unlikely that caveolae biogenesis is affected in cells expressing mutated phosphorylation sites of PTRF because no effects were seen on caveolin-1 protein expression in these cells (data not shown). However, it would be interesting to determine whether PTRF phosphorylation has an effect on caveolae morphology and dynamics.
Our study represents the first direct demonstration of nutritional and hormonal control of PTRF expression and phosphorylation in adipose tissue in mouse. We provide evidence for a novel adipose tissue-specific function of PTRF as a critical mediator of lipolysis, which is a central function of the adipocytes.