Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylum-belliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets.
"The amount of perilipin A detected in the floating fat-cake fraction was similar between WT and PRIP-DKO mice under fed and fasting conditions (Fig. 2B, D). It was reported that phosphorylation of perilipin A at Ser492 is required for maximal lipolysis and triggers a massive remodeling of lipid droplets that increases the surface area of lipid droplets available to lipases , . Therefore, phosphorylation of perilipin A Ser492 was examined using a phosphospecific antibody; the results showed that PRIP-DKO mice exhibited higher phosphorylation of perilipin A Ser492 in adipose tissues under both fed and fasting conditions (Fig. 2B, D), indicating that more phosphorylated (active) perilipin A was present on lipid droplets in PRIP-DKO adipocytes. "
[Show abstract][Hide abstract] ABSTRACT: Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP-KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis.
PLoS ONE 06/2014; 9(6):e100559. DOI:10.1371/journal.pone.0100559 · 3.23 Impact Factor
"This process leads to the formation of the single LD in mature adipocytes.68 Conversely, TG hydrolysis induces LD fragmentation and shrinking69,70 with a rapid formation of small and micro LDs (<1 μm) in adipocytes.71–73 Although, both small and micro LD originate during lipolysis, small LDs would be generated from the fission of large LDs, whereas micro LDs would be formed with TG produced by the re-esterification of FAs.72 Micro LDs and small LDs are considered physiologically relevant in the response to energy demands.72,73 "
[Show abstract][Hide abstract] ABSTRACT: The main cells of the adipose tissue of animals, adipocytes, are characterized by the presence of large cytosolic lipid droplets (LDs), which store triglyceride (TG) and cholesterol. However, most cells have LDs and the ability to store lipids. LDs have a well-known central role in storage and provision of fatty acids and cholesterol. However, the complexity of the regulation of lipid metabolism on the surface of the LDs is still a matter of intense study. Beyond this role, a number of recent studies have suggested that LDs have major functions in other cellular processes, such as protein storage and degradation, and infection and immunity. Thus, our perception of LDs, from simple globules of fat to highly dynamic organelles of unexpected complexity, has been radically transformed. Here we compiled some recent evidence supporting the emerging view that LDs act as platforms connecting a number of relevant metabolic and cellular functions.
"Perilipin 1 itself is phosphorylated during this event, and mutation of one single phosphorylation site within perilipin 1, serine 492, is sufficient to prevent the declustering of LDs upon lipolytic stimulation . Yet, direct evidence that phosphorylation of perilipin 1 at serine 492 is sufficient to drive the dispersion of LDs is lacking, since substitution of serine 492 for negatively charged glutamic acid, mimicking the phosphorylated state of serine 492, is not sufficient to drive the dispersion of LD clusters . Regarding the driving force of the process, a recent study suggested that levels of free fatty acids are a major regulator of the LD remodeling during lipogenesis and lipolysis . "
[Show abstract][Hide abstract] ABSTRACT: Lipid droplets, the intracellular storage organelles for neutral lipids, exist in a wide range of sizes and of morphologically distinct organization, from loosely dispersed lipid droplets to tightly packed lipid droplet clusters. We show that the lipid droplet protein AUP1 induces cluster formation. A fraction of AUP1 is monoubiquitinated at various lysine residues. This process depends on its internal CUE domain, which is a known ubiquitin-binding domain. AUP1 with a deleted or point mutagenized CUE domain, as well as a lysine-free mutant, are not ubiquitinated and do not induce lipid droplet clustering. When such ubiquitination deficient mutants are fused to ubiquitin, clustering is restored. AUP1 mutants with defective droplet targeting fail to induce clustering. Also, another lipid droplet protein, NSDHL, with a fused ubiquitin does not induce clustering. The data indicate that monoubiquitinated AUP1 on the lipid droplet surface specifically induces clustering, and suggest a homophilic interaction with a second AUP1 molecule or a heterophilic interaction with another ubiquitin-binding protein.
PLoS ONE 09/2013; 8(9):e72453. DOI:10.1371/journal.pone.0072453 · 3.23 Impact Factor
M.D. Cordero, E. Alcocer-Gómez, L. Román Malo, N. Casas-Barquero, P. Bullón, A.M. Carrion
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.