6 results on '"Greenberg, Andrew S."'
Search Results
2. AMP-activated Protein Kinase Is Activated as a Consequence of Lipolysis in the Adipocyte POTENTIAL MECHANISM AND PHYSIOLOGICAL RELEVANCE.
- Author
-
Gauthier, Marie-Soleil, Miyoshi, Hideaki, Souza, Sandra C., Cacicedo, José M., Saha, Asish K., Greenberg, Andrew S., and Ruderman, Neil B.
- Subjects
- *
PROTEIN kinases , *LIPOLYSIS , *FAT cells , *RNA , *OXIDATIVE stress - Abstract
AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its phys- iological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with cAMP-inducing agents (iso- proterenol, forskolin, and isobutylinethyixanthine), which stimu- late lipolysis and activate AMPK. When lipolysis was partially inhibited with the general lipase inhibitor orlistat, AMPK activa- tion by these agents was also partially reduced, but the increases in cAMP levels and cAMP-dependent protein kinase (PKA) activity were unaffected. Likewise, small hairpin RNA-mediated silencing of adipose tissue triglyceride lipase inhibited both forskolin-stim- ulated lipolysis and AMPK activation but not that of PKA. Forsko- lin treatment increased the AMP:ATP ratio, and this too was reduced by orlistat. When acyl-CoA synthetase, which catalyzes the conversion of fatty acids to fatty acyl-CoA, was inhibited with triacsin C, the increases in both AMPK activity and AMP:ATP ratio were blunted. Isoproterenol-stimulated lipolysis was accompanied by an increase in oxidative stress, an effect that was quintupled in cells incubated with the AMPK inhibitor compound C. The isopro- terenol-induced increase in the AMP:ATP ratio was also much greater in these cells. In conclusion, the results indicate that activa- tion of AMPK in adipocytes by cAMP-inducing agents is a conse- quence of lipolysis and not of PICA activation. They suggest that AMPK activation in this setting is caused by an increase in the AMP:ATP ratio that appears to be due, at least in part, to the acy- lation of fatty acids. Finally, this AMPK activation appears to restrain the energy depletion and oxidative stress caused by lipolysis. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
3. Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes.
- Author
-
Granneman, James G., Moore, Hsiao-Ping H., Granneman, Rachel L., Greenberg, Andrew S., Obin, Martin S., and Zhengxian Zhu
- Subjects
- *
LIPOLYSIS , *PROTEIN-protein interactions , *FAT cells , *PROTEIN kinases , *BIOCHEMISTRY - Abstract
This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormone-sensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Pun and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
4. Control of Adipose Triglyceride Lipase Action by Serine 517 of Perilipin A Globally Regulates Protein Kinase A-stimulated Lipolysis in Adipocytes.
- Author
-
Miyoshi, Hideaki, Perfield II, James W., Souza, Sandra C., Wen-Jun Shen, Hui-Hong Zhang, Stancheva, Zlatina S., Kraemer, Fredric B., Obin, Martin S., and Greenberg, Andrew S.
- Subjects
- *
FAT cells , *LIPOLYSIS , *LIPASES , *PHOSPHORYLATION , *PROTEIN kinases , *GENETIC mutation - Abstract
Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the ‘downstream’ lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (fonskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a ‘phosphomimetic’ (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Pen A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Pen A regulates ATGL-dependent lipolysis and identify serine 517 as the Pen A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphonylated or phosphomimetic serine 517. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
5. Lipase-selective Functional Domains of Perilipin A Differentially Regulate Constitutive and Protein Kinase A-stimulated Lipolysis.
- Author
-
Zhang, Hui H., Souza, Sandra C., Muliro, Kizito V., Kraemer, Fredric B., Obin, Martin S., and Greenberg, Andrew S.
- Subjects
- *
PHOSPHOPROTEINS , *LIPOLYSIS , *PROTEIN kinases , *LIPASES , *ADENOVIRUSES , *FIBROBLASTS - Abstract
Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMPdependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser[sup 81], Ser[sup 222], and Ser[sup 276]). PKAstimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser[sup 433], Ser[sup 492], and Ser[sup 517]). Peri 301-517 prorooted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and nonHSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
- View/download PDF
6. Lipolysis Decreases the Energy State and Activates AMP-Activated Protein Kinase (AMPK) in the Adipocyte.
- Author
-
Gauthier, Marie-Soleil, Miyoshi, Hideaki, Souza, Sandra C., Saha, Asish K., Greenberg, Andrew S., and Ruderman, Neil B.
- Subjects
- *
LIPOLYSIS , *ADENOSINE monophosphate , *PROTEIN kinases , *FAT cells , *TYPE 2 diabetes , *OBESITY - Abstract
Activation of the fuel-sensing enzyme AMPK has been shown to improve various conditions associated with obesity and type 2 diabetes. In adipocytes, beta-adrenergic agonists activate AMPK; however the mechanism(s) by which they do so is not known. In the present study we examined whether the effect of these agents is dependent on their ability to stimulate lipolysis. Incubation of 3T3-L1 adipocytes with isoproterenol, isobutylmethylxanthine or forskolin increased AMPK activity, as reflected by a 3-fold increase in P-ACC Ser79 and a 2-fold increase in P-AMPK T172-within 1 hour. ShRNA-mediated silencing of adipose tissue triglyceride lipase (ATGL), a key regulator of triglyceride hydrolysis, totally inhibited the stimulation of both lipolysis and AMPK activation by forskolin. Likewise, co-incubation of the adipocytes with the general lipase inhibitor orlistat caused a 50% inhibition of both forskolin-stimulated lipolysis and AMPK activation. In contrast, orlistat did not diminish forskolin-induced increases in the abundance of P-CREB Ser 133 or P-LKB1 Ser431, indicating that it did not alter signaling events caused by PKA activation. We then assessed whether a change in cellular energy state (AMP/ATP ratio) mediated the activation of AMPK caused by lipolysis. In support of this notion, incubation with forskolin caused a 4-fold increase in the cellular AMP/ATP ratio. Conversely, when forskolin stimulated-lipolysis was partially inhibited by orlistat, both the activation of AMPK, and the increase in the AMP/ATP ratio were diminished by 50%. Fatty acyl CoA synthase (ACS) uses ATP and generates AMP when it catalyzes the conversion of flee fatty acids to fatty acyl CoA. When We inhibited ACS with triacsin C, activation of AMPK by forskolin was completely abrogated despite the fact that lipolysis was increased to the same extent as in control conditions. In conclusion, the results indicate that the activation of AMPK following beta-adrenergic stimulation of the adipocyte is secondary to lipolysis and is not the direct result of increases in cAMP abundance or PKA activity. They suggest that a decrease in energy state most likely explains how lipolysis activates AMPK and that the energy-consuming enzyme ACS is involved in this mechanism. [ABSTRACT FROM AUTHOR]
- Published
- 2007
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.