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Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2.

Grimsey N, Han GS, O'Hara L, Rochford JJ, Carman GM, Siniossoglou S - J. Biol. Chem. (2008)

Bottom Line: Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis.Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis.We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Hills Road, CB2 0XY Cambridge, United Kingdom.

ABSTRACT
Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.

Show MeSH
shRNA-mediated silencing of lipin 1 and 2 in differentiating adipocytes. 3T3-L1 preadipocytes stably transfected with retroviral vectors expressing shRNA targeting lipin 1 (Lipin 1), lipin 2 (Lipin 2) or a control sequence (Control), were induced to differentiate for 8 days. At the indicated time points, the cell lysates were prepared, the protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies.
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fig6: shRNA-mediated silencing of lipin 1 and 2 in differentiating adipocytes. 3T3-L1 preadipocytes stably transfected with retroviral vectors expressing shRNA targeting lipin 1 (Lipin 1), lipin 2 (Lipin 2) or a control sequence (Control), were induced to differentiate for 8 days. At the indicated time points, the cell lysates were prepared, the protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies.

Mentions: To examine whether expression of the different lipins is co-regulated during adipocyte differentiation, we silenced lipin 1 or lipin 2 by infecting 3T3-L1 preadipocytes with retroviruses carrying shRNAs specifically targeting their mRNA sequences. For each lipin, two cell lines were constructed, each expressing a different shRNA (see “Experimental Procedures”), and the results described below were reproduced with both. Infection of the cells with shRNA targeting either lipin 1 or lipin 2 resulted in an efficient decrease in the levels of the respective protein (Fig. 6). Notably, in lipin 1 knock-down cells, lipin 2 protein levels significantly increased at all time points examined and persisted for longer during differentiation (Fig. 6). Despite the increased lipin 2 levels, depletion of lipin 1 inhibited the differentiation of preadipocytes to mature adipocytes as judged by the failure to induce aP2 expression (Fig. 6) or to accumulate intracellular lipid (triacylglycerol/steryl esters), followed by Nile Red staining.4 In contrast, depletion of lipin 2 did not inhibit adipogenesis. Indeed, knock-down of lipin 2 led to a more rapid and robust induction of aP2, suggesting that adipogenesis may be accelerated in these cells (Fig. 6). Together, these data are consistent with the reciprocal regulation of the two lipins observed in HeLa M cells and also indicate that they perform distinct functions in differentiating adipocytes.


Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2.

Grimsey N, Han GS, O'Hara L, Rochford JJ, Carman GM, Siniossoglou S - J. Biol. Chem. (2008)

shRNA-mediated silencing of lipin 1 and 2 in differentiating adipocytes. 3T3-L1 preadipocytes stably transfected with retroviral vectors expressing shRNA targeting lipin 1 (Lipin 1), lipin 2 (Lipin 2) or a control sequence (Control), were induced to differentiate for 8 days. At the indicated time points, the cell lysates were prepared, the protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570901&req=5

fig6: shRNA-mediated silencing of lipin 1 and 2 in differentiating adipocytes. 3T3-L1 preadipocytes stably transfected with retroviral vectors expressing shRNA targeting lipin 1 (Lipin 1), lipin 2 (Lipin 2) or a control sequence (Control), were induced to differentiate for 8 days. At the indicated time points, the cell lysates were prepared, the protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies.
Mentions: To examine whether expression of the different lipins is co-regulated during adipocyte differentiation, we silenced lipin 1 or lipin 2 by infecting 3T3-L1 preadipocytes with retroviruses carrying shRNAs specifically targeting their mRNA sequences. For each lipin, two cell lines were constructed, each expressing a different shRNA (see “Experimental Procedures”), and the results described below were reproduced with both. Infection of the cells with shRNA targeting either lipin 1 or lipin 2 resulted in an efficient decrease in the levels of the respective protein (Fig. 6). Notably, in lipin 1 knock-down cells, lipin 2 protein levels significantly increased at all time points examined and persisted for longer during differentiation (Fig. 6). Despite the increased lipin 2 levels, depletion of lipin 1 inhibited the differentiation of preadipocytes to mature adipocytes as judged by the failure to induce aP2 expression (Fig. 6) or to accumulate intracellular lipid (triacylglycerol/steryl esters), followed by Nile Red staining.4 In contrast, depletion of lipin 2 did not inhibit adipogenesis. Indeed, knock-down of lipin 2 led to a more rapid and robust induction of aP2, suggesting that adipogenesis may be accelerated in these cells (Fig. 6). Together, these data are consistent with the reciprocal regulation of the two lipins observed in HeLa M cells and also indicate that they perform distinct functions in differentiating adipocytes.

Bottom Line: Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis.Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis.We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Hills Road, CB2 0XY Cambridge, United Kingdom.

ABSTRACT
Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.

Show MeSH