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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.

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Reciprocal pattern of lipin 1 and 2 protein expression during adipogenesis. A, 3T3-L1 preadipocytes were induced to differentiate for 12 days. At the indicated time points, the cell lysates were prepared, protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using lipin 1 and 2 antibodies. Adipocyte differentiation was monitored by following αP2 expression. B, lipin 2 is phosphorylated in 3T3-L1 cells. Endogenous lipin 2 was immunoprecipitated (IP) from extracts of 3T3-L1 preadipocytes. Immune pellets were incubated with or without λ-phosphatase (λPPase) and analyzed by SDS-PAGE followed by immunoblotting using lipin 2 antibodies.
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fig4: Reciprocal pattern of lipin 1 and 2 protein expression during adipogenesis. A, 3T3-L1 preadipocytes were induced to differentiate for 12 days. At the indicated time points, the cell lysates were prepared, protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using lipin 1 and 2 antibodies. Adipocyte differentiation was monitored by following αP2 expression. B, lipin 2 is phosphorylated in 3T3-L1 cells. Endogenous lipin 2 was immunoprecipitated (IP) from extracts of 3T3-L1 preadipocytes. Immune pellets were incubated with or without λ-phosphatase (λPPase) and analyzed by SDS-PAGE followed by immunoblotting using lipin 2 antibodies.

Mentions: Reciprocal Regulation of Lipin 1 and 2 in Differentiating Adipocytes—The reciprocal regulation of the levels of the two lipins in HeLa M cells prompted us to test how their expression is coordinated during adipogenesis where PAP1 activity is required for the biosynthesis of the storage lipid triacylglycerol. To address this question, 3T3-L1 preadipocytes were induced to differentiate and protein extracts were prepared at various time points afterward. As seen in Fig. 4A, lipin 2 is expressed in preadipocytes and also during the early stages of adipogenesis before its levels decrease almost to undetectable levels in mature adipocytes. Lipin 1 shows a reciprocal pattern with no detectable expression in preadipocytes and a gradual increase after 48 h of differentiation, which coincides with a decrease in lipin 2 levels (Fig. 4A). Lipin 1 is expressed first as a low and subsequently as a higher molecular weight protein band on SDS-PAGE that persists in mature adipocytes, probably corresponding to the two isoforms of lipin 1 generated by alternative splicing (12). Interestingly, lipin 2 is also expressed as a high and low molecular weight protein band on SDS-PAGE, but in this case the higher band is due to phosphorylation as it resolves to the lower form following phosphatase treatment of the immunoprecipitated protein (Fig. 4B).


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)

Reciprocal pattern of lipin 1 and 2 protein expression during adipogenesis. A, 3T3-L1 preadipocytes were induced to differentiate for 12 days. At the indicated time points, the cell lysates were prepared, protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using lipin 1 and 2 antibodies. Adipocyte differentiation was monitored by following αP2 expression. B, lipin 2 is phosphorylated in 3T3-L1 cells. Endogenous lipin 2 was immunoprecipitated (IP) from extracts of 3T3-L1 preadipocytes. Immune pellets were incubated with or without λ-phosphatase (λPPase) and analyzed by SDS-PAGE followed by immunoblotting using lipin 2 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2570901&req=5

fig4: Reciprocal pattern of lipin 1 and 2 protein expression during adipogenesis. A, 3T3-L1 preadipocytes were induced to differentiate for 12 days. At the indicated time points, the cell lysates were prepared, protein concentrations were measured by Bradford assay, and equal protein amounts/time point were analyzed by SDS-PAGE followed by immunoblotting using lipin 1 and 2 antibodies. Adipocyte differentiation was monitored by following αP2 expression. B, lipin 2 is phosphorylated in 3T3-L1 cells. Endogenous lipin 2 was immunoprecipitated (IP) from extracts of 3T3-L1 preadipocytes. Immune pellets were incubated with or without λ-phosphatase (λPPase) and analyzed by SDS-PAGE followed by immunoblotting using lipin 2 antibodies.
Mentions: Reciprocal Regulation of Lipin 1 and 2 in Differentiating Adipocytes—The reciprocal regulation of the levels of the two lipins in HeLa M cells prompted us to test how their expression is coordinated during adipogenesis where PAP1 activity is required for the biosynthesis of the storage lipid triacylglycerol. To address this question, 3T3-L1 preadipocytes were induced to differentiate and protein extracts were prepared at various time points afterward. As seen in Fig. 4A, lipin 2 is expressed in preadipocytes and also during the early stages of adipogenesis before its levels decrease almost to undetectable levels in mature adipocytes. Lipin 1 shows a reciprocal pattern with no detectable expression in preadipocytes and a gradual increase after 48 h of differentiation, which coincides with a decrease in lipin 2 levels (Fig. 4A). Lipin 1 is expressed first as a low and subsequently as a higher molecular weight protein band on SDS-PAGE that persists in mature adipocytes, probably corresponding to the two isoforms of lipin 1 generated by alternative splicing (12). Interestingly, lipin 2 is also expressed as a high and low molecular weight protein band on SDS-PAGE, but in this case the higher band is due to phosphorylation as it resolves to the lower form following phosphatase treatment of the immunoprecipitated protein (Fig. 4B).

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