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Distinct roles of the phosphatidate phosphatases lipin 1 and 2 during adipogenesis and lipid droplet biogenesis in 3T3-L1 cells.

Sembongi H, Miranda M, Han GS, Fakas S, Grimsey N, Vendrell J, Carman GM, Siniossoglou S - J. Biol. Chem. (2013)

Bottom Line: We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3.This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell.We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.

View Article: PubMed Central - PubMed

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

ABSTRACT
Lipins are evolutionarily conserved Mg(2+)-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.

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Combined down-regulation of lipins 1 and 2 in 3T3-L1 cells.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or both lipin 1 and lipin 2 (L1 + 2) siRNAs at days 4 and 6 after induction of differentiation. qPCR analysis was performed at the indicated days to quantify lipin 1α, 1β, 2, and 3 mRNA levels. The data are normalized to cyclophilin A mRNA and to control at day 1. The values are means ± S.D. of three independent experiments, and within each experiment siRNA transfections were performed in triplicate. B, extracts from cells in A were prepared at the indicated time points, and 5 μg of each sample was analyzed by Western blot with the specified antibodies. The blot shown is representative of six experiments. C, PAP1 and PAP2 activities were measured in 3T3-L1 cell extracts transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNAs as described under “Experimental Procedures.” The values are means ± S.D. of four experiments.
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Figure 4: Combined down-regulation of lipins 1 and 2 in 3T3-L1 cells.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or both lipin 1 and lipin 2 (L1 + 2) siRNAs at days 4 and 6 after induction of differentiation. qPCR analysis was performed at the indicated days to quantify lipin 1α, 1β, 2, and 3 mRNA levels. The data are normalized to cyclophilin A mRNA and to control at day 1. The values are means ± S.D. of three independent experiments, and within each experiment siRNA transfections were performed in triplicate. B, extracts from cells in A were prepared at the indicated time points, and 5 μg of each sample was analyzed by Western blot with the specified antibodies. The blot shown is representative of six experiments. C, PAP1 and PAP2 activities were measured in 3T3-L1 cell extracts transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNAs as described under “Experimental Procedures.” The values are means ± S.D. of four experiments.

Mentions: To assess the roles of lipin 1 after the initiation of adipogenesis, we set up a siRNA transfection protocol to down-regulate lipin 1 expression at days 4 and 6 of differentiation before assaying its function at day 8, when control 3T3-L1 adipocytes are filled by large lipid droplets. We chose day 4 as the initial time point for the lipin 1 knockdown, because this is the latest point after the start of adipogenesis when cells are still devoid of medium or large sized droplets. Transfection of nontargeting siRNAs were applied to control adipocytes at the same time points. Surprisingly, when lipin 1 was knocked down at this time point, there was only a minor change in neutral lipid levels, and cells expressed comparable levels of FABP4 at day 8 as control cells (Fig. 3). However, we noticed that the protein levels of lipin 2 were significantly elevated when lipin 1 was knocked down at this stage of differentiation (Fig. 3A). Although up-regulation of lipin 2 cannot compensate for the loss of lipin 1 during initiation of 3T3-L1 adipogenesis (24), it is not known to what extent lipin 2 can perform such a role at later stages, when cells form lipid droplets and accumulate TAG. To rule out the possibility of compensatory effects that could mask some of the phenotypes of lipin 1 knockdown, we performed a combined transfection of both lipin 1 and 2 siRNAs at day 4, followed by a second transfection of both lipin siRNAs at day 6 of differentiation. Applying this protocol, mRNA levels of lipin 1α, lipin 1β, and lipin 2 were efficiently down-regulated (Fig. 4A). Lipin 3 mRNA could be detected in control cells transfected with the nontargeting siRNAs at very low levels, as judged by the observed Ct values, which decreased further in the double lipin1/2 knockdown cells (Fig. 4A). The basis of this change of lipin 3 mRNA is currently not clear. However, at the protein level, lipin 3 was undetectable in control cells, and no change was observed in the double lipin 1 and 2 knockdown cells (Fig. 4B). To validate the effective down-regulation of lipin 1 and 2 function, we assayed PAP activity in adipocyte extracts following the transfection of the lipin 1 and 2 siRNA oligonucleotides. Consistent with previous data (24), Mg2+-dependent PAP activity (PAP1), which is lipin-dependent, increased during differentiation of control adipocytes (Fig. 4C). Importantly, this activity was almost undetectable in the lipin 1/2 knockdown adipocytes (Fig. 4C), whereas the Mg2+-independent (PAP2) activity did not change. Taken together, these data showed that the protein and activity levels of lipins 1 and 2 are efficiently down-regulated in 3T3-L1 adipocytes when their expression was reduced after day 4 of differentiation.


Distinct roles of the phosphatidate phosphatases lipin 1 and 2 during adipogenesis and lipid droplet biogenesis in 3T3-L1 cells.

Sembongi H, Miranda M, Han GS, Fakas S, Grimsey N, Vendrell J, Carman GM, Siniossoglou S - J. Biol. Chem. (2013)

Combined down-regulation of lipins 1 and 2 in 3T3-L1 cells.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or both lipin 1 and lipin 2 (L1 + 2) siRNAs at days 4 and 6 after induction of differentiation. qPCR analysis was performed at the indicated days to quantify lipin 1α, 1β, 2, and 3 mRNA levels. The data are normalized to cyclophilin A mRNA and to control at day 1. The values are means ± S.D. of three independent experiments, and within each experiment siRNA transfections were performed in triplicate. B, extracts from cells in A were prepared at the indicated time points, and 5 μg of each sample was analyzed by Western blot with the specified antibodies. The blot shown is representative of six experiments. C, PAP1 and PAP2 activities were measured in 3T3-L1 cell extracts transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNAs as described under “Experimental Procedures.” The values are means ± S.D. of four experiments.
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Figure 4: Combined down-regulation of lipins 1 and 2 in 3T3-L1 cells.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or both lipin 1 and lipin 2 (L1 + 2) siRNAs at days 4 and 6 after induction of differentiation. qPCR analysis was performed at the indicated days to quantify lipin 1α, 1β, 2, and 3 mRNA levels. The data are normalized to cyclophilin A mRNA and to control at day 1. The values are means ± S.D. of three independent experiments, and within each experiment siRNA transfections were performed in triplicate. B, extracts from cells in A were prepared at the indicated time points, and 5 μg of each sample was analyzed by Western blot with the specified antibodies. The blot shown is representative of six experiments. C, PAP1 and PAP2 activities were measured in 3T3-L1 cell extracts transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNAs as described under “Experimental Procedures.” The values are means ± S.D. of four experiments.
Mentions: To assess the roles of lipin 1 after the initiation of adipogenesis, we set up a siRNA transfection protocol to down-regulate lipin 1 expression at days 4 and 6 of differentiation before assaying its function at day 8, when control 3T3-L1 adipocytes are filled by large lipid droplets. We chose day 4 as the initial time point for the lipin 1 knockdown, because this is the latest point after the start of adipogenesis when cells are still devoid of medium or large sized droplets. Transfection of nontargeting siRNAs were applied to control adipocytes at the same time points. Surprisingly, when lipin 1 was knocked down at this time point, there was only a minor change in neutral lipid levels, and cells expressed comparable levels of FABP4 at day 8 as control cells (Fig. 3). However, we noticed that the protein levels of lipin 2 were significantly elevated when lipin 1 was knocked down at this stage of differentiation (Fig. 3A). Although up-regulation of lipin 2 cannot compensate for the loss of lipin 1 during initiation of 3T3-L1 adipogenesis (24), it is not known to what extent lipin 2 can perform such a role at later stages, when cells form lipid droplets and accumulate TAG. To rule out the possibility of compensatory effects that could mask some of the phenotypes of lipin 1 knockdown, we performed a combined transfection of both lipin 1 and 2 siRNAs at day 4, followed by a second transfection of both lipin siRNAs at day 6 of differentiation. Applying this protocol, mRNA levels of lipin 1α, lipin 1β, and lipin 2 were efficiently down-regulated (Fig. 4A). Lipin 3 mRNA could be detected in control cells transfected with the nontargeting siRNAs at very low levels, as judged by the observed Ct values, which decreased further in the double lipin1/2 knockdown cells (Fig. 4A). The basis of this change of lipin 3 mRNA is currently not clear. However, at the protein level, lipin 3 was undetectable in control cells, and no change was observed in the double lipin 1 and 2 knockdown cells (Fig. 4B). To validate the effective down-regulation of lipin 1 and 2 function, we assayed PAP activity in adipocyte extracts following the transfection of the lipin 1 and 2 siRNA oligonucleotides. Consistent with previous data (24), Mg2+-dependent PAP activity (PAP1), which is lipin-dependent, increased during differentiation of control adipocytes (Fig. 4C). Importantly, this activity was almost undetectable in the lipin 1/2 knockdown adipocytes (Fig. 4C), whereas the Mg2+-independent (PAP2) activity did not change. Taken together, these data showed that the protein and activity levels of lipins 1 and 2 are efficiently down-regulated in 3T3-L1 adipocytes when their expression was reduced after day 4 of differentiation.

Bottom Line: We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3.This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell.We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.

View Article: PubMed Central - PubMed

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

ABSTRACT
Lipins are evolutionarily conserved Mg(2+)-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.

Show MeSH
Related in: MedlinePlus