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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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Opposing effects of lipin 1 and lipin 2 depletion in lipid droplet biogenesis of 3T3-L1 cells.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNA at days 4 and 6 after induction of differentiation. Day 8 adipocytes were fixed, stained with BODIPY 493/503, and imaged with an LSM 710 confocal microscope. Three-dimensional reconstruction from Z-stacks to visualize lipid droplets was performed as described under “Experimental Procedures.” Representative three-dimensional reconstructions from control or L1 + 2 adipocytes are shown. Rectangle side size was 100 μm. B, scatter plot of lipid droplet number (x axis) versus average lipid droplet volume (y axis) per cell in control or L1 + 2 3T3-L1 day 8 cells. Representative data from three separate experiments are shown. C, distribution of lipid droplet numbers in 3T3-L1 day 8 cells transfected with nontargeting (control, ctr), lipin 1 and lipin 2 (L1 + 2), lipin 1 only (L1), or lipin 2 only (L2) siRNA at days 4 and 6. D, average total lipid droplet volumes per cell for the experiments shown in C. The values are normalized to the lipid droplet volumes per cell of the control (ctr) 3T3-L1 transfectants, set at 1. The experiments in C and D represent means ± S.D. of at least three (control and L1 + 2) or two (L1 and L2) experiments. The total number of cells analyzed: control, 423; L1 + 2, 334; L1 203; and L2, 235. *, p < 0.05 for the comparison with control. ns, not significant for the comparison with control (p = 0.2 for L1 + 2; p = 0.5 for L1). LD, lipid droplets.
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Figure 7: Opposing effects of lipin 1 and lipin 2 depletion in lipid droplet biogenesis of 3T3-L1 cells.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNA at days 4 and 6 after induction of differentiation. Day 8 adipocytes were fixed, stained with BODIPY 493/503, and imaged with an LSM 710 confocal microscope. Three-dimensional reconstruction from Z-stacks to visualize lipid droplets was performed as described under “Experimental Procedures.” Representative three-dimensional reconstructions from control or L1 + 2 adipocytes are shown. Rectangle side size was 100 μm. B, scatter plot of lipid droplet number (x axis) versus average lipid droplet volume (y axis) per cell in control or L1 + 2 3T3-L1 day 8 cells. Representative data from three separate experiments are shown. C, distribution of lipid droplet numbers in 3T3-L1 day 8 cells transfected with nontargeting (control, ctr), lipin 1 and lipin 2 (L1 + 2), lipin 1 only (L1), or lipin 2 only (L2) siRNA at days 4 and 6. D, average total lipid droplet volumes per cell for the experiments shown in C. The values are normalized to the lipid droplet volumes per cell of the control (ctr) 3T3-L1 transfectants, set at 1. The experiments in C and D represent means ± S.D. of at least three (control and L1 + 2) or two (L1 and L2) experiments. The total number of cells analyzed: control, 423; L1 + 2, 334; L1 203; and L2, 235. *, p < 0.05 for the comparison with control. ns, not significant for the comparison with control (p = 0.2 for L1 + 2; p = 0.5 for L1). LD, lipid droplets.

Mentions: Because lipin 1/2 knockdown cells still contain significant TAG at day 8 and recent evidence implicates the yeast lipin Pah1p in lipid droplet biogenesis (31), we examined droplet morphology in these cells using the lipophilic dye BODIPY 493/503. This revealed that double lipin knockdown cells exhibited a dramatic increase in the number of smaller lipid droplets (Fig. 7A). Differentiated adipocytes treated with the control siRNA contained between 4 and 20 droplets, whereas in lipin-depleted cells this number increased drastically with a concurrent decrease of individual droplet volume (Fig. 7, B and C). However, when quantified by three-dimensional image reconstruction of confocal Z-slices, the total lipid droplet volume per adipocyte did not change significantly in lipin-depleted cells (80% of the volume of control adipocytes, p = 0.2; Fig. 7D). To examine whether droplet fragmentation was due to an additive effect of depleting the two lipins, we down-regulated individually either lipin 1 or lipin 2 at days 4 and 6 and quantified droplet numbers and total volume at day 8. We found that depletion of lipin 1 caused a similar defect as the double knockdown, whereas depletion of lipin 2 resulted in an increase of total droplet volume per cell without significantly affecting droplet numbers (Fig. 7D). Moreover, combining the two lipin siRNAs did cause a modest but reproducible increase of droplet numbers when compared with the single lipin 1 knockdown cells (Fig. 7C). Similar results in lipid droplet volume and numbers were obtained when both single and double lipin 1 and/or 2 knockdowns were performed with a different combination of siRNA oligonucleotides (data not shown).


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)

Opposing effects of lipin 1 and lipin 2 depletion in lipid droplet biogenesis of 3T3-L1 cells.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNA at days 4 and 6 after induction of differentiation. Day 8 adipocytes were fixed, stained with BODIPY 493/503, and imaged with an LSM 710 confocal microscope. Three-dimensional reconstruction from Z-stacks to visualize lipid droplets was performed as described under “Experimental Procedures.” Representative three-dimensional reconstructions from control or L1 + 2 adipocytes are shown. Rectangle side size was 100 μm. B, scatter plot of lipid droplet number (x axis) versus average lipid droplet volume (y axis) per cell in control or L1 + 2 3T3-L1 day 8 cells. Representative data from three separate experiments are shown. C, distribution of lipid droplet numbers in 3T3-L1 day 8 cells transfected with nontargeting (control, ctr), lipin 1 and lipin 2 (L1 + 2), lipin 1 only (L1), or lipin 2 only (L2) siRNA at days 4 and 6. D, average total lipid droplet volumes per cell for the experiments shown in C. The values are normalized to the lipid droplet volumes per cell of the control (ctr) 3T3-L1 transfectants, set at 1. The experiments in C and D represent means ± S.D. of at least three (control and L1 + 2) or two (L1 and L2) experiments. The total number of cells analyzed: control, 423; L1 + 2, 334; L1 203; and L2, 235. *, p < 0.05 for the comparison with control. ns, not significant for the comparison with control (p = 0.2 for L1 + 2; p = 0.5 for L1). LD, lipid droplets.
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Figure 7: Opposing effects of lipin 1 and lipin 2 depletion in lipid droplet biogenesis of 3T3-L1 cells.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNA at days 4 and 6 after induction of differentiation. Day 8 adipocytes were fixed, stained with BODIPY 493/503, and imaged with an LSM 710 confocal microscope. Three-dimensional reconstruction from Z-stacks to visualize lipid droplets was performed as described under “Experimental Procedures.” Representative three-dimensional reconstructions from control or L1 + 2 adipocytes are shown. Rectangle side size was 100 μm. B, scatter plot of lipid droplet number (x axis) versus average lipid droplet volume (y axis) per cell in control or L1 + 2 3T3-L1 day 8 cells. Representative data from three separate experiments are shown. C, distribution of lipid droplet numbers in 3T3-L1 day 8 cells transfected with nontargeting (control, ctr), lipin 1 and lipin 2 (L1 + 2), lipin 1 only (L1), or lipin 2 only (L2) siRNA at days 4 and 6. D, average total lipid droplet volumes per cell for the experiments shown in C. The values are normalized to the lipid droplet volumes per cell of the control (ctr) 3T3-L1 transfectants, set at 1. The experiments in C and D represent means ± S.D. of at least three (control and L1 + 2) or two (L1 and L2) experiments. The total number of cells analyzed: control, 423; L1 + 2, 334; L1 203; and L2, 235. *, p < 0.05 for the comparison with control. ns, not significant for the comparison with control (p = 0.2 for L1 + 2; p = 0.5 for L1). LD, lipid droplets.
Mentions: Because lipin 1/2 knockdown cells still contain significant TAG at day 8 and recent evidence implicates the yeast lipin Pah1p in lipid droplet biogenesis (31), we examined droplet morphology in these cells using the lipophilic dye BODIPY 493/503. This revealed that double lipin knockdown cells exhibited a dramatic increase in the number of smaller lipid droplets (Fig. 7A). Differentiated adipocytes treated with the control siRNA contained between 4 and 20 droplets, whereas in lipin-depleted cells this number increased drastically with a concurrent decrease of individual droplet volume (Fig. 7, B and C). However, when quantified by three-dimensional image reconstruction of confocal Z-slices, the total lipid droplet volume per adipocyte did not change significantly in lipin-depleted cells (80% of the volume of control adipocytes, p = 0.2; Fig. 7D). To examine whether droplet fragmentation was due to an additive effect of depleting the two lipins, we down-regulated individually either lipin 1 or lipin 2 at days 4 and 6 and quantified droplet numbers and total volume at day 8. We found that depletion of lipin 1 caused a similar defect as the double knockdown, whereas depletion of lipin 2 resulted in an increase of total droplet volume per cell without significantly affecting droplet numbers (Fig. 7D). Moreover, combining the two lipin siRNAs did cause a modest but reproducible increase of droplet numbers when compared with the single lipin 1 knockdown cells (Fig. 7C). Similar results in lipid droplet volume and numbers were obtained when both single and double lipin 1 and/or 2 knockdowns were performed with a different combination of siRNA oligonucleotides (data not shown).

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