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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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Effects of combined lipins 1 and 2 down-regulation on the masses of lipids in 3T3-L1 adipocytes.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNAs at days 4 and 6 after induction of differentiation. Cells from days 0, 4, 6, and 8 were collected, and total TAG was determined as described under “Experimental Procedures.” The values are means ± S.D. of four experiments. ns, not significant for the comparison with ctr (p = 0.091). B, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2), lipin 1 only (L1), or lipin 2 only (L2) siRNAs as in A. Total PA levels were calculated enzymatically as described under “Experimental Procedures.” The values are means ± S.D. of seven experiments. ***, p < 0.005 for the comparison with control. C, 3T3-L1 adipocytes were differentiated and transfected with siRNAs as in A. Lipid extracts were prepared from day 8 adipocytes as described under “Experimental Procedures,” and the amounts of TAG and major phospholipid classes were analyzed by high performance liquid chromatography-mass spectroscopy. The values are means ± S.D. of two experiments. *, p < 0.05 for the comparison with control.
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Figure 6: Effects of combined lipins 1 and 2 down-regulation on the masses of lipids in 3T3-L1 adipocytes.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNAs at days 4 and 6 after induction of differentiation. Cells from days 0, 4, 6, and 8 were collected, and total TAG was determined as described under “Experimental Procedures.” The values are means ± S.D. of four experiments. ns, not significant for the comparison with ctr (p = 0.091). B, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2), lipin 1 only (L1), or lipin 2 only (L2) siRNAs as in A. Total PA levels were calculated enzymatically as described under “Experimental Procedures.” The values are means ± S.D. of seven experiments. ***, p < 0.005 for the comparison with control. C, 3T3-L1 adipocytes were differentiated and transfected with siRNAs as in A. Lipid extracts were prepared from day 8 adipocytes as described under “Experimental Procedures,” and the amounts of TAG and major phospholipid classes were analyzed by high performance liquid chromatography-mass spectroscopy. The values are means ± S.D. of two experiments. *, p < 0.05 for the comparison with control.

Mentions: Taken together, these data suggest that adipocyte maturation is impaired when lipins 1 and 2 are down-regulated at day 4 of differentiation. Despite these defects, FACS-based quantification showed that the lipin1/2 knockdowns accumulate neutral lipid during differentiation (data not shown). To investigate the lipid changes in these cells, we performed a time course of TAG buildup during differentiation with or without lipin 1 and 2 down-regulation. We found that mature adipocytes could still accumulate TAG after day 4, when lipins 1 and 2 were first down-regulated, although there was a modest, but not significant, decrease when compared with the TAG levels of control adipocytes transfected with the nontargeting siRNA (L1 + 2 cells: 77% of control TAG, p = 0,091; Fig. 6A). Analysis of total cellular PA levels using an enzymatic assay showed that lipin1/2 knockdown cells contained more PA than control adipocytes, consistent with the loss of PAP activity, and that this difference was due to the depletion of lipin 1 (Fig. 6B). To further examine in more detail how the lipin depletion affects the masses of TAG and phospholipids and their respective fatty acyl compositions, lipid extracts from either knockdown or control adipocytes, both at day 8, were analyzed by high performance liquid chromatography-mass spectroscopy. Surprisingly, the levels of the abundant structural phospholipid classes, PE and PC, did not show any major changes (Fig. 6C). PA mass levels exhibited an increase of 229% following lipin down-regulation. The depletion of lipins 1/2 did not cause any striking changes in the fatty acyl moieties of these phospholipids (data not shown). Consistent with the time course data above, the lipin 1/2 knockdown cells accumulated 67% of total day 8 TAG mass when compared with the control adipocytes (p = 0.057; Fig. 6C) and showed no significant differences in their fatty acyl chain composition (data not shown). These data raise the possibility that another pathway may be able to compensate and provide DAG for TAG synthesis in these cells. However, mRNA levels of PPAP2a1, PPAPa2, and PPAP2c, all encoding Mg2+-independent PAP enzymes, did not considerably increase following lipin depletion (Fig. 5C, with the exception of a 30% increase of PPAP2a1 at day 6), consistent with the PAP2 activity data (Fig. 4C). Similarly, mRNA levels of MOGAT1 and 2, belonging to the monoacylglycerol acyltransferase family involved in TAG production in enterocytes, did not increase (Fig. 5C). Alternative mechanisms may thus provide TAG in the lipin-depleted adipocytes (see “Discussion”).


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)

Effects of combined lipins 1 and 2 down-regulation on the masses of lipids in 3T3-L1 adipocytes.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNAs at days 4 and 6 after induction of differentiation. Cells from days 0, 4, 6, and 8 were collected, and total TAG was determined as described under “Experimental Procedures.” The values are means ± S.D. of four experiments. ns, not significant for the comparison with ctr (p = 0.091). B, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2), lipin 1 only (L1), or lipin 2 only (L2) siRNAs as in A. Total PA levels were calculated enzymatically as described under “Experimental Procedures.” The values are means ± S.D. of seven experiments. ***, p < 0.005 for the comparison with control. C, 3T3-L1 adipocytes were differentiated and transfected with siRNAs as in A. Lipid extracts were prepared from day 8 adipocytes as described under “Experimental Procedures,” and the amounts of TAG and major phospholipid classes were analyzed by high performance liquid chromatography-mass spectroscopy. The values are means ± S.D. of two experiments. *, p < 0.05 for the comparison with control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Effects of combined lipins 1 and 2 down-regulation on the masses of lipids in 3T3-L1 adipocytes.A, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2) siRNAs at days 4 and 6 after induction of differentiation. Cells from days 0, 4, 6, and 8 were collected, and total TAG was determined as described under “Experimental Procedures.” The values are means ± S.D. of four experiments. ns, not significant for the comparison with ctr (p = 0.091). B, 3T3-L1 adipocytes were transfected with nontargeting (control, ctr) or lipin 1 and lipin 2 (L1 + 2), lipin 1 only (L1), or lipin 2 only (L2) siRNAs as in A. Total PA levels were calculated enzymatically as described under “Experimental Procedures.” The values are means ± S.D. of seven experiments. ***, p < 0.005 for the comparison with control. C, 3T3-L1 adipocytes were differentiated and transfected with siRNAs as in A. Lipid extracts were prepared from day 8 adipocytes as described under “Experimental Procedures,” and the amounts of TAG and major phospholipid classes were analyzed by high performance liquid chromatography-mass spectroscopy. The values are means ± S.D. of two experiments. *, p < 0.05 for the comparison with control.
Mentions: Taken together, these data suggest that adipocyte maturation is impaired when lipins 1 and 2 are down-regulated at day 4 of differentiation. Despite these defects, FACS-based quantification showed that the lipin1/2 knockdowns accumulate neutral lipid during differentiation (data not shown). To investigate the lipid changes in these cells, we performed a time course of TAG buildup during differentiation with or without lipin 1 and 2 down-regulation. We found that mature adipocytes could still accumulate TAG after day 4, when lipins 1 and 2 were first down-regulated, although there was a modest, but not significant, decrease when compared with the TAG levels of control adipocytes transfected with the nontargeting siRNA (L1 + 2 cells: 77% of control TAG, p = 0,091; Fig. 6A). Analysis of total cellular PA levels using an enzymatic assay showed that lipin1/2 knockdown cells contained more PA than control adipocytes, consistent with the loss of PAP activity, and that this difference was due to the depletion of lipin 1 (Fig. 6B). To further examine in more detail how the lipin depletion affects the masses of TAG and phospholipids and their respective fatty acyl compositions, lipid extracts from either knockdown or control adipocytes, both at day 8, were analyzed by high performance liquid chromatography-mass spectroscopy. Surprisingly, the levels of the abundant structural phospholipid classes, PE and PC, did not show any major changes (Fig. 6C). PA mass levels exhibited an increase of 229% following lipin down-regulation. The depletion of lipins 1/2 did not cause any striking changes in the fatty acyl moieties of these phospholipids (data not shown). Consistent with the time course data above, the lipin 1/2 knockdown cells accumulated 67% of total day 8 TAG mass when compared with the control adipocytes (p = 0.057; Fig. 6C) and showed no significant differences in their fatty acyl chain composition (data not shown). These data raise the possibility that another pathway may be able to compensate and provide DAG for TAG synthesis in these cells. However, mRNA levels of PPAP2a1, PPAPa2, and PPAP2c, all encoding Mg2+-independent PAP enzymes, did not considerably increase following lipin depletion (Fig. 5C, with the exception of a 30% increase of PPAP2a1 at day 6), consistent with the PAP2 activity data (Fig. 4C). Similarly, mRNA levels of MOGAT1 and 2, belonging to the monoacylglycerol acyltransferase family involved in TAG production in enterocytes, did not increase (Fig. 5C). Alternative mechanisms may thus provide TAG in the lipin-depleted adipocytes (see “Discussion”).

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