Cell autonomous lipin 1 function is essential for development and maintenance of white and brown adipose tissue

Abstract

Through analysis of mice with spatially and temporally restricted inactivation of Lpin1, we characterized its cell autonomous function in both white (WAT) and brown (BAT) adipocyte development and maintenance. We observed that the lipin 1 inactivation in adipocytes of aP2(Cre/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice resulted in lipodystrophy and the presence of adipocytes with multilocular lipid droplets. We further showed that time-specific loss of lipin 1 in mature adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice led to their replacement by newly formed Lpin1-positive adipocytes, thus establishing a role for lipin 1 in mature adipocyte maintenance. Importantly, we observed that the presence of newly formed Lpin1-positive adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice protected these animals against WAT inflammation and hepatic steatosis induced by a high-fat diet. Loss of lipin 1 also affected BAT development and function, as revealed by histological changes, defects in the expression of peroxisome proliferator-activated receptor alpha (PPARα), PGC-1α, and UCP1, and functionally by altered cold sensitivity. Finally, our data indicate that phosphatidic acid, which accumulates in WAT of animals lacking lipin 1 function, specifically inhibits differentiation of preadipocytes. Together, these observations firmly demonstrate a cell autonomous role of lipin 1 in WAT and BAT biology and indicate its potential as a therapeutical target for the treatment of obesity.

Fig 1

Adipocyte-specific deletion of Lpin1 using an aP2^Cre/+ transgenic mouse strain. (A) Schematic overview of the wild-type Lpin1 genomic locus (Lpin1⁺), the floxed Lpin1 allele (Lpin1^fEx2-3), and the Lpin1-null allele (Lpin1^ΔEx2-3), obtained after aP2^Cre-mediated excision. Exons are labeled as described previously (36). The F1, F2, and R1 primers were used for PCR amplification of genomic DNA. (B) Lpin1 deletion was absent in control animals (aP2^+/+) and was observed in white adipose tissue (WAT) and the perineurial/epineurial (P/E) compartment of sciatic nerves but not in the endoneurium (Endo) from aP2^Cre/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre/+) mice. PCR amplification, using a combination of the primers F1, F2, and R1, was used to detect the floxed allele (Lpin1^fEx2-3; 1,449 bp) and the Lpin1-null allele (Lpin1^ΔEx2-3; 1,074 bp). (C) Quantitative PCR showed that Lpin1 expression is decreased in epididymal (Epi) and subcutaneous (Sc) WAT whereas its expression in heart and liver was not affected in aP2^Cre/+ mice (n = 3; ∗, P < 0.001). (D) Quantitative PCR showed that Lpin1 expression is almost abolished in purified aP2^Cre/+ adipocytes (n = 4; ∗, P < 0.05). (E) PAP1 activity was substantially decreased in WAT, but not in the liver, of aP2^Cre/+ mice compared to results for control aP2^+/+ mice. However, no significant difference in both WAT and liver PAP2 activity was observed between genotypes (n = 8; ∗, P < 0.001).

Fig 2

WAT lipodystrophy in aP2^Cre/+/Lpin1^{fEx2-3/fEx2-3} mice. (A, B, and C) Body weight and epididymal WAT (eWAT) weight measurements in aP2^+/+/Lpin1^{fEx2-3/fEx2-3} (aP2^+/+) and aP2^Cre/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre/+) mice with a regular (Chow; n = 5) (A and C) or high-fat (HFD; n = 6) (B and C) diet (∗, P < 0.05; ∗∗, P < 0.001). (D) Paraffin sections of eWAT from aP2^+/+ and aP2^Cre/+ mice on chow or HFD, stained with hematoxylin and eosin. Scale bars, 100 μm (in panel iv) and 20 μm (in panel v). (E and F) BrdU immunofluorescence staining (red) (E) and determination of percentage (%) of BrdU-labeled cells (F) in WAT sections from 4-month-old aP2^+/+ and aP2^Cre/+ mice. For panel E, AF indicates the autofluorescence of adipocyte cell membranes (green). Cell nuclei are counterstained with DAPI (blue). The asterisk indicates a multilocular adipocyte labeled with BrdU. Scale bar: 10 μm. For panel F, results are expressed as mean (± SD) percentages of the number of BrdU-positive cells (n = 4 to 5; ∗, P < 0.01). (G and H) Blood glucose concentrations during an intraperitoneal glucose tolerance test (G) or an insulin tolerance test (H) in 4-month-old aP2^+/+ and aP2^Cre/+ mice maintained on a chow diet. In panel H, the area under the curve (AUC) represents 558 ± 87 for aP2^+/+ mice and 740 ± 178 for aP2^Cre/+ mice. Data represent means ± SD (n = 11 or 12; ∗, P < 0.05).

Fig 3

In vivo and ex vivo characterizations of aP2^Cre/+/Lpin1^{fEx2-3/fEx2-3} adipocytes. (A and B) Quantitative PCR measurement of Pparg, Fabp4, Dgat1, and Cd36 expression in purified eWAT adipocytes from aP2^+/+ and aP2^Cre/+ mice on chow (n = 5) (A) or an HFD (n = 6) (B) (∗, P < 0.05). (C) Confluent aP2^+/+/Lpin1^{fEx2-3/fEx2-3} (aP2^+/+) and aP2^Cre/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre/+) MEFs were exposed to differentiation medium for 2 days (D) and then cultured for 0, 2, 4, 6, 8, and 10 days in the growth medium containing insulin. Oil Red O staining of aP2^+/+ and aP2^Cre/+ MEFs at 0, 2, 6, 8, and 10 days of differentiation is shown. Scale bars, 100 μm and 1 cm. (D) Differentiation of aP2^Cre/+ MEFs into adipocytes reproduced in vitro the multilocular phenotype. Bright-field pictures of aP2^+/+ (i) or aP2^Cre/+ (ii) MEFs after 10 days (D10) of differentiation are presented together with adipose differentiation-related protein (ADFP) immunostaining (red) on aP2^+/+ (iii) or aP2^Cre/+ (iv and v) MEFs. Neutral lipid droplets in the MEFs were detected by green staining with Bodipy (493/503), and DAPI (blue) was used as a nuclear counterstain. Scale bars, 100 μm (in panels i to iv) and 20 μm (in panel v). (E) Quantitative PCR measurement of the Cre, Lpin1, Pparg, Dgat1, UCP1, PGC-1a, HSL, ATGL, and Cd36 genes in differentiation-induced aP2^+/+ and aP2^Cre/+ MEFs at D10 (data are represented as means ± SD for three independent experiments; ∗, P < 0.01; ∗∗, P < 0.001).

Fig 4

Consequences of Lpin1 inactivation in mature adipocytes in aP2^Cre-ERT2/+/Lpin1^{fEx2-3/fEx2-3} mice. (A) Timing of Tamox administration and phenotypic analysis. D0 to D7, days of Tamox injection; P30 to P160, age in postnatal days; Chow, regular diet; HFD, high-fat diet. (B) Lpin1 deletion was absent in control animals (aP2^+/+) and was detectable in WAT at D7 and to a lower extent at D22 and D60 in aP2^Cre-ERT2/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre-ERT2/+) mice. (C) eWAT weight measurements in aP2^+/+/Lpin1^{fEx2-3/fEx2-3} (aP2^+/+) and aP2^Cre-ERT2/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre-ERT2/+) mice on a chow diet at D7, D22, and D60 (n = 5 per time point; ∗, P < 0.05; ∗∗, P < 0.001). (D) Paraffin sections of eWAT from aP2^+/+ and aP2^Cre-ERT2/+ mice on a chow diet, stained with hematoxylin and eosin. Arrowheads indicate multilocular adipocytes. Scale bar, 10 μm. (E) Quantitative PCR measurement of Fabp4, Cre, Lpin1, Dgat1, Pparg, HSL, ATGL, Bcl2, Bcl2l1, and Wisp2 expression in eWAT from aP2^+/+ and aP2^Cre-ERT2/+ mice on a chow diet at D7, D22, and D60 (n = 5 per time point; ∗, P < 0.05; ∗∗, P < 0.001). (F) Quantitative PCR measurement of F4/80 and CD68 expression in eWAT from aP2^+/+ and aP2^Cre-ERT2/+ mice on a chow diet at D7, D22, and D60 (n = 5; ∗∗, P < 0.001). Data represent means ± SD.

Fig 5

aP2^Cre-ERT2/+/Lpin1^{fEx2-3/fEx2-3} mice are resistant to HFD-induced obesity. (A and B) Body weight and eWAT weight measurements in aP2^+/+/Lpin1^{fEx2-3/fEx2-3} (aP2^+/+) and aP2^Cre-ERT2/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre-ERT2/+) mice on a high-fat diet (HFD) (n = 6) (∗, P < 0.05; ∗∗, P < 0.001). (C) Paraffin sections of eWAT (i and ii) and liver (iii and iv) from P160 (D130) aP2^+/+ and aP2^Cre-ERT2/+ mice on a HFD, stained with hematoxylin and eosin. Scale bar, 100 μm. (D) eWAT adipocyte area determined in aP2^+/+ and aP2^Cre-ERT2/+ mice on an HFD at D130 (n = 5 per time point; ∗∗, P < 0.001). (E) Quantitative PCR measurement of F4/80 and CD68 expression in eWAT at D130 in HFD from aP2^+/+ and aP2^Cre-ERT2/+ mice (n = 7 or 8; ∗∗, P < 0.001). (F) Plasma insulin levels at D7, D22, D60 (chow), and D130 (HFD) in aP2^+/+ and aP2^Cre-ERT2/+ mice (n = 5 per time point; ∗, P < 0.05). (G and H) Blood glucose concentrations during an intraperitoneal glucose tolerance test (G) or an insulin tolerance test (H) in P160 aP2^+/+ and aP2^Cre-ERT2/+ mice maintained on an HFD. Data represent means ± SD (n = 7 or 8).

Fig 6

Phosphatidic acid prevents adipocyte differentiation. (A) PA levels in eWAT of aP2^Cre/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre/+) mice and aP2^+/+/Lpin1^{fEx2-3/fEx2-3} (aP2^+/+) control mice (age, P56; n = 5 or 6; ∗, P < 0.01). (B) i. 3T3-L1 cells were induced to differentiate (D) in the presence of either 50 μM PA (D+PA) or in the presence of PA and 20 μM MEK-Erk pathway inhibitor U0126 (D+PA+U). ND, nondifferentiated cells. Cells were stained with Oil Red O. (ii) Higher magnification images of cells shown in panel i. Scale bar, 1 cm (i) or 100 μm (ii). (C) Quantitative PCR analysis of the expression of the adipocyte markers Cd36, Fabp4, Fasn, Lpin1, and Pparg in 3T3-L1 cells grown under conditions described for panel B. The data represent the means ± SD of triplicate measurements (∗, P < 0.001). (D) Representative images of human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cells induced to differentiate in the presence (PA) or absence (Control) of 50 μM PA. Scale bar, 100 μm. (E) Expression of the adipocyte marker Fabp4, analyzed by quantitative PCR in SGBS cells grown under conditions described for panel D. (F) Representative images of 3T3-L1 cells grown under conditions described for panel B in the absence [Control (C)] or in the presence (Prop) or absence (Control) of 100 μM propranolol. Cells were stained with Oil Red O. Scale bar, 100 μm. (G) Expression of adipocyte markers Cd36, Fabp4, Fasn, Lpin1, and Pparg was analyzed by quantitative PCR in 3T3-L1 cells grown under conditions described for panel F. The data represent the means ± SD of triplicate measurements (∗∗, P < 0.001). (H) Representative images of 3T3-L1 cells grown under conditions described for panel B in the absence [Control (C)] or in the presence of 50 μM PA and treated either with empty vector (50 μM PA) or lipin 1β (β) [PA + Lpin1β (β)] expression-inducing viruses. Cells were stained with Oil Red O. Scale bar, 100 μm. (I) After 8 days of differentiation, the expression of adipocyte markers Fabp4 and Cd36 was analyzed by quantitative PCR in 3T3-L1 cells grown under conditions described for panel H. The data represent the means ± SD of triplicate measurements (∗, P < 0.01; ∗∗, P < 0.001).

Fig 7

PA effect in adipocytes is independent of G_i protein coupled receptor. (A) 3T3-L1 cells were grown in the presence of the indicated concentration of PA or LPA or pretreated with 50 ng/ml of pertussis toxin before addition of the indicated concentration of phosphatidic acid (PA + PTX) or lysophosphatidic acid (LPA + PTX). The activation of Erk1/2 was evaluated by Western blotting using a specific antibody recognizing its phosphorylated form (P-Erk1/2). An antibody against Erk1/2 revealed its total amount in the lysate. (B) 3T3-L1 cells were treated for 1 h with increasing concentrations (0, 1, 10, and 100 μM) of PA or 100 μM of propranolol (Prop). Activation of Erk1/2 and Akt was evaluated by specific antibodies recognizing their phosphorylated forms (P-Erk1/2 and P-Akt).

Fig 8

Lipin 1 is required for brown adipocyte development and function. (A) Representative macroscopic view of BAT isolated from 3-month-old (P90) aP2^+/+/Lpin1^{fEx2-3/fEx2-3} (aP2^+/+) and aP2^Cre/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre/+) male mice. Scale bar, 5 mm. (B) BAT weight measurements in aP2^+/+ and aP2^Cre/+ P10 and P90 mice on a regular diet (Chow; n = 5; ∗∗, P < 0.001). (C) Paraffin sections prepared from BAT of aP2^+/+ and aP2^Cre/+ mice, stained with hematoxylin and eosin. Scale bar, 100 μm in (i and ii) or 25 μm (iii and iv). (D) Body temperature in aP2^+/+ and aP2^Cre/+ mice exposed to 24°C or 4°C for the indicated period of time (n = 9; ∗, P < 0.01; ∗∗, P < 0.001). (E) Quantitative PCR measurement of Lpin1, Ppara, PGC-1α, Ucp1, Acox1, Cpt1b, Acsl1, and Acadm expression in BAT from aP2^+/+ and aP2^Cre/+ mice exposed to 24°C or 4°C for 12 h (n = 5; ∗, P < 0.01; ∗∗, P < 0.001). (F) Wild-type mouse embryonic fibroblasts treated either with control (empty vector) or lipin 1β (Lpin1β) expression-inducing viruses were induced to differentiate. After 8 days of differentiation, the levels of Ucp1, PGC-1α, Elovl3, Acox1, Cpt1b, Acsl1, and Acadm expression were determined by quantitative PCR. The data represent the means ± SD for triplicate measurements (∗, P < 0.01; ∗∗, P < 0.001; control versus Lpin1β under differentiation).

Fig 9

Lipin1 is crucial for brown adipocyte maintenance. (A) BAT weight in aP2^+/+/Lpin1^{fEx2-3/fEx2-3} (aP2^+/+) and aP2^Cre-ERT2/+/Lpin1^{fEx2-3/fEx2-3} (aP2^Cre-ERT2/+) mice on a chow diet at D7, D22, and D60 (n = 5 per time point; ∗∗, P < 0.001). (B) Quantitative PCR measurement of Fabp4, Cre, Lpin1, Ucp1, PGC1-α, Ppara, and Pparg expression in BAT from aP2^+/+ and aP2^Cre-ERT2/+ mice on a chow diet at D7, D22, and D60 (n = 5 per time point; ∗, P < 0.001). Data represent means ± SD. (C) Paraffin sections prepared from BAT of aP2^+/+ and aP2^Cre-ERT2/+ mice on a chow diet at D7, D22, and D60, stained with hematoxylin and eosin. Scale bar, 100 μm.

Fig 10

Role of lipin 1 in development and survival of adipocytes. (A) With a chow diet, the wild-type adipocytes accumulate lipids, leading to their increased size. This phenotype is accentuated with an HFD, leading to adipocyte inflammation, represented by the presence of macrophages (red cells). The cellular WAT morphology observed in both aP2^Cre/+/Lp^{fEx2-3/fEx2-3} (aP2^Cre/+ at P30 to P160) and aP2^Cre-ERT2/+/Lp^{fEx2-3/fEx2-3} (aP2^Cre-ERT2/+ at D7 to D22) mice consists of large numbers of smaller adipocytes filled with multilocular lipid droplets (yellow) accompanied by macrophage infiltration. Lpin1 inactivation also affected the BAT structure, leading to a decreased capacity to accumulate lipids (yellow). The lipodystrophy phenotype present at D7 to D22 in WAT and BAT of aP2^Cre-ERT2/+/Lp^{fEx2-3/fEx2-3} mice is partially recovered at D130, indicating that Lpin1-deficient adipocytes were replaced by newly differentiated adipocytes. Importantly, the presence of newly formed Lpin1-positive adipocytes in aP2^Cre-ERT2/+/Lp^{fEx2-3/fEx2-3} mice protected them against HFD-induced adipocyte inflammation. For timing of Tamox administration (aP2^Cre-ERT2/+/Lp^{fEx2-3/fEx2-3} mice) and phenotypic analysis, D0 (day 0) represents the first injection of Tamox which was performed at P30; P30 to P160, age in postnatal days; Chow, regular diet. (B) PA inhibits adipocyte differentiation. Accumulation of intracellular PA, either as a consequence of biochemical (propranolol) or genetic (Lpin1^fld/fld) lipin 1 inactivation, leads to activation of the MEK-Erk pathway and cell proliferation. The exogenous PA can also potentially be converted to LPA and affect adipocyte function through the G_i/o-protein LPA receptor (LPA-R) (also called EDG-2). However, biochemical inactivation (pertussis toxin) of G_i/o-protein LPA receptor does not affect the ability of extracellular PA to activate the MEK-Erk pathway, arguing against this possibility. Based on the proposed model, lipin 1/PAP-1 inactivation affects adipocytes via two mechanisms: (i) the accumulation of intracellular PA, which leads to sustained activation of the MEK-Erk pathway, affecting adipocyte differentiation, and (ii) the impairment of TAG biosynthesis.