AGPAT2 is essential for postnatal development and maintenance of white and brown adipose tissue

Abstract

Objective: Characterize the cellular and molecular events responsible for lipodystrophy in AGPAT2 deficient mice.

Methods: Adipose tissue and differentiated MEF were assessed using light and electron microscopy, followed by protein (immunoblots) and mRNA analysis (qPCR). Phospholipid profiling was determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Results: In contrast to adult Agpat2 (-/-) mice, fetuses and newborn Agpat2 (-/-) mice have normal mass of white and brown adipose tissue. Loss of both the adipose tissue depots occurs during the first week of postnatal life as a consequence of adipocyte death and inflammatory infiltration of the adipose tissue. At the ultrastructural level, adipose tissue of newborn Agpat2 (-/-) mice is virtually devoid of caveolae and has abnormal mitochondria and lipid droplets. Autophagic structures are also abundant. Consistent with these findings, differentiated Agpat2 (-/-) mouse embryonic fibroblasts (MEFs) also have impaired adipogenesis, characterized by a lower number of lipid-laden cells and ultrastructural abnormalities in lipid droplets, mitochondria and plasma membrane. Overexpression of PPARγ, the master regulator of adipogenesis, increased the number of Agpat2 (-/-) MEFs that differentiated into adipocyte-like cells but did not prevent morphological abnormalities and cell death. Furthermore, differentiated Agpat2 (-/-) MEFs have abnormal phospholipid compositions with 3-fold increased levels of phosphatidic acid.

Conclusion: We conclude that lipodystrophy in Agpat2 (-/-) mice results from postnatal cell death of adipose tissue in association with acute local inflammation. It is possible that AGPAT2 deficient adipocytes have an altered lipid filling or a reduced capacity to adapt the massive lipid availability associated with postnatal feeding.

Keywords: AGPAT2; Adipogenesis; Adipose tissue; Lipodystrophy; Phospholipid.

Graphical abstract

Supplementary Figure 1

(A) Oil red O/hematoxylin stained transversal sections at the inguinal level of newborn Agpat2^−/− mice. Bilateral subcutaneous inguinal WAT depots (left and middle images) are located between the skin and muscle fascia just anterior to the lower segment of the hind limbs. Higher magnification of the inguinal WAT depots stained with Oil red O (right image). (B) Perilipin-1 immunofluorescence staining (green) of inguinal WAT. F-actin was detected with phalloidin/rhodamine (red), indicating skeletal muscle. Nuclei were stained with DAPI (blue). (C) Development of hepatic steatosis matches adipose tissue destruction in Agpat2^−/− mice. Histological analysis (hematoxylin-eosin staining) shows growing lipids build up in the liver of Agpat2^−/− mice starting at P2.5. No differences between female and male Agpat2^−/− mice were detected.

Supplementary Figure 2

(A) Immunoblot analysis of whole-cell protein extracts from Agpat2^+/+ and Agpat2^−/− MEFs at different days of differentiation. 50 μg of proteins were loaded, total Akt was used as loading control. (B) At the indicated times, the number of lipid-laden cells were counted as previously described. Graph shows the percentage of lipid-laden cells. (C) TUNEL assay in 10 days differentiated MEFs.

Figure 1

Late fetuses and newborn Agpat2^−/−mice have normal adipose tissue mass, anatomical distribution and Perilipin-1 expression. (A) Oil red O/hematoxylin stained transversal thoracic sections revealed normal distribution of adipose tissue in E18.5 fetuses and newborn Agpat2^+/+ and Agpat2^−/− mice. (B) Higher magnification of the dorsal areas stained with Oil red O showed a thin layer of lipid-laden cells in the hypodermis corresponding to developing subcutaneous white adipose tissue (scWAT) in animals of both genotypes. scWAT is separated from the interscapular brown adipose tissue (iBAT) by the panniculus carnosus muscle (outlined by dashed line) (C) Perilipin-1 immunofluorescence staining (green) revealed that lipid-laden cells correspond to adipocytes expressing Perilipin-1. F-actin was detected with Phalloidin/rhodamine (red), indicating skeletal muscle. Nuclei were stained with DAPI (blue). Images are representative of 6 embryos or fetuses per genotype and age.

Figure 2

Postnatal adipose tissue development and growth are impaired in Agpat2^−/−mice. (A–B) Paraffin-embedded sections of anterior scWAT and iBAT from Agpat2^+/+ and Agpat2^−/− mice were obtained at different days of postnatal life and stained with Hematoxylin and Eosin. Slides were photographed at 40× magnification. Images are representative of N > 6 per genotype and age (C, F) Three-dimension digital reconstruction of Perilipin-1 stained (green) adipose tissue depots from Agpat2^+/+ and Agpat2^−/− mice at P0.5 (C) and P4.5 (F), DAPI was used to stain nuclei (blue). (D) Histogram shows adipocyte size distribution in scWAT of Agpat2^+/+ and Agpat2^−/− newborn mice (P0.5). (E) Quantitative comparison of total number of adipocyte per field in scWAT of newborn and P4.5 Agpat2^−/− mice. Adipocyte number and size were analyzed using Adiposoft software. At least 1000 adipocytes and 5–6 different areas per mouse (N = 4) were analyzed. (G–H) Adipose tissue mRNA markers were assessed by qPCR in scWAT and iBAT depots at different pre and postnatal time points. Graphs represent the relative abundance of each transcript normalized to 36B4 mRNA. The bars show the mean ± standard deviation (SD) of N = 6. ***p < 0.001 compared to Agpat2^+/+ mice at E18.5.

Figure 3

Transmission electron microscopy reveals ultrastructural abnormalities in scWAT from newborn Agpat2^−/−mice. (A–D) Representatives images of scWAT of Agpat2^+/+ and Agpat2^−/− mice. Caveolae are depicted by red arrowhead. (E) Quantification of caveolae normalized against membrane length. (F–I) scWAT of Agpat2^−/− mice show small adipocytes with numerous lipid droplets (LD), abnormal mitochondria (m), autophagic structures (enclosed by dashed red lines for clarity), and absence of plasma membrane caveolae. (J) Representative quantification of autophagic structures per adipocyte on section. For image comparative analysis and quantification, 20 adipocytes per sample were analyzed with a total of N = 3 samples per experimental group. (K) Representative confocal co-immunofluorescence of caveolin-1 (red) and CD31 (magenta) on scWAT of newborn mice. Asterisks indicate Agpat2^−/− adipocytes with plasma membrane-associated Caveolin-1. (L) Quantitation of caveolin-1 immunofluorescence signal in adipocytes.

Figure 4

The loss of adipose tissue in Agpat2^−/−mice is associated with massive cell death and adipose inflammation. (A) Paraffin-embedded sections of scWAT and iBAT from Agpat2^+/+ and Agpat2^−/− littermates were assessed by TUNEL staining. (B) 3D slide reconstruction and surface rendering of confocal data from TUNEL/Perilipin-1 double-labeled scWAT and iBAT from P2.5 mice. Volume-rendering in yellow color highlights TUNEL and DAPI co-localization. (C) TEM of degenerating adipocytes in scWAT and iBAT from Agpat2^−/− mice at P4.5. scWAT: red arrows indicate apoptotic bodies, yellow arrows indicate free lipid material (free “LDs”), red arrowhead indicates a phagocytic cell, blood vessel is outlined by a red dashed line. iBAT: red dashed areas show trace of extensive cell disintegration and leakage of intracellular components including lipid material. (D) Paraffin-embedded sections of scWAT and iBAT from Agpat2^+/+ and Agpat2^−/− littermates were stained with anti-MAC-2 and detected by immunofluorescence. (E) The mRNA level of genes involved in inflammatory response were assessed by qPCR at different postnatal time points in iBAT of Agpat2^+/+ and Agpat2^−/− mice. Graphs show the relative abundance of each transcript normalized to 36B4 mRNA. The bars show the means ± SD of N = 8. *** denote significant difference (p < 0.001) compared to Agpat2^+/+ mice at E18.5.

Figure 5

Adipogenic differentiation of Agpat2^−/−MEFs results in significantly fewer neutral lipid-laden cells after 6 days of differentiation. (A) Representative images (N > 10) from 6 days differentiated MEFs stained with the neutral lipid dye BODIPY and DAPI. (B) Graph shows the grade of adipogenic differentiation expressed as percentage of BODIPY stained cells. (C) Graph shows total cellular triglycerides quantified by an enzymatic-colorimetric method. (D) mRNA levels of adipogenic transcription factors and adipocyte related markers were quantified by qPCR. Gene expression was normalized to 36B4 mRNA levels and presented as fold-change relative to non-differentiated Agpat2^+/+ MEFs. Data correspond to the means ± SD of six independent experiments (N > 10). ***p < 0.001 and **p < 0.01 denote statistically significant difference compared to differentiated Agpat2^+/+ MEFs after 6 days of differentiation. (E) Three dimensional digital reconstruction of fluorescence stacks of differentiated Agpat2^+/+ and Agpat2^−/− MEFs stained with BODIPY and anti-Perilipin-1 antibody. (F) Representative confocal images of Caveolin-1 (red) and lipid droplets (green) in MEFs after 6 days of adipogenic differentiation. Caveolin-1 preferentially marks the plasma membrane but also intracellular structures, possibly the Golgi complex (white arrowheads).

Figure 6

AGPAT2 deficiency is associated with ultrastructural abnormalities in differentiated MEFs. Transmission electron microscopy of differentiated Agpat2^+/+ and Agpat2^−/− MEFs at day 6 post adipogenic induction. (A) Comparative images show lipid-laden cells from differentiated Agpat2^−/− MEFs (right panel) have aberrant mitochondria (m) and numerous autophagic structures (yellow arrowheads) in comparison with differentiated Agpat2^+/+ MEFs (left panel). (B) Representative imaging of mitochondria from differentiated Agpat2^−/− MEFs. (C) Representative imaging of autophagic structures that were observed only in differentiated Agpat2^−/− MEFs. (D) Relative percentage of lipid-laden cells containing more than 20 autophagic compartments per whole cell. (E) Representative imaging of plasma membrane contrasting the differences in the density of caveolae (red arrowheads) in differentiated MEFs of both genotypes. (F) Quantification of plasma membrane-associated caveloae normalized against membrane area. For image analysis and quantification, 10 differentiated lipid-laden cells per independent sample were imaged in each experiment with a total of N = 4 samples per experimental group).

Figure 7

Overexpression of PPARγ2 increases the proportion of lipid laden-cells and the gene expression profile but not the morphology of differentiated Agpat2^−/−MEFs. (A) qPCR quantification of C/EBPs and PPARγ mRNA levels at early and late stages of adipogenic differentiation. Data were normalized to 36B4 mRNA levels and expressed as relative fold changes to non-differentiated Agpat2^+/+ MEFs at day 0. (B) Immunoblot analysis of whole-cell protein extracts from Agpat2^+/+ and Agpat2^−/− MEFs at different days of differentiation. 50 μg of proteins were loaded, β-actin was used as loading control. (C) Confocal immunofluorescence analysis of PPARγ in Agpat2^+/+ and Agpat2^−/− MEFs at two different stages of adipocyte differentiation. Graphs show the relative total immunofluorescence signal of nuclear PPARγ per cell. (D) Time-line showing the adipogenic differentiation protocol used on primary cultures of Agpat2^+/+ and Agpat2^−/− MEFs previously infected with recombinant adenoviruses. (E) Adipogenic differentiation expressed as percentage of BODIPY stained cell after 6 days of differentiation. (F) Total cellular triglycerides quantified by an enzymatic-colorimetric method. (G) mRNA levels of adipogenic transcription factors and adipocyte related markers were quantified by qPCR. Gene expression was normalized to 36B4 mRNA levels and presented as fold-change relative to non-differentiated Agpat2^+/+ MEFs. (H) Three dimensional digital reconstruction of fluorescence stacks of differentiated Agpat2^+/+ and Agpat2^−/− MEFs immunostained with anti-Perilipin-1 antibody (pink). Neutral lipids were stained with BODIPY (green). All bar graphs show mean ± SD of three independent experiments (N > 6). *** (p < 0.001), **(p < 0.01) and *(p < 0.05) denote significant difference compared to Agpat2^+/+ MEFs.

Figure 8

Phospholipid quantification in differentiated Agpat2^+/+and Agpat2^−/−MEFs. (A) Pathways for PA and phospholipid synthesis in mammals. (B–C) Electron spray ionization-mass spectrometric analysis of phospholipids in differentiated Agpat2^+/+ and Agpat2^−/− MEFs. AGPAT: 1-acyl-sn-glycerol-3-phosphate acyltransferase; CL: cardiolipin; DAG: diacylglycerol; LPA: lysophosphatidic acid; PA: phosphatidic acid; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PI: phosphatidylinositol; PS: phosphatidylserine; TAG: triacylglycerol. All bar graphs show mean ± SD of three independent experiments (N > 6). ***p < 0.001, **p < 0.01 and *p < 0.05 denote significant difference compared to differentiated Agpat2^+/+ MEFs.