Mechanisms involved in the loss of brown adipose tissue in the AGPAT2 deficient mouse

Abstract

1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2) is an enzyme that participates in the synthesis of triacylglycerols and glycerophospholipids. In the differentiation of adipocytes (adipogenesis) the storage of lipids in lipid droplets of adipocytes plays a fundamental role. Mutations in the gene coding for this enzyme generate generalized congenital lipodystrophy, a disease characterized by severe reduction of white and brown adipose tissue, as well as severe metabolic complications. Mice deficient for AGPAT2 (Agpat2-/-) are born with brown adipose tissue of morphology and abundance similar to that of normal mice, however, this tissue degenerates completely during the first 6 days after birth. The causes of this phenomenon are currently unknown. The hypothesis of this thesis is that brown adipocytes of Agpat2-/- mice have transcriptional abnormalities that determine defective adipogenesis and greater susceptibility to lipotoxic cell death. To evaluate this hypothesis, brown preadipocytes from the interscapular adipose tissue of Agpat2-/- and wild type mice were differentiated adipogenically in cell culture and compared in various morphological and molecular parameters. Differentiated preadipocyte cultures Agpat2-/- showed a lower proportion of lipid-laden cells throughout the differentiation and a decreased expression of the pro-adipogenic transcriptional regulators PPARy, PPARα, C/EBPα and PGC1α. Importantly, the transcription factors PRDM16 and C/EBPβ, previously identified as mediators of brown adipogenic differentiation, had normal levels in the Agpat2-/- brown adipocytes differentiated in vitro. Accordingly, the UCP1 protein, a specific marker of mature brown adipocyte, remained undetectable in the differentiated Agpat2-/- adipocytes. On the contrary, their levels were strongly induced in wild type differentiated adipocytes in vitro. Ultrastructural analysis of the differentiated adipocytes showed that the mitochondria of the15 differentiated Agpat2-/- adipocytes have notoriously altered morphology and are not physically associated with lipid droplets, unlike what happens in differentiated wild type adipocytes. The overall transcriptome analysis of adipocytes differentiated in vitro showed significant divergence in the abundance of mRNA encoded by several groups of genes. These include, lower expression of genes related to the accumulation of lipids and mitochondrial structure and function, and greater expression of genes stimulated by interferon. These results were independently verified by qRT-PCR analysis. Additionally, differentiated Agpat2-/- adipocyte cultures have lower levels of Elovl3 and mitoNEET, both previously identified as regulators of differentiation and adipose function. Importantly, forced overexpression of these by means of adenoviral vectors did not normalize the accumulation of lipid droplets in differentiated Agpat2-/- adipocytes. Finally, to evaluate the increased susceptibility to cell death by lipotoxicity in differentiated Agpat2-/- adipocytes, the concentration and composition of fatty acids in the plasma of newborn Agpat2-/- mice were quantified in comparison with wild type mice of the same age, showing a marked elevation of all classes of fatty acids, particularly saturated and monounsaturated, from the second postnatal day. Among the first ones, palmitic acid stood out, whose concentration was ~5 times higher in the newborn Agpat2-/- mice compared with the wild type. The incubation of Agpat2-/- and wild type preadipocytes with palmitic acid, showed similar levels of cell death caused by lipotoxicity. However, when adipocytes differentiated in vitro were exposed to palmitic acid, Agpat2-/- cultures had significantly higher cell death levels compared to wild typE