Abstract
Lipodystrophy is a major disease involving severe alterations of adipose tissue distribution and metabolism. Mutations in genes encoding the nuclear envelope protein lamin A or its processing enzyme, the metalloproteinase Zmpste24, cause diverse human progeroid syndromes that are commonly characterized by a selective loss of adipose tissue. Similarly to humans, mice deficient in Zmpste24 accumulate prelamin A and display phenotypic features of accelerated aging, including lipodystrophy. Herein, we report the proteome and phosphoproteome of adipose tissue as well as serum metabolome in lipodystrophy by using Zmpste24(-/-) mice as experimental model. We show that Zmpste24 deficiency enhanced lipolysis, fatty acid biogenesis and β-oxidation as well as decreased fatty acid re-esterification, thus pointing to an increased partitioning of fatty acid toward β-oxidation and away from storage that likely underlies the observed size reduction of Zmpste24-null adipocytes. Besides the mitochondrial proteins related to lipid metabolism, other protein networks related to mitochondrial function, including those involved in tricarboxylic acid cycle and oxidative phosphorylation, were up-regulated in Zmpste24(-/-) mice. These results, together with the observation of an increased mitochondrial response to oxidative stress, support the relationship between defective prelamin A processing and mitochondrial dysfunction and highlight the relevance of oxidative damage in lipoatrophy and aging. We also show that absence of Zmpste24 profoundly alters the processing of the cytoskeletal protein vimentin and identify a novel protein dysregulated in lipodystrophy, High-Mobility Group Box-1 Protein. Finally, we found several lipid derivates with important roles in energy balance, such as Lysophosphatidylcholine or 2-arachidonoylglycerol, to be dysregulated in Zmpste24(-/-) serum. Together, our findings in Zmpste24(-/-) mice may be useful to unveil the mechanisms underlying adipose tissue dysfunction and its overall contribution to body homeostasis in progeria and other lipodystrophy syndromes as well as to develop novel strategies to prevent or ameliorate these diseases.
Highlights
From the ‡Department of Cell Biology, Physiology and Immunology, University of Cordoba
We have observed that they suffer from a marked reduction of the visceral fat depot (Fig. 2A), which in some cases reached more than a sixfold decrease when compared with that observed in their wild-type littermates
Analysis of Mitochondrial Function in Zmpste24Ϫ/Ϫ Adipose Tissue—Given the finding that two proteins potentially involved in mitochondrial fatty acid -oxidation, long-chain acyl-CoA synthetase-1 (ACSL1) and acyl-coenzymeA dehydrogenase, short chain (ACADS), were up-regulated in adipose tissue of Zmpste24Ϫ/Ϫ mice, we investigated whether mitochondrial fatty acid import could be affected in these animals by quantifying protein levels of the key enzyme mediating this process, carnitine palmitoyltransferase I (CPT1) [34]
Summary
Animals—Mutant mice deficient in Zmpste metalloproteinase have been previously described [26]. Histological Analysis—For histological analysis, adipose tissue samples from the visceral fat pad obtained from four Zmpste24Ϫ/Ϫ and three wild-type animals were fixed in 4% paraformaldehyde in PBS and stored in 70% ethanol. Immunoblotting—Frozen fat samples from four additional wild-type and Zmpste24Ϫ/Ϫ mice distinct from those employed for 2-DE were disrupted in Triton buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% Triton, and complete protease inhibitor) and incubated in the presence of 30 units of DNase I (Sigma) for 15 min on ice. Thirty to 70 g of protein were loaded on 10% SDS-PAGE and transferred to nitrocellulose membranes (Biotrace, Pall, Germany).
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