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Proteome characterization of mitochondria, ER, and mitochondrial-associated membrane from BAT and liver

ABSTRACT: Mitochondria are the provider of cellular energy from aerobic respiration in all tissues. However, only brown adipose tissue (BAT) mitochondria are able to convert chemical energy in the form triglycerides into heat (thermogenesis). To ask if cellular respiration or thermogenic function require a specific lipidomic environment, we investigated the lipid species composition of brown adipose tissue mitochondria, and compared it those purified from liver, skeletal muscle and white adipose tissue. We emplyed mass spectrometry-based proteomics to evalaute the purity of the mitochondia fractions isolated from murine BAT and liver tissue by differential centrifugation, by comparison of its full proteome with endoplasmatic reticulum, mitochondrial-associated membrane, and mitochondria fractions isolated by density gradient centrifugation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brown Adipose Tissue, Liver

SUBMITTER: Piero Giansanti

LAB HEAD: Bernhard Kuster

PROVIDER: PXD028128 | Pride | 2024-05-24

REPOSITORIES: Pride

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			Action	DRS
	3935_GA10_043571_S00_T16_R1.raw	Raw
	3935_GA11_043571_S00_T17_R1.raw	Raw
	3935_GA12_043571_S00_T32_R1.raw	Raw
	3935_GA1_043569_S00_T01_R1.raw	Raw
	3935_GA2_043569_S00_T16_R1.raw	Raw

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Project description:Thoracic perivascular adipose tissue (PVAT) is a unique adipose depot that likely influences vascular function and susceptibility to pathogenesis in obesity and metabolic syndrome. Surprisingly, PVAT has been reported to share characteristics of both brown and white adipose, but a detailed direct comparison to interscapular brown adipose tissue (BAT) has not been performed. Here we show by full genome DNA microarray analysis that global gene expression profiles of PVAT are virtually identical to BAT, with equally high expression of Ucp-1, Cidea and other genes known to be uniquely or very highly expressed in BAT. PVAT and BAT also displayed nearly identical phenotypes upon immunohistochemical analysis, and electron microscopy confirmed that PVAT contained multilocular lipid droplets and abundant mitochondria. Compared to white adipose tissue (WAT), PVAT and BAT from C57BL/6 mice fed a high fat diet for 13 weeks had markedly lower expression of immune cell-enriched mRNAs, suggesting resistance to obesity-induced inflammation. Indeed, staining of BAT and PVAT for macrophage markers (F4/80, CD68) in obese mice showed virtually no macrophage infiltration, and FACS analysis of BAT confirmed the presence of very few CD11b+/CD11c+ macrophages in BAT (1.0%) in comparison to WAT (31%). In summary, murine PVAT from the thoracic aorta is virtually identical to interscapular BAT, is resistant to diet-induced macrophage infiltration, and thus may play an important role in protecting the vascular bed from thermal and inflammatory stress. 8-week-old male C57BL6/J mice were fed a normal (ND) or high fat diet (HFD) (Research Diets 12451, 45 kcal% fat) for 13 weeks. Mice were then euthanized and four different adipose depots were harvested for RNA analysis: perivascular fat from the lesser curvature of the aortic arch (PVAT), interscapular brown adipose (BAT), inguinal adipose tissue (SAT), and epididymal adipose tissue (VAT). 250 ng total RNA pooled from two mice was used for cDNA synthesis; 3 biological replicates per tissue and diet were performed for a total of 24 hybridizations.

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Proteome characterization of mitochondria, ER, and mitochondrial-associated membrane from BAT and liver

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