Exosomal proteins constitute an essential part of the human adipose tissue secretome

Highlights

• Exosomes from primary human adipocytes contribute essential to the secretome.

• Proteome analysis identified 884 exosomal derived proteins → Exoadipokines.

• 212 proteins are found in both the secretome and the exosomal fraction.

• Exoadipokines are assigned to signaling pathways and membrane-mediated processes.

Abstract

Adipose tissue is an endocrine organ, secreting various adipokines, either directly or via extracellular vesicles, including exosomes. Exosomes are vesicles of 40–150 nm size that represent a novel concept of biomolecule release. We purified exosomes from isolated primary human preadipocytes differentiated to mature adipocytes. The analyses of these exosomal preparations by LC-MS identified 884 proteins, so called exoadipokines. The comparison of exoadipokines with previously identified human exosome-associated proteins in ExoCarta database show an overlap of 817 proteins, but also revealed 67 proteins not assigned to human exosomes, yet. We further compared all exoadipokines to our previously reported reference secretome of human adipose tissue (http://diabesityprot.org/), finding 212 common proteins, whereas 672 proteins were specific for the exosomal fraction. Bioinformatic analyses revealed that the 212 common proteins can be assigned to all major functions of adipose tissue secreted proteins e.g. molecules involved in fibrotic processes or inflammation. In contrast, the exosome-specific proteins were rather assigned to signaling pathways and membrane-mediated processes. In conclusion, the isolation of exosomes allows to further specify the functionality of adipokines and exoadipokines as part of the adipocyte secretome in signaling and interorgan crosstalk.

Introduction

The prevalence of obesity has reached pandemic dimensions, with dramatic consequences for public health [1]. Imbalanced energy supply and consumption coupled with a sedentary lifestyle favor obesity, which represents a major risk factor for developing various disorders such as type 2 diabetes mellitus, non-alcoholic liver and cardiovascular diseases [[2], [3], [4], [5]]. One of the major developments in obesity research over the past decade is the recognition of adipose tissue as a major endocrine organ. Adipocytes release a multiplicity of protein hormones and signaling factors, so called adipokines [6], by which adipose tissue communicates with other organs including skeletal muscle and liver. Alterations of these complex protein signatures are suggested to play a crucial role in the pathophysiology of metabolic diseases [7]. Detailed characterization adipokine secretion profiles will likely improve the understanding of the molecular pathophysiology of metabolic disorders. Accordingly, several attempts have been made to elucidate the complex nature of adipose tissue secretomes utilizing different proteomic approaches [8]. In this context, we identified several hundred proteins, which were annotated to be putatively secreted by primary human adipocytes [9] and can be found at http://diabesityprot.org/.

Using web-based computational filter algorithms like SignalP and SecretomeP, a relevant proportion of consistently identified adipokines has been assigned to intracellular non-secretory proteins [10,11]. There is evidence that these proteins might be shuttled using unconventional protein secretion pathways [[12], [13], [14]]. In addition to conventional secretion via the ER and trans-Golgi network (TGN), cytosolic proteins can be directly secreted via carrier proteins or can be directly transported to the plasma membrane or lysosomal compartments bypassing Golgi processing [15]. Furthermore, some proteins are secreted via microvesicles or exosomes [16]. Microvesciles, sized 50 nm to 1000 nm, bud from the plasma membrane, and are known to be involved in protein secretion since the late 90s when interleukin-1ß release could be associated to microvesicles [17]. In many publications, unconventional protein secretion (UPS) is only associated with cellular stress, e.g. apoptotic bodies, but more and more evidence exists that it is a form of regular intercellular communication [18]. Exosomes are vesicles of 40–150 nm of size mainly released via multivesicular bodies (MVBs), which can contain microRNAs and numerous molecules involved in signal transduction, such as protein kinases and heterotrimeric G-proteins, various metabolic enzymes and heat-shock proteins Hsp70 and Hsp90 [19]. Experimental data indicates that transport of intracellular proteins to the extracellular compartment via exosomes and its uptake by other cells constitutes an important component of the overall secretome composition with distant effects of the shuttled cargo [20]. This notion should be considered for comprehensive secretome analysis approaches.

Therefore, the current study aims at elucidating the nature of proteins released by exosomes from primary human adipocytes in comparions with the overall secretome from the same cells. The evaluation of exosomal signatures with the corresponding reference secretome [9] indicate a significant contribution of exosomal messenging within the overall secretomes derived from adipose tissue.