Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions.

BACKGROUND: Excessive lipid accumulation in the adipose tissue in obesity alters the endocrine and energy storage functions of adipocytes. Adipocyte lipid droplets represent key organelles coordinating lipid storage and mobilization in these cells. Recently, we identified the small GTPase, Rab34, in the lipid droplet proteome of adipocytes. Herein, we have characterized the distribution, intracellular transport, and potential contribution of this GTPase to adipocyte physiology and its regulation in obesity. METHODS: 3T3-L1 and human primary preadipocytes were differentiated in vitro and Rab34 distribution and trafficking were analyzed using markers of cellular compartments. 3T3-L1 adipocytes were transfected with expression vectors and/or Rab34 siRNA and assessed for secretory activity, lipid accumulation and expression of proteins regulating lipid metabolism. Proteomic and protein interaction analyses were employed for the identification of the Rab34 interactome. These studies were combined with functional analysis to unveil the role played by the GTPase in adipocytes, with a focus on the actions conveyed by Rab34 interacting proteins. Finally, Rab34 regulation in response to obesity was also evaluated. RESULTS: Our results show that Rab34 localizes at the Golgi apparatus in preadipocytes. During lipid droplet biogenesis, Rab34 translocates from the Golgi to endoplasmic reticulum-related compartments and then reaches the surface of adipocyte lipid droplets. Rab34 exerts distinct functions related to its intracellular location. Thus, at the Golgi, Rab34 regulates cisternae integrity as well as adiponectin trafficking and oligomerization. At the lipid droplets, this GTPase controls lipid accumulation and lipolysis through its interaction with the E1-ubiquitin ligase, UBA1, which induces the ubiquitination and proteasomal degradation of the fatty acid transporter and member of Rab34 interactome, FABP5. Finally, Rab34 levels in the adipose tissue and adipocytes are regulated in response to obesity and related pathogenic insults (i.e., fibrosis). CONCLUSIONS: Rab34 plays relevant roles during adipocyte differentiation, including from the regulation of the oligomerization (i.e., biological activity) and secretion of a major adipokine with insulin-sensitizing actions, adiponectin, to lipid storage and mobilization from lipid droplets. Rab34 dysregulation in obesity may contribute to the altered adipokine secretion and lipid metabolism that characterize adipocyte dysfunction in conditions of excess adiposity.

Rab18 Drift in Lipid Droplet and Endoplasmic Reticulum Interactions of Adipocytes under Obesogenic Conditions.

The adipose tissue stores excess energy in the form of neutral lipids within adipocyte lipid droplets (LDs). The correct function of LDs requires the interaction with other organelles, such as the endoplasmic reticulum (ER) as well as with LD coat-associated proteins, including Rab18, a mediator of intracellular lipid trafficking and ER-LD interaction. Although perturbations of the inter-organelle contact sites have been linked to several diseases, such as cancer, no information regarding ER-LD contact sites in dysfunctional adipocytes from the obese adipose tissue has been published to date. Herein, the ER-LD connection and Rab18 distribution at ER-LD contact sites are examined in adipocytes challenged with fibrosis and inflammatory conditions, which represent known hallmarks of the adipose tissue in obesity. Our results show that adipocytes differentiated in fibrotic conditions caused ER fragmentation, the expansion of ER-LD contact sites, and modified Rab18 dynamics. Likewise, adipocytes exposed to inflammatory conditions favored ER-LD contact, Rab18 accumulation in the ER, and Rab18 redistribution to large LDs. Finally, our studies in human adipocytes supported the suggestion that Rab18 transitions to the LD coat from the ER. Taken together, our results suggest that obesity-related pathogenic processes alter the maintenance of ER-LD interactions and interfere with Rab18 trafficking through these contact sites.

Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.

Adipose tissue dysregulation in obesity strongly influences systemic metabolic homeostasis and is often linked to insulin resistance (IR). However, the molecular mechanisms underlying adipose tissue dysfunction in obesity are not fully understood. Herein, a proteomic analysis of subcutaneous (SC) and omental (OM) fat from lean subjects and obese individuals with different degrees of insulin sensitivity was performed to identify adipose tissue biomarkers related to obesity-associated metabolic disease. Our results suggest that dysregulation of both adipose tissue extracellular matrix (ECM) organization and intracellular trafficking processes may be associated with IR in obesity. Thus, abnormal accumulation of the small leucine-rich proteoglycan, lumican, as observed in SC fat of IR obese individuals, modifies collagen I organization, impairs adipogenesis and activates stress processes [endoplasmic reticulum and oxidative stress] in adipocytes. In OM fat, IR is associated with increased levels of the negative regulator of the Rab family of small GTPases, GDI2, which alters lipid storage in adipocytes by inhibiting insulin-stimulated binding of the Rab protein, Rab18, to lipid droplets. Together, these results indicate that lumican and GDI2 might play depot-dependent, pathogenic roles in obesity-associated IR. Our findings provide novel insights into the differential maladaptive responses of SC and OM adipose tissue linking obesity to IR.

Interrelation of adipose tissue macrophages and fibrosis in obesity.

Obesity is characterized by adipose tissue expansion, extracellular matrix remodelling and unresolved inflammation that contribute to insulin resistance and fibrosis. Adipose tissue macrophages represent the most abundant class of immune cells in adipose tissue inflammation and could be key mediators of adipocyte dysfunction and fibrosis in obesity. Although macrophage activation states are classically defined by the M1/M2 polarization nomenclature, novel studies have revealed a more complex range of macrophage phenotypes in response to external condition or the surrounding microenvironment. Here, we discuss the plasticity of adipose tissue macrophages (ATMs) in response to their microenvironment in obesity, with special focus on macrophage infiltration and polarization, and their contribution to adipose tissue fibrosis. A better understanding of the role of ATMs as regulators of adipose tissue remodelling may provide novel therapeutic strategies against obesity and associated metabolic diseases.

Understanding the adipose tissue acetylome in obesity and insulin resistance.

Obesity is a widely prevalent pathology with a high exponential growth worldwide. Altered lipid accumulation by adipose tissue is one of the main causes of obesity and exploring lipid homeostasis in this tissue may represent a source for the identification of possible therapeutic targets. The study of the proteome and the post-translational modifications of proteins, specifically acetylation due to its involvement in energy metabolism, may be of great interest to understand the molecular mechanisms involved in adipose tissue dysfunction in obesity. The objective of this study was to characterize the subcutaneous and omental adipose tissue acetylome in conditions of obesity and insulin resistance and to describe the importance of acetylation of key molecules in adipose tissue to use them as therapeutic targets. The results describe for the first time the acetylome of subcutaneous and omental adipose tissue under physiological and physiopathological conditions such as obesity and insulin resistance. New evidence showed different acetylation patterns between two main depots and highlight the molecular complexity of adipose tissue. Results showed changes in FABP4 acetylation in subcutaneous fat in relation to insulin resistance, thus unveiling a potential marker of depot-specific dysfunctional expansion in obesity-associated metabolic disease. Furthermore, it is shown that the acetylation of FABP4 affects its function, modulating the capacity of differentiation in adipocytes. In conclusion, this study demonstrates a profound, depot-specific alteration of adipose tissue acetylome, wherein the acetylation of FABP4 may play a key role in adipocyte differentiation and lipid accumulation.

Influence of Protein Carbonylation on Human Adipose Tissue Dysfunction in Obesity and Insulin Resistance.

BACKGROUND: Obesity is characterized by adipose tissue dysregulation and predisposes individuals to insulin resistance and type 2 diabetes. At the molecular level, adipocyte dysfunction has been linked to obesity-triggered oxidative stress and protein carbonylation, considering protein carbonylation as a link between oxidative stress and metabolic dysfunction. The identification of specific carbonylated proteins in adipose tissue could provide novel biomarkers of oxidative damage related to metabolic status (i.e prediabetes). Thus, we aimed at characterizing the subcutaneous and omental human adipose tissue carbonylome in obesity-associated insulin resistance. METHODS: 2D-PAGE was used to identify carbonylated proteins, and clinical correlations studies and molecular biology approaches including intracellular trafficking, reactive oxygen species assay, and iron content were performed using in vitro models of insulin resistance. RESULTS: The carbonylome of human adipose tissue included common (serotransferrin, vimentin, actin, and annexin A2) and depot-specific (carbonic anhydrase and α-crystallin B in the subcutaneous depot; and α-1-antitrypsin and tubulin in the omental depot) differences that point out the complexity of oxidative stress at the metabolic level, highlighting changes in carbonylated transferrin expression. Posterior studies using in vitro prediabetic model evidence alteration in transferrin receptor translocation, linked to the prediabetic environment. Finally, ligand-receptor molecular docking studies showed a reduced affinity for carbonylated transferrin binding to its receptor compared to wild-type transferrin, emphasizing the role of transferrin carbonylation in the link between oxidative stress and metabolic dysfunction. CONCLUSIONS: The adipose tissue carbonylome contributes to understanding the molecular mechanism driving adipocyte dysfunction and identifies possible adipose tissue carbonylated targets in obesity-associated insulin resistance.