Whole-body deletion of Endospanin 1 protects from obesity-associated deleterious metabolic alterations.

The importance of the proper localization of most receptors at the cell surface is often underestimated, although this feature is essential for optimal receptor response. Endospanin 1 (Endo1) (also known as OBRGRP or LEPROT) is a protein generated from the same gene as the human leptin receptor and regulates the trafficking of proteins to the surface, including the leptin receptor. The systemic role of Endo1 on whole-body metabolism has not been studied so far. Here, we report that general Endo1-KO mice fed a high-fat diet develop metabolically healthy obesity with lipid repartitioning in organs and preferential accumulation of fat in adipose tissue, limited systematic inflammation, and better controlled glucose homeostasis. Mechanistically, Endo1 interacts with the lipid translocase CD36, thus regulating its surface abundance and lipid uptake in adipocytes. In humans, the level of Endo1 transcripts is increased in the adipose tissue of patients with obesity, but low levels rather correlate with a profile of metabolically healthy obesity. We suggest here that Endo1, most likely by controlling CD36 cell surface abundance and lipid uptake in adipocytes, dissociates obesity from diabetes and that its absence participates in metabolically healthy obesity.

Interferons are key cytokines acting on pancreatic islets in type 1 diabetes.

AIMS/HYPOTHESIS: The proinflammatory cytokines IFN-α, IFN-γ, IL-1ß and TNF-α may contribute to innate and adaptive immune responses during insulitis in type 1 diabetes and therefore represent attractive therapeutic targets to protect beta cells. However, the specific role of each of these cytokines individually on pancreatic beta cells remains unknown. METHODS: We used deep RNA-seq analysis, followed by extensive confirmation experiments based on reverse transcription-quantitative PCR (RT-qPCR), western blot, histology and use of siRNAs, to characterise the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by type 1 diabetes. RESULTS: IFN-α and IFN-γ had a greater impact on the beta cell transcriptome when compared with IL-1ß and TNF-α. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from individuals with type 1 diabetes, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to 'danger signals' such as viral infections. Zinc finger NFX1-type containing 1 (ZNFX1), a double-stranded RNA sensor, was identified as highly induced by IFNs and shown to play a key role in the antiviral response in beta cells. CONCLUSIONS/INTERPRETATION: These data suggest that IFN-α and IFN-γ are key cytokines at the islet level in human type 1 diabetes, contributing to the triggering and amplification of autoimmunity.

Inhibition of the type 1 diabetes candidate gene PTPN2 aggravates TNF-α-induced human beta cell dysfunction and death.

AIMS/HYPOTHESIS: TNF-α plays a role in pancreatic beta cell loss in type 1 diabetes mellitus. In clinical interventions, TNF-α inhibition preserves C-peptide levels in early type 1 diabetes. In this study we evaluated the crosstalk of TNF-α, as compared with type I IFNs, with the type 1 diabetes candidate gene PTPN2 (encoding protein tyrosine phosphatase non-receptor type 2 [PTPN2]) in human beta cells. METHODS: EndoC-ßH1 cells, dispersed human pancreatic islets or induced pluripotent stem cell (iPSC)-derived islet-like cells were transfected with siRNAs targeting various genes (siCTRL, siPTPN2, siJNK1, siJNK3 or siBIM). Cells were treated for 48 h with IFN-α (2000 U/ml) or TNF-α (1000 U/ml). Cell death was evaluated using Hoechst 33342 and propidium iodide staining. mRNA levels were assessed by quantitative reverse transcription PCR (qRT-PCR) and protein expression by immunoblot. RESULTS: PTPN2 silencing sensitised beta cells to cytotoxicity induced by IFN-α and/or TNF-α by 20-50%, depending on the human cell model utilised; there was no potentiation between the cytokines. We silenced c-Jun N-terminal kinase (JNK)1 or Bcl-2-like protein 2 (BIM), and this abolished the proapoptotic effects of IFN-α, TNF-α or the combination of both after PTPN2 inhibition. We further observed that PTPN2 silencing increased TNF-α-induced JNK1 and BIM phosphorylation and that JNK3 is necessary for beta cell resistance to IFN-α cytotoxicity. CONCLUSIONS/INTERPRETATION: We show that the type 1 diabetes candidate gene PTPN2 is a key regulator of the deleterious effects of TNF-α in human beta cells. It is conceivable that people with type 1 diabetes carrying risk-associated PTPN2 polymorphisms may particularly benefit from therapies inhibiting TNF-α.

Visceral and subcutaneous adipose tissue express and secrete functional alpha2hsglycoprotein (fetuin a) especially in obesity.

The secretion of the hepatokine alpha-2-Heremans-Schmid glycoprotein/Fetuin A, implicated in pathological processes including systemic insulin resistance, by adipose tissue has been recently described. Thus, we have recently identified its presence in white adipose tissue secretomes by mass spectrometry. However, the secretion pattern and function of adipose-derived alpha-2-Heremans-Schmid glycoprotein are poorly understood. The aim of this study is to evaluate the expression and secretion of total and active phosphorylated alpha-2-Heremans-Schmid glycoprotein by adipose tissue from visceral and subcutaneous localizations in animals at different physiological and nutritional status including anorexia and obesity. Alpha-2-Heremans-Schmid glycoprotein expression and secretion in visceral adipose tissue and subcutaneous adipose tissue explants from animals under fasting and exercise training, at pathological situations such as anorexia and obesity, and from human obese individuals were assayed by immunoblotting, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. We reveal that visceral adipose tissue expresses and secretes more alpha-2-Heremans-Schmid glycoprotein than subcutaneous adipose tissue, and that this secretion is diminished after fasting and exercise training. Visceral adipose tissue from anorectic animals showed reduced alpha-2-Heremans-Schmid glycoprotein secretion; on the contrary, alpha-2-Heremans-Schmid glycoprotein is over-secreted by visceral adipose tissue in the occurrence of obesity. While secretion of active-PhophoSer321α2HSG by visceral adipose tissue is independent of body mass index, we found that the fraction of active-alpha-2-Heremans-Schmid glycoprotein secreted by subcutaneous adipose tissue increments significantly in situations of obesity. Functional studies show that the inhibition of adipose-derived alpha-2-Heremans-Schmid glycoprotein increases insulin sensitivity in differentiated adipocytes. In conclusion, visceral adipose tissue secretes more alpha-2-Heremans-Schmid glycoprotein than subcutaneous adipose tissue and this secretion is more sensitive to nutritional and physiological changes. The over-secretion of alpha-2-Heremans-Schmid glycoprotein by visceral adipose tissue, the increased secretion of the active phosphorylated form by subcutaneous adipose tissuein obese animals, and the adipose-derived alpha-2-Heremans-Schmid glycoprotein capacity to inhibit the insulin pathway suggest the participation of adipose-derived alpha-2-Heremans-Schmid glycoprotein in the deleterious effects of obesity.

Comparative secretome analysis of rat stomach under different nutritional status.

The fact that gastric surgery is at the moment the most effective treatment to fight against obesity highlights the relevance of gastric derived proteins as potential targets to treat this pathology. Taking advantage of a previously established gastric explant model for endocrine studies, the proteomic analysis of gastric secretome was performed. To validate this gastric explant system for proteomic analysis, the identification of ghrelin, a classical gastric derived peptide, was performed by MS. In addition, the differential analysis of gastric secretomes under differential nutritional status (control feeding vs fasting vs re-feeding) was performed. The MS identified proteins are showed in the present manuscript. The data supplied in this article is related to the research article entitled "Comparative secretome analysis of rat stomach under different nutritional status" [1].

CILAIR-Based Secretome Analysis of Obese Visceral and Subcutaneous Adipose Tissues Reveals Distinctive ECM Remodeling and Inflammation Mediators.

In the context of obesity, strong evidences support a distinctive pathological contribution of adipose tissue depending on its anatomical site of accumulation. Therefore, subcutaneous adipose tissue (SAT) has been lately considered metabolically benign compared to visceral fat (VAT), whose location is associated to the risk of developing cardiovascular disease, insulin resistance, and other associated comorbidities. Under the above situation, the chronic local inflammation that characterizes obese adipose tissue, has acquired a major role on the pathogenesis of obesity. In this work, we have analyzed for the first time human obese VAT and SAT secretomes using an improved quantitative proteomic approach for the study of tissue secretomes, Comparison of Isotope-Labeled Amino acid Incorporation Rates (CILAIR). The use of double isotope-labeling-CILAIR approach to analyze VAT and SAT secretomes allowed the identification of location-specific secreted proteins and its differential secretion. Additionally to the very high percentage of identified proteins previously implicated in obesity or in its comorbidities, this approach was revealed as a useful tool for the study of the obese adipose tissue microenvironment including extracellular matrix (ECM) remodeling and inflammatory status. The results herein presented reinforce the fact that VAT and SAT depots have distinct features and contribute differentially to metabolic disease.

Comparative secretome analysis of rat stomach under different nutritional status.

Obesity is a major public health threat for many industrialised countries. Bariatric surgery is the most effective treatment against obesity, suggesting that gut derived signals are crucial for energy balance regulation. Several descriptive studies have proven the presence of gastric endogenous systems that modulate energy homeostasis; however, these systems and the interactions between them are still not well known. In the present study, we show for the first time the comparative 2-DE gastric secretome analysis under different nutritional status. We have identified 38 differently secreted proteins by comparing stomach secretomes from tissue explant cultures of rats under feeding, fasting and re-feeding conditions. Among the proteins identified, glyceraldehyde-3-phosphate dehydrogenase was found to be more abundant in gastric secretome and plasma after re-feeding, and downregulated in obesity. Additionally, two calponin-1 species were decreased in feeding state, and other were modulated by nutritional and metabolic conditions. These and other secreted proteins identified in this work may be considered as potential gastrokines implicated in food intake regulation. BIOLOGICAL SIGNIFICANCE: The present work has an important impact in the field of obesity, especially in the regulation of body weight maintenance by the stomach. Nowadays, the most effective treatment in the fight against obesity is bariatric surgery, which suggests that stomach derived signals might be crucial for the regulation of the energy homeostasis. However, until now, the knowledge about the gastrokines and its mechanism of action has been poorly elucidated. In the present work, we had updated a previously validated explant secretion model for proteomic studies; this analysis allowed us, for the first time, to study the gastric secretome without interferences from other organs. We had identified 38 differently secreted proteins comparing ex vivo cultured stomachs from rats under feeding, fasting and re-feeding regimes. The results in the present article provide novel targets to study the role of the stomach in body weight and appetite regulation, and suggest new potential therapeutic targets for treating obesity.

The gastric CB1 receptor modulates ghrelin production through the mTOR pathway to regulate food intake.

Over the years, the knowledge regarding the relevance of the cannabinoid system to the regulation of metabolism has grown steadily. A central interaction between the cannabinoid system and ghrelin has been suggested to regulate food intake. Although the stomach is the main source of ghrelin and CB1 receptor expression in the stomach has been described, little information is available regarding the possible interaction between the gastric cannabinoid and ghrelin systems in the integrated control of energy homeostasis. The main objective of the present work was to assess the functional interaction between these two systems in terms of food intake using a combination of in vivo and in vitro approaches. The present work demonstrates that the peripheral blockade of the CB1 receptor by rimonabant treatment decreased food intake but only in food-deprived animals. This anorexigenic effect is likely a consequence of decreases in gastric ghrelin secretion induced by the activation of the mTOR/S6K1 intracellular pathway in the stomach following treatment with rimonabant. In support of this supposition, animals in which the mTOR/S6K1 intracellular pathway was blocked by chronic rapamycin treatment, rimonabant had no effect on ghrelin secretion. Vagal communication may also be involved because rimonabant treatment was no longer effective when administered to animals that had undergone surgical vagotomy. In conclusion, to the best of our knowledge, the present work is the first to describe a CB1 receptor-mediated mechanism that influences gastric ghrelin secretion and food intake through the mTOR pathway.

FNDC5/irisin is not only a myokine but also an adipokine.

Exercise provides clear beneficial effects for the prevention of numerous diseases. However, many of the molecular events responsible for the curative and protective role of exercise remain elusive. The recent discovery of FNDC5/irisin protein that is liberated by muscle tissue in response to exercise might be an important finding with regard to this unsolved mechanism. The most striking aspect of this myokine is its alleged capacity to drive brown-fat development of white fat and thermogenesis. However, the nature and secretion form of this new protein is controversial. The present study reveals that rat skeletal muscle secretes a 25 kDa form of FNDC5, while the 12 kDa/irisin theoretical peptide was not detected. More importantly, this study is the first to reveal that white adipose tissue (WAT) also secretes FNDC5; hence, it may also behave as an adipokine. Our data using rat adipose tissue explants secretomes proves that visceral adipose tissue (VAT), and especially subcutaneous adipose tissue (SAT), express and secrete FNDC5. We also show that short-term periods of endurance exercise training induced FNDC5 secretion by SAT and VAT. Moreover, we observed that WAT significantly reduced FNDC5 secretion in fasting animals. Interestingly, WAT of obese animals over-secreted this hormone, which might suggest a type of resistance. Because 72% of circulating FNDC5/irisin was previously attributed to muscle secretion, our findings suggest a muscle-adipose tissue crosstalk through a regulatory feedback mechanism.

Muscle tissue as an endocrine organ: comparative secretome profiling of slow-oxidative and fast-glycolytic rat muscle explants and its variation with exercise.

The notion that skeletal muscle is a secretory organ capable to release proteins that can act locally in an autocrine/paracrine manner or even in an endocrine manner to communicate with distant tissues has now been recognized. Under this context, a new paradigm has arisen implicating the muscle in metabolism regulation. Considering the evidences that give exercise a protective role against illnesses associated to physical inactivity, it becomes of especial relevance to characterize muscle secreted proteins. In the present study we show for the first time the secretome characterization and the comparative 2-DE secretome analysis among fast-glycolytic (gastrocnemius) and slow-oxidative (soleus) rat muscle explants and its variation after exercise intervention. We have identified 19 differently secreted proteins when comparing soleus and gastrocnemius secretomes, and 10 in gastrocnemius and 17 in soleus distinctive secreted proteins after 1 week of endurance exercise training. Among identified proteins, DJ-1 was found to be more abundant in fast-glycolytic fiber secretomes. On the contrary, FABP-3 was elevated in slow-oxidative fiber secretomes, although its secretion from gastrocnemius muscle increased in exercised animals. These and other secreted proteins identified in this work may be considered as potential myokines.

Obesidomics: contribution of adipose tissue secretome analysis to obesity research.

Obesity is presently reaching pandemic proportions and it is becoming a major health concern in developed and developing countries due to its comorbidities like type II diabetes, cardiovascular pathologies, and some cancers. The discovery of the adipose tissue role as an endocrine gland able to secrete adipokines that affects whole-body energy homeostasis has become a key break-through toward a better molecular understanding of obesity. Among the known adipokines involved in the regulation of energy metabolism very few have been clearly seen as central regulators of insulin sensitivity, metabolism, and energy homeostasis. Thus, the discovery and characterization of new adipocyte-derived factors is still in progress. Proteomics technology has emerged as a useful tool to analyze adipose tissue secretion (secretome) dynamics giving a wider picture into the molecular events that control body weight. Besides the identification of new secreted proteins, the advantage of using this approach is the possibility to detect post-translational modifications and protein interactions that generally cannot be predicted by genome studies. In this review, we summarize the recent efforts to identify new bioactive adipokines by proteomics especially in pathological situations such as obesity.

Secretome analysis of rat adipose tissues shows location-specific roles for each depot type.

Obesity prevalence is reaching pandemic proportions becoming a major public health threat for many industrialized nations. It is especially worrying as it causes a higher risk of premature death due to associated diseases such as type 2 diabetes, cardiovascular disease, and some cancers. Current evidence shows biological and genetic differences between adipose tissues depending on its anatomical location. Particularly, upper body/visceral fat distribution in obesity is closely linked to metabolic complications. In this report, we characterize for the first time the secretome of rat adipose tissue explants from different anatomical localizations and its differential analysis. Visceral, subcutaneous, and gonadal fat specific secretomes and differentially secreted proteins among the three fat depots were analyzed by 2-DE and MS. Reference maps for location-specific adipose tissue secretomes are shown and the 45 most significant differences are listed. Identified proteins include classical adipokines and novel secreted proteins. Interestingly, our results show that the type of proteins and their role in different biological processes diverge significantly when comparing the set of proteins identified from visceral, subcutaneous and gonadal fat explants. This study emphasizes and supports the differential role of adipose tissue in accordance to its anatomical localization.

Obestatin as a regulator of adipocyte metabolism and adipogenesis.

The role of obestatin, a 23-amino-acid peptide encoded by the ghrelin gene, on the control of the metabolism of pre-adipocyte and adipocytes as well as on adipogenesis was determined. For in vitro assays, pre-adipocyte and adipocyte 3T3-L1 cells were used to assess the obestatin effect on cell metabolism and adipogenesis based on the regulation of the key enzymatic nodes, Akt and AMPK and their downstream targets. For in vivo assays, white adipose tissue (WAT) was obtained from male rats under continuous subcutaneous infusion of obestatin. Obestatin activated Akt and its downstream targets, GSK3α/ß, mTOR and S6K1, in 3T3-L1 adipocyte cells. Simultaneously, obestatin inactivated AMPK in this cell model. In keeping with this, ACC phosphorylation was also decreased. This fact was confirmed in vivo in white adipose tissue (omental, subcutaneous and gonadal) obtained from male rats under continuous sc infusion of obestatin (24 and 72 hrs). The relevance of obestatin as regulator of adipocyte metabolism was supported by AS160 phosphorylation, GLUT4 translocation and augment of glucose uptake in 3T3-L1 adipocyte cells. In contrast, obestatin failed to modify translocation of fatty acid transporters, FATP1, FATP4 and FAT/CD36, to plasma membrane. Obestatin treatment in combination with IBMX and DEX showed to regulate the expression of C/EBPα, C/EBPß, C/EBPδ and PPARγ promoting adipogenesis. Remarkable, preproghrelin expression, and thus obestatin expression, increased during adipogenesis being sustained throughout terminal differentiation. Neutralization of endogenous obestatin secreted by 3T3-L1 cells by anti-obestatin antibody decreased adipocyte differentiation. Furthermore, knockdown experiments by preproghrelin siRNA supported that obestatin contributes to adipogenesis. In summary, obestatin promotes adipogenesis in an autocrine/paracrine manner, being a regulator of adipocyte metabolism. These data point to a putative role in the pathogenesis of metabolic syndrome.

Quorum quenching in cultivable bacteria from dense marine coastal microbial communities.

Acylhomoserine lactone (AHLs)-mediated quorum-sensing (QS) processes seem to be common in the marine environment and among marine pathogenic bacteria, but no data are available on the prevalence of bacteria capable of interfering with QS in the sea, a process that has been generally termed 'quorum quenching' (QQ). One hundred and sixty-six strains isolated from different marine dense microbial communities were screened for their ability to interfere with AHL activity. Twenty-four strains (14.4%) were able to eliminate or significantly reduce N-hexanoyl-l-homoserine lactone activity as detected by the biosensor strain Chromobacterium violaceum CV026, a much higher percentage than that reported for soil isolates, which reinforces the ecological role of QS and QQ in the marine environment. Among these, 15 strains were also able to inhibit N-decanoyl-l-homoserine lactone activity and all of them were confirmed to enzymatically inactivate the AHL signals by HPLC-MS. Active isolates belonged to nine different genera of prevalently or exclusively marine origin, including members of the Alpha- and Gammaproteobacteria (8), Actinobacteria (2), Firmicutes (4) and Bacteroidetes (1). Whether the high frequency and diversity of cultivable bacteria with QQ activity found in near-shore marine isolates reflects their prevalence among pelagic marine bacterial communities deserves further investigation in order to understand the ecological importance of AHL-mediated QS and QQ processes in the marine environment.

Peripheral leptin and ghrelin receptors are regulated in a tissue-specific manner in activity-based anorexia.

The aim of this research was to investigate the effect of long-term exposure to low leptin and high ghrelin levels, inherent to activity-based anorexia (ABA), on peripheral metabolism-implicated tissues such as muscle and fat depots. For this purpose, rats under ABA were submitted to a global study which included the characterization of body weight and composition change, the evaluation of leptin and ghrelin levels as well as their receptors expression at peripheral level. Our results confirm that feeding restriction to 1 h per day, and particularly the combination of this fasting regime with exercise (ABA), significantly reduces fat mass, decreases leptin circulating levels, increases ghrelin levels strikingly and enhances insulin sensitivity. By direct in vitro assays, we show that visceral and gonadal fat participate more than subcutaneous fat in the hypoleptinemia of these animals. The study of ghrelin (GHS-R1a) and leptin (LEPR) receptors at peripheral level exhibits a tissue-specific expression pattern. Concretely, oxidative-soleus type of muscle appears to be more susceptible to ghrelin and leptin circulating levels than glycolytic-gastrocnemius type under exercise and food restriction situations. In relation to adipose tissue, chronic hyperghrelinemia induces GHS-R1a expression on visceral and subcutaneous fat which might suggest the prevention of lipid loss. On the other hand, only subcutaneous fat express the active long form of LEPR compared to visceral and gonadal fat under low leptin levels in ABA animals. All together, these findings indicate tissue-specific mechanisms for the control of energy homeostasis in response to nutrient and energy availability.