Year: 2021
Type: dissertation
Abstract: Obesity is the pandemic of the 21st century. Overall, about 15% of the world’s adult population is obese, and if the rising trend continues, it is estimated that global prevalence reaches 20% by 2025. Obesity poses a major public health issue due to its elevated risks for adverse health consequences, including several serious chronic diseases, such as insulin resistance (IR), type 2 diabetes (T2D), non-alcoholic fatty liver disease (NAFLD), cardiovascular disease, dyslipidemia, hypertension, stroke, hypercholesterolemia, hypertriglyceridemia, arthritis, asthma, neurodegenerative diseases, and even certain forms of cancer. Many factors and mechanisms have been implicated in obesity pathogenesis, but their trigger is uncertain, and the causal relationship between them and the complications of obesity remains in question. However, there is little doubt that obesity is closely associated with adipose tissue dysfunction, and that this condition leads to the development of metabolic diseases. Adipose tissue is a complex organ with primary roles in energy homeostasis control. Thus, adipose tissue not only acts as a reservoir for energy storage and utilization, but also it senses energy demands and secretes signalling factors to regulate other metabolic tissues. However, in obesity, adipose tissue may become severely dysfunctional and not expand properly to store the energy excess. This induces ectopic fat deposition in other tissues involved in the maintenance of glucose homeostasis, an event commonly defined as “lipotoxicity”. It has been extensively demonstrated that excessive lipid accumulation in ectopic tissues leads to local inflammation and IR. Numerous pathogenic processes have been associated with the unhealthy expansion of the adipose tissue, including inflammation, fibrosis, hypoxia, altered adipokines secretion, mitochondrial dysfunction, hyperinsulinemia, endoplasmic reticulum (ER), and oxidative stress. Dietary and lifestyle, as well as therapeutic interventions, can be adequate to treat obesity and prevent metabolic alterations. Nevertheless, the exact molecular and cellular mechanisms underlying adipose tissue dysfunction and its implications in the development of metabolic disturbances are broadly unknown. Improving our understanding in this field might lead to the development of new approaches and the identification of therapeutic targets for treating obesity. In this Doctoral Thesis, we aimed at identifying potential factors and pathway markers relevant to the loss of adipose tissue function in obesity and their relationship to obesityrelated IR/T2D. To this end, we analysed human adipose tissue samples in combination with both human and murine cell lines, using state-of-the-art methodologies to investigate adipose tissue functionality in obesity and in response to weight loss upon bariatric surgery (BS), and their corresponding mechanisms associated with metabolic alterations or recovery, respectively. Specifically, to reach this general goal, we have carried out two separate studies, which are depicted in detail below. 1. Study 1: Identification of pathogenic markers of metabolic disease in preadipocytes of obese individuals Adipose precursor cells, the preadipocytes, are essential for the maintenance of adipose tissue homeostasis, regeneration, and expansion. Preadipocytes differentiation into adipocytes (i.e., adipogenesis) enables adipocyte turnover and adipose tissue growth, and ensures adipose tissue plasticity to accommodate surplus energy. It has been proposed that the inability for recruiting new adipose cells, together with the functional impairment of hypertrophied adipocytes that occur in obesity, contributes to lipid spillover from the adipose tissue. Thus, increasing adipogenesis appears as a valuable strategy to facilitate healthy adipose tissue expansion and ensure metabolic health. Adipocyte differentiation relies on major changes in gene expression programs regulating mRNA and protein production. An increasing body of evidence shows that mRNA processing and, in particular, alternative splicing, is crucial for genome reprogramming during cell differentiation. However, the splicing components relevant to adipogenesis and the cellular events regulated by alternative splicing during adipocyte differentiation have been scarcely explored, and it is yet to be established whether alternative splicing is modified in human obesity. Another crucial mechanism preserving precursor cell function relates to protein homeostasis (i.e., proteostasis), which maintains the capacity of cells to expand in order to sustain tissue growth and regeneration. Several lines of evidence support an important role for the endoplasmic reticulum (ER) protein quality control system in the regulation of adipogenesis. In fact, the unfolded protein response (UPR) is perturbed in the obese adipose tissue, and it has been proposed to contribute to the pathology of obesity. By contrast, it is still unknown whether the other component of the protein control system, the ER-associated protein degradation (ERAD), which is crucial for protecting cells against the accumulation of misfolded/unfolded proteins and proteotoxicity, is altered in the obese adipose tissue. Here, in order to identify altered molecular pathways that may contribute to metabolic disease in obesity, we set out an iTRAQ-LC-MS/MS proteomic approach for the analysis of subcutaneous (SC) and omental (OM) preadipocytes from obese individuals with normoglycaemia (NG) and T2D. Down-regulation of multiple components of the splicing machinery was observed in SC preadipocytes from obese individuals with insulin resistance (IR) or T2D, as compared to NG obesity. This, together with the observation that adipogenesis can be modulated by regulating the expression levels of a key spliceosome component, PRFP8/PRP8, supports a role for alternative splicing in the development of obesity-associated metabolic complications. In addition, our studies show that not only the UPR is altered in human SC and OM preadipocytes from IR/T2D obese subjects, but also that the ERAD system is hyperactivated. This condition, when mimicked in vitro, prevented adipogenesis. Our results provide novel mechanistic explanations for the impaired adipogenic capacity observed in IR/T2D obesity that relates to both mRNA and ER-proteostasis disturbances. 2. Study 2: Characterization of miRNAs as markers of dysfunctional adipose tissue in type 2 diabetes (T2D) It is increasingly accepted that, besides adipokines, the adipose tissue is a major physiological source of circulating microRNAs (miRNAs). miRNAs, a class of small noncoding RNAs that post-transcriptionally regulate gene expression, have emerged as key molecules for cell function. Anomalous miRNA levels and alterations in their biogénesis machinery have been related to several metabolic diseases, including obesity as well as T2D, and dyslipidemia. miRNAs are also actively secreted into the circulation and have been proposed to act as messengers for intercellular communication. This characteristic has pointed out circulating miRNAs as potential biomarkers for disease and altered circulating levels of numerous miRNAs have been associated with metabolic disorders. In this scenario, in order to design targeted preventive therapeutic strategies, it is critical to identify the early mechanisms that precede disease onset. Recently, the predictive value of a number of miRNAs for the diagnosis of T2D incidence was assessed in the CORonary Diet Intervention with Olive oil and cardiovascular PREVention (CORDIOPREV) study (ClinicalTrial.gov ID: NTC00924937), a prospective study carried out in 1,002 patients with coronary heart disease and high cardiovascular risk. These studies demonstrated that, when combined with HbA1c, a group of nine miRNAs (miR-9, miR-28-3p, miR-29a, miR-30a-5p, miR- 103, miR-126, miR-150, miR-223-3p, and miR-375), provided a higher predictive value in T2D diagnosis than clinical parameters. The relationship between baseline levels of these miRNAs with markers of beta-cell function and systemic and peripheral IR was also investigated. However, their potential association with adipose tissue deregulation was not analyzed. This is of interest as adipose tissue dysfunction has been proposed as a major contributing factor for the development of T2D, and adipose tissue miRNA expression profile is altered in obesity and T2D. In this line, several cross-sectional human studies have shown that the obesity-related expression pattern of specific circulating miRNAs reflects their miRNA adipose tissue expression profiles, supporting a role for circulating miRNAs as adipose tissue biomarkers. However, the relationship between circulating miRNAs and adipose tissue functional state is not fully understood, yet it might be useful to identify adipose tissue-related metabolic complications. In this scenario, we aimed at analyzing the relationship between previously established predictive miRNAs for T2D onset in the CORDIOPREV cohort (Jiménez- Lucena et al., 2018) and the loss of adipose tissue insulin sensitivity. Once established that circulating miR-223-3p was dysregulated in relation to adipose tissue function, we performed functional analysis to elucidate both the potential of preadipocytes and adipocytes as miR-223-3p secreting cells, and the consequences of miR-223-3p dysregulation on adipocyte biology. Our results indicate that miR-223-3p secretion by preadipocytes and adipocytes is prevented under inflammatory conditions, and that its pathogenic accumulation leads to alterations in both glucose and lipid metabolism in these cells. These observations could answer to the lower circulating levels on this miRNA found in those patients who are going to develop T2D, as well as explain its underlying role as a potential predictor of adipose dysfunction related to T2D development. We have also demonstrated that the changes that occur in the circulatin
Author: Sánchez Ceinos, Julia
Language: English
Subfield: Endocrinology, Diabetes and Metabolism
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7011421050