Proteomic Profiling of Plasma- and Gut-Derived Extracellular Vesicles in Obesity.

Obesity entails metabolic alterations across multiple organs, highlighting the role of inter-organ communication in its pathogenesis. Extracellular vesicles (EVs) are communication agents in physiological and pathological conditions, and although they have been associated with obesity comorbidities, their protein cargo in this context remains largely unknown. To decipher the messages encapsulated in EVs, we isolated plasma-derived EVs from a diet-induced obese murine model. Obese plasma EVs exhibited a decline in protein diversity while control EVs revealed significant enrichment in protein-folding functions, highlighting the importance of proper folding in maintaining metabolic homeostasis. Previously, we revealed that gut-derived EVs' proteome holds particular significance in obesity. Here, we compared plasma and gut EVs and identified four proteins exclusively present in the control state of both EVs, revealing the potential for a non-invasive assessment of gut health by analyzing blood-derived EVs. Given the relevance of post-translational modifications (PTMs), we observed a shift in chromatin-related proteins from glycation to acetylation in obese gut EVs, suggesting a regulatory mechanism targeting DNA transcription during obesity. This study provides valuable insights into novel roles of EVs and protein PTMs in the intricate mechanisms underlying obesity, shedding light on potential biomarkers and pathways for future research.

Pre-Diabetes-Linked miRNA miR-193b-3p Targets PPARGC1A, Disrupts Metabolic Gene Expression Profile and Increases Lipid Accumulation in Hepatocytes: Relevance for MAFLD.

Distinct plasma microRNA profiles associate with different disease features and could be used to personalize diagnostics. Elevated plasma microRNA hsa-miR-193b-3p has been reported in patients with pre-diabetes where early asymptomatic liver dysmetabolism plays a crucial role. In this study, we propose the hypothesis that elevated plasma hsa-miR-193b-3p conditions hepatocyte metabolic functions contributing to fatty liver disease. We show that hsa-miR-193b-3p specifically targets the mRNA of its predicted target PPARGC1A/PGC1α and consistently reduces its expression in both normal and hyperglycemic conditions. PPARGC1A/PGC1α is a central co-activator of transcriptional cascades that regulate several interconnected pathways, including mitochondrial function together with glucose and lipid metabolism. Profiling gene expression of a metabolic panel in response to overexpression of microRNA hsa-miR-193b-3p revealed significant changes in the cellular metabolic gene expression profile, including lower expression of MTTP, MLXIPL/ChREBP, CD36, YWHAZ and GPT, and higher expression of LDLR, ACOX1, TRIB1 and PC. Overexpression of hsa-miR-193b-3p under hyperglycemia also resulted in excess accumulation of intracellular lipid droplets in HepG2 cells. This study supports further research into potential use of microRNA hsa-miR-193b-3p as a possible clinically relevant plasma biomarker for metabolic-associated fatty liver disease (MAFLD) in dysglycemic context.

Leveraging the future of diagnosis and management of diabetes: From old indexes to new technologies.

BACKGROUND: Diabetes is a heterogeneous and multifactorial disease. However, glycemia and glycated hemoglobin have been the focus of diabetes diagnosis and management for the last decades. As diabetes management goes far beyond glucose control, it has become clear that assessment of other biochemical parameters gives a much wider view of the metabolic state of each individual, enabling a precision medicine approach. METHODS: In this review, we summarize and discuss indexes that have been used in epidemiological studies and in the clinical practice. RESULTS: Indexes of insulin secretion, sensitivity/resistance and metabolism have been developed and validated over the years to account also with insulin, C-peptide, triglycerides or even anthropometric measures. Nevertheless, each one has their own objective and consequently, advantages and disadvantages for specific cases. Thus, we discuss how new technologies, namely new sensors but also new softwares/applications, can improve the diagnosis and management of diabetes, both for healthcare professionals but also for caretakers and, importantly, to promote the empowerment of people living with diabetes. CONCLUSIONS: In long-term, the solution for a better diabetes management would be a platform that allows to integrate all sorts of relevant information for the person with diabetes and for the healthcare practitioners, namely glucose, insulin and C-peptide or, in case of need, other parameters/indexes at home, sometimes more than once a day. This solution would allow a better and simpler disease management, more adequate therapeutics thereby improving patients' quality of life and reducing associated costs.

Big data and machine learning to tackle diabetes management.

BACKGROUND: Type 2 Diabetes (T2D) diagnosis is based solely on glycaemia, even though it is an endpoint of numerous dysmetabolic pathways. Type 2 Diabetes complexity is challenging in a real-world scenario; thus, dissecting T2D heterogeneity is a priority. Cluster analysis, which identifies natural clusters within multidimensional data based on similarity measures, poses a promising tool to unravel Diabetes complexity. METHODS: In this review, we scrutinize and integrate the results obtained in most of the works up to date on cluster analysis and T2D. RESULTS: To correctly stratify subjects and to differentiate and individualize a preventive or therapeutic approach to Diabetes management, cluster analysis should be informed with more parameters than the traditional ones, such as etiological factors, pathophysiological mechanisms, other dysmetabolic co-morbidities, and biochemical factors, that is the millieu. Ultimately, the above-mentioned factors may impact on Diabetes and its complications. Lastly, we propose another theoretical model, which we named the Integrative Model. We differentiate three types of components: etiological factors, mechanisms and millieu. Each component encompasses several factors to be projected in separate 2D planes allowing an holistic interpretation of the individual pathology. CONCLUSION: Fully profiling the individuals, considering genomic and environmental factors, and exposure time, will allow the drive to precision medicine and prevention of complications.

Distinct impacts of fat and fructose on the liver, muscle, and adipose tissue metabolome: An integrated view.

Objective: In the last years, changes in dietary habits have contributed to the increasing prevalence of metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). The differential burden of lipids and fructose on distinct organs needs to be unveiled. Herein, we hypothesized that high-fat and high-fructose diets differentially affect the metabolome of insulin-sensitive organs such as the liver, muscle, and different adipose tissue depots. Methods: We have studied the impact of 12 weeks of a control (11.50% calories from fat, 26.93% from protein, and 61.57% from carbohydrates), high-fat/sucrose (HFat), or high-fructose (HFruct) feeding on C57Bl/6J male mice. Besides glucose homeostasis, we analyzed the hepatic levels of glucose and lipid-metabolism-related genes and the metabolome of the liver, the muscle, and white (WAT) and brown adipose tissue (BAT) depots. Results: HFat diet led to a more profound impact on hepatic glucose and lipid metabolism than HFruct, with mice presenting glucose intolerance, increased saturated fatty acids, and no glycogen pool, yet both HFat and HFruct presented hepatic insulin resistance. HFat diet promoted a decrease in glucose and lactate pools in the muscle and an increase in glutamate levels. While HFat had alterations in BAT metabolites that indicate increased thermogenesis, HFruct led to an increase in betaine, a protective metabolite against fructose-induced inflammation. Conclusions: Our data illustrate that HFat and HFruct have a negative but distinct impact on the metabolome of the liver, muscle, WAT, and BAT.

Fibrosis nonalcoholic steatohepatitis index validation and applicability considering glycaemic severity and T2D duration.

Nonalcoholic fatty liver disease (NAFLD) diagnosis without using invasive methods is extremely challenging, highlighting the need for simple indexes for this end. Recently, the fibrotic nonalcoholic steatohepatitis index (FNI) was developed and proposed as an affordable non-invasive score calculated with aspartate aminotransferase, high-density lipoprotein cholesterol and haemoglobin A1c. Herein, and given the link between NAFLD and diabetes, we aimed at validating FNI in a population with type 2 diabetes (T2D), also considering diabetes duration and glycaemic severity. The performance of FNI was higher than FIB-4 (AUROC = 0.89 vs 0.67, respectively). Additionally, using 0.1 as the rule-out cut-off of FNI, the sensitivity was 0.99 and the positive predictive value was 0.19. Both duration of diabetes and A1c did not impact FNI performance. In sum, FNI is a valuable score for predicting fibrotic nonalcoholic steatohepatitis not only for primary care units but also for diabetes specialized care.

Messages from the Small Intestine Carried by Extracellular Vesicles in Prediabetes: A Proteomic Portrait.

Extracellular vesicles (EVs) mediate communication in physiological and pathological conditions. In the pathogenesis of type 2 diabetes, inter-organ communication plays an important role in its progress and metabolic surgery leads to its remission. Moreover, gut dysbiosis is emerging as a diabetogenic factor. However, it remains unclear how the gut senses metabolic alterations and whether this is transmitted to other tissues via EVs. Using a diet-induced prediabetic mouse model, we observed that protein packaging in gut-derived EVs (GDE), specifically the small intestine, is altered in prediabetes. Proteins related to lipid metabolism and to oxidative stress management were more abundant in prediabetic GDE compared to healthy controls. On the other hand, proteins related to glycolytic activity, as well as those responsible for the degradation of polyubiquitinated composites, were depleted in prediabetic GDE. Together, our findings show that protein packaging in GDE is markedly modified during prediabetes pathogenesis, thus suggesting that prediabetic alterations in the small intestine are translated into modified GDE proteomes, which are dispersed into the circulation where they can interact with and influence the metabolic status of other tissues. This study highlights the importance of the small intestine as a tissue that propagates prediabetic metabolic dysfunction throughout the body and the importance of GDE as the messengers. Data are available via ProteomeXchange with identifier PXD028338.

Prediabetes blunts DPP4 genetic control of postprandial glycaemia and insulin secretion.

AIMS/HYPOTHESIS: Imbalances in glucose metabolism are hallmarks of clinically silent prediabetes (defined as impaired fasting glucose and/or impaired glucose tolerance) representing dysmetabolism trajectories leading to type 2 diabetes. CD26/dipeptidyl peptidase 4 (DPP4) is a clinically proven molecular target of diabetes-controlling drugs but the DPP4 gene control of dysglycaemia is not proven. METHODS: We dissected the genetic control of post-OGTT and insulin release responses by the DPP4 gene in a Portuguese population-based cohort of mainly European ancestry that comprised individuals with normoglycaemia and prediabetes, and in mouse experimental models of Dpp4 deficiency and hyperenergetic diet. RESULTS: In individuals with normoglycaemia, DPP4 single-nucleotide variants governed glycaemic excursions (rs4664446, p=1.63x10-7) and C-peptide release responses (rs2300757, p=6.86x10-5) upon OGTT. Association with blood glucose levels was stronger at 30 min OGTT, but a higher association with the genetic control of insulin secretion was detected in later phases of the post-OGTT response, suggesting that the DPP4 gene directly senses glucose challenges. Accordingly, in mice fed a normal chow diet but not a high-fat diet, we found that, under OGTT, expression of Dpp4 is strongly downregulated at 30 min in the mouse liver. Strikingly, no genetic association was found in prediabetic individuals, indicating that post-OGTT control by DPP4 is abrogated in prediabetes. Furthermore, Dpp4 KO mice provided concordant evidence that Dpp4 modulates post-OGTT C-peptide release in normoglycaemic but not dysmetabolic states. CONCLUSIONS/INTERPRETATION: These results showed the DPP4 gene as a strong determinant of post-OGTT levels via glucose-sensing mechanisms that are abrogated in prediabetes. We propose that impairments in DPP4 control of post-OGTT insulin responses are part of molecular mechanisms underlying early metabolic disturbances associated with type 2 diabetes.

Insights into Macrophage/Monocyte-Endothelial Cell Crosstalk in the Liver: A Role for Trem-2.

Liver disease accounts for millions of deaths worldwide annually being a major cause of global morbidity. Hepatotoxic insults elicit a multilayered response involving tissue damage, inflammation, scar formation, and tissue regeneration. Liver cell populations act coordinately to maintain tissue homeostasis and providing a barrier to external aggressors. However, upon hepatic damage, this tight regulation is disrupted, leading to liver pathology which spans from simple steatosis to cirrhosis. Inflammation is a hallmark of liver pathology, where macrophages and endothelial cells are pivotal players in promoting and sustaining disease progression. Understanding the drivers and mediators of these interactions will provide valuable information on what may contribute to liver resilience against disease. Here, we summarize the current knowledge on the role of macrophages and liver sinusoidal endothelial cells (LSEC) in homeostasis and liver pathology. Moreover, we discuss the expanding body of evidence on cell-to-cell communication between these two cell compartments and present triggering receptor expressed on myeloid cells-2 (Trem-2) as a plausible mediator of this cellular interlink. This review consolidates relevant knowledge that might be useful to guide the pursue of successful therapeutic targets and pharmacological strategies for controlling liver pathogenesis.

Gut-Pancreas-Liver Axis as a Target for Treatment of NAFLD/NASH.

Non-alcoholic fatty liver disease (NAFLD) represents the most common form of chronic liver disease worldwide. Due to its association with obesity and diabetes and the fall in hepatitis C virus morbidity, cirrhosis in NAFLD is becoming the most frequent indication to liver transplantation, but the pathogenetic mechanisms are still not completely understood. The so-called gut-liver axis has gained enormous interest when data showed that its alteration can lead to NAFLD development and might favor the occurrence of non-alcoholic steatohepatitis (NASH). Moreover, several therapeutic approaches targeting the gut-pancreas-liver axis, e.g., incretins, showed promising results in NASH treatment. In this review, we describe the role of incretin hormones in NAFLD/NASH pathogenesis and treatment and how metagenomic/metabolomic alterations in the gut microbiota can lead to NASH in the presence of gut barrier modifications favoring the passage of bacteria or bacterial products in the portal circulation, i.e., bacterial translocation.

Insulin: Trigger and Target of Renal Functions.

Kidney function in metabolism is often underestimated. Although the word "clearance" is associated to "degradation", at nephron level, proper balance between what is truly degraded and what is redirected to de novo utilization is crucial for the maintenance of electrolytic and acid-basic balance and energy conservation. Insulin is probably one of the best examples of how diverse and heterogeneous kidney response can be. Kidney has a primary role in the degradation of insulin released in the bloodstream, but it is also incredibly susceptible to insulin action throughout the nephron. Fluctuations in insulin levels during fast and fed state add another layer of complexity in the understanding of kidney fine-tuning. This review aims at revisiting renal insulin actions and clearance and to address the association of kidney dysmetabolism with hyperinsulinemia and insulin resistance, both highly prevalent phenomena in modern society.

Insights from qualitative research on NAFLD awareness with a cohort of T2DM patients: time to go public with insulin resistance?

BACKGROUND: It is estimated that around 70% of Type 2 Diabetes Mellitus patients (T2DM) have Non-Alcoholic Fatty Liver Disease (NAFLD). Awareness and education are amongst the major shortcomings of the public health response to the increasing threat of NAFLD. Characterizing the specific NAFLD-related information needs of particular high-risk metabolic communities, for instance, T2DM patients, might aid in the development of evidence-based health promotion strategies, ultimately promoting NAFLD-awareness, treatment adherence and therapeutic success rates. METHODS: Semi-structured interviews with T2DM patients were conducted to gain insight into their awareness of NAFLD, including its relationship with insulin resistance and T2DM. RESULTS: Awareness of NAFLD as a disease entity, as well as its progression to end-stage liver disease or its relationship with other metabolic conditions, including insulin resistance and T2DM was low. Surveillance behaviours were also suboptimal and perceptions on the self-management knowledge and praxis regarding lifestyle intervention components of T2DM treatment seemed detached from those of NAFLD. CONCLUSIONS: Our findings could inform the integration of NAFLD-related content in T2DM health promotion strategies. Rising awareness on NAFLD progression and its relationship with T2DM using culturally and community-relevant constructs might facilitate the development of primary and secondary prevention programmes to promote the adherence to lifestyle interventions by influencing NAFLD threat perceptions.

Review of methods for detecting glycemic disorders.

Prediabetes (intermediate hyperglycemia) consists of two abnormalities, impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) detected by a standardized 75-gram oral glucose tolerance test (OGTT). Individuals with isolated IGT or combined IFG and IGT have increased risk for developing type 2 diabetes (T2D) and cardiovascular disease (CVD). Diagnosing prediabetes early and accurately is critical in order to refer high-risk individuals for intensive lifestyle modification. However, there is currently no international consensus for diagnosing prediabetes with HbA1c or glucose measurements based upon American Diabetes Association (ADA) and the World Health Organization (WHO) criteria that identify different populations at risk for progressing to diabetes. Various caveats affecting the accuracy of interpreting the HbA1c including genetics complicate this further. This review describes established methods for detecting glucose disorders based upon glucose and HbA1c parameters as well as novel approaches including the 1-hour plasma glucose (1-h PG), glucose challenge test (GCT), shape of the glucose curve, genetics, continuous glucose monitoring (CGM), measures of insulin secretion and sensitivity, metabolomics, and ancillary tools such as fructosamine, glycated albumin (GA), 1,5- anhydroglucitol (1,5-AG). Of the approaches considered, the 1-h PG has considerable potential as a biomarker for detecting glucose disorders if confirmed by additional data including health economic analysis. Whether the 1-h OGTT is superior to genetics and omics in providing greater precision for individualized treatment requires further investigation. These methods will need to demonstrate substantially superiority to simpler tools for detecting glucose disorders to justify their cost and complexity.

Urine-Derived Stem Cells: Applications in Regenerative and Predictive Medicine.

Despite being a biological waste, human urine contains a small population of cells with self-renewal capacity and differentiation potential into several cell types. Being derived from the convoluted tubules of nephron, renal pelvis, ureters, bladder and urethra, urine-derived stem cells (UDSC) have a similar phenotype to mesenchymal stroma cells (MSC) and can be reprogrammed into iPSC (induced pluripotent stem cells). Having simple, safer, low-cost and noninvasive collection procedures, the interest in UDSC has been growing in the last decade. With great potential in regenerative medicine applications, UDSC can also be used as biological models for pharmacology and toxicology tests. This review describes UDSC biological characteristics and differentiation potential and their possible use, including the potential of UDSC-derived iPSC to be used in drug discovery and toxicology, as well as in regenerative medicine. Being a new cellular platform amenable to noninvasive collection for disease stratification and personalized therapy could be a future application for UDSC.

Trem-2 Promotes Emergence of Restorative Macrophages and Endothelial Cells During Recovery From Hepatic Tissue Damage.

Macrophages are pivotal in mounting liver inflammatory and tissue repair responses upon hepatic injury, showing remarkable functional plasticity. The molecular mechanisms determining macrophage transition from inflammatory to restorative phenotypes in the damaged liver remain unclear. Using mouse models of acute (APAP) and chronic (CCl4) drug-induced hepatotoxic injury we show that the immune receptor Trem-2 controls phenotypic shifts of liver macrophages and impacts endothelial cell differentiation during tissue recovery. Trem-2 gene ablation led to a delayed re-population of Kupffer cells correlating with deterred resolution of hepatic damage following acute and chronic injury. During tissue recovery, we found that macrophages transitioning to Kupffer cells expressed high levels of Trem-2. Acquisition of the transition phenotype was associated with a unique transcriptomic profile denoting strong responsiveness to oxidative stress and downmodulation of the pro-inflammatory phenotype, which was not observed in absence of Trem-2. During tissue recovery, lack of Trem-2 favored accumulation of a liver-damage associated endothelial cell population (LDECs), whose transcriptional program was compatible with endothelial de-differentiation. Accordingly, LDECs precursor potential is supported by the downregulation of surface endothelial cell markers and by striking in vitro morphological changes towards typical endothelial cells. In conclusion, we found that the dynamics of liver macrophages in response to liver injury are critically controlled by Trem-2 and this regulation is interlinked with the de-differentiation of endothelial cells and heightened liver pathology. We propose that Trem-2 promotes the transition from pro-inflammatory to tissue repair phase by driving the acquisition of restorative properties in phagocytic macrophages.

Clustering Clinical Data in R.

We are currently witnessing a paradigm shift from evidence-based medicine to precision medicine, which has been made possible by the enormous development of technology. The advances in data mining algorithms will allow us to integrate trans-omics with clinical data, contributing to our understanding of pathological mechanisms and massively impacting on the clinical sciences. Cluster analysis is one of the main data mining techniques and allows for the exploration of data patterns that the human mind cannot capture.This chapter focuses on the cluster analysis of clinical data, using the statistical software, R. We outline the cluster analysis process, underlining some clinical data characteristics. Starting with the data preprocessing step, we then discuss the advantages and disadvantages of the most commonly used clustering algorithms and point to examples of their applications in clinical work. Finally, we briefly discuss how to perform validation of clusters. Throughout the chapter we highlight R packages suitable for each computational step of cluster analysis.

Transfer of glucose hydrogens via acetyl-CoA, malonyl-CoA, and NADPH to fatty acids during de novo lipogenesis.

Deuterated water (2H2O) is widely used for measuring de novo lipogenesis (DNL). 2H is incorporated into fatty acids via exchange between body water and the hydrogens of acetyl-CoA, malonyl-CoA, and NADPH. Previous studies concluded that these exchanges are incomplete; therefore, fatty acid 2H enrichment requires correcting. In mice, we measured the 2H enrichment of fatty acid positions 2 and 3 and methyl hydrogens from [U-2H7]glucose to determine 2H transfer from glucose to fatty acid via malonyl-CoA, NADPH, and acetyl-CoA, respectively. Positional fatty acid 2H enrichments were compared with 13C enrichment of the same sites from an equivalent amount of [U-13C6]glucose provided alongside the [U-2H7]glucose tracer. Transfer of glucose 2H to fatty acid position 2 and methyl sites was low (2H enrichment of 0.06 ± 0.01 and 0.14 ± 0.01 relative to 13C) indicating extensive exchange at both malonyl- and acetyl-CoA, respectively. Transfer of glucose 2H into fatty acid position 3 was more extensive (0.46 ± 0.04 relative to 13C, P < 10-5 vs. position 2), indicating a more limited exchange of those glucose hydrogens that were transferred via NADPH. However, mice provided with [U-13C6]glucose and 2H2O had equivalent 2H enrichments of fatty acid positions 2 and 3, suggesting that in this setting, NADPH and body water 2H had exchanged extensively. This is explained by contributions of substrates other than exogenous glucose to DNL coupled with their extensive 2H enrichment from 2H2O prior to DNL. Under such conditions, 2H enrichment of fatty acids from 2H2O does not need correction.

Determining contributions of exogenous glucose and fructose to de novo fatty acid and glycerol synthesis in liver and adipose tissue.

The de novo synthesis of triglyceride (TG) fatty acids (FA) and glycerol can be measured with stable isotope tracers. However, these methods typically do not inform the contribution of a given substrate to specific pathways on these synthetic processes. We integrated deuterated water (2H2O) measurement of de novo lipogenesis (DNL) and glycerol-3-phosphate (GLY) synthesis from all substrates with a 13C nuclear magnetic resonance (NMR) method that quantifies TG FA and glycerol enrichment from a specific [U-13C]precursor. This allowed the [U-13C]precursor contribution to DNL and GLY to be estimated. We applied this method in mice to determine the contributions of fructose and glucose supplemented in the drinking water to DNL and GLY in liver, mesenteric adipose tissue (MAT) and subcutaneous adipose tissue (SCAT). In liver, fructose contributed significantly more to DNL of saturated fatty acids (SFA) and oleate as well as to GLY compared to glucose. Moreover, its contribution to SFA synthesis was significantly higher compared to that of oleate. MAT and SCAT had lower fractional rates of total DNL and GLY compared to liver and glucose was utilized more predominantly than fructose for TG synthesis in these tissues. This novel 2H2O/13C integrated method revealed for the first time, tissue specific selection of substrates for DNL, particularly fructose in regard to glucose in liver. Also, this approach was able to resolve the distribution of specific FAs into the TG sn2 and sn1,3 sites. This stable isotope integrated approach yielded information so far uncovered by other lipidomic tools and should powerfully assist in other nutritional, pathological or environmental contexts.

Paraoxonase-1 as a Regulator of Glucose and Lipid Homeostasis: Impact on the Onset and Progression of Metabolic Disorders.

Metabolic disorders are characterized by an overall state of inflammation and oxidative stress, which highlight the importance of a functional antioxidant system and normal activity of some endogenous enzymes, namely paraoxonase-1 (PON1). PON1 is an antioxidant and anti-inflammatory glycoprotein from the paraoxonases family. It is mainly expressed in the liver and secreted to the bloodstream, where it binds to HDL. Although it was first discovered due to its ability to hydrolyze paraoxon, it is now known to have an antiatherogenic role. Recent studies have shown that PON1 plays a protective role in other diseases that are associated with inflammation and oxidative stress, such as Type 1 and Type 2 Diabetes Mellitus and Non-Alcoholic Fatty Liver Disease. The aim of this review is to elucidate the physiological role of PON1, as well as the impact of altered PON1 levels in metabolic disorders.