Tissue-specific inflammation and insulin sensitivity in subjects with obesity.

Obesity is associated with low-grade inflammation and insulin resistance (IR). The contribution of adipose tissue (AT) and hepatic inflammation to IR remains unclear. We conducted a study across three cohorts to investigate this relationship. The first cohort consists of six women with normal weight and twenty with obesity. In women with obesity, we found an upregulation of inflammatory markers in subcutaneous and visceral adipose tissue, isolated AT macrophages, and the liver, but no linear correlation with tissue-specific insulin sensitivity. In the second cohort, we studied 24 women with obesity in the upper vs lower insulin sensitivity quartile. We demonstrated that several omental and mesenteric AT inflammatory genes and T cell-related pathways are upregulated in IR, independent of BMI. The third cohort consists of 23 women and 18 men with obesity, studied before and one year after bariatric surgery. Weight loss following surgery was associated with downregulation of multiple immune pathways in subcutaneous AT and skeletal muscle, alongside notable metabolic improvements. Our results show that obesity is characterised by systemic and tissue-specific inflammation. Subjects with obesity and IR show a more pronounced inflammation phenotype, independent of BMI. Bariatric surgery-induced weight loss is associated with reduced inflammation and improved metabolic health.

Dysregulation of macrophage PEPD in obesity determines adipose tissue fibro-inflammation and insulin resistance.

Resulting from impaired collagen turnover, fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance (IR). Prolidase, also known as peptidase D (PEPD), plays a vital role in collagen turnover by degrading proline-containing dipeptides but its specific functional relevance in AT is unknown. Here we show that in human and mouse obesity, PEPD expression and activity decrease in AT, and PEPD is released into the systemic circulation, which promotes fibrosis and AT IR. Loss of the enzymatic function of PEPD by genetic ablation or pharmacological inhibition causes AT fibrosis in mice. In addition to its intracellular enzymatic role, secreted extracellular PEPD protein enhances macrophage and adipocyte fibro-inflammatory responses via EGFR signalling, thereby promoting AT fibrosis and IR. We further show that decreased prolidase activity is coupled with increased systemic levels of PEPD that act as a pathogenic trigger of AT fibrosis and IR. Thus, PEPD produced by macrophages might serve as a biomarker of AT fibro-inflammation and could represent a therapeutic target for AT fibrosis and obesity-associated IR and type 2 diabetes.

Extracellular vesicles released by steatotic hepatocytes alter adipocyte metabolism.

The composition of extracellular vesicles (EVs) is altered in many pathological conditions, and their molecular content provides essential information on features of parent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome (MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dyslipidemia and hypertension that increases the risk of cardiovascular disease and type 2 diabetes mellitus. Here, we investigated the crosstalk between liver and adipocytes by characterizing EVs secreted by primary hepatocytes isolated from Zucker rat model, and studied the effect they have on 3T3-L1 adipocytes. We found that steatotic hepatocytes secrete EVs with significantly reduced exosomal markers in comparison with their lean counterpart. Moreover, proteomic analysis revealed that those EVs reflect the metabolic state of the parent cell in that the majority of proteins upregulated relate to fat metabolism, fatty acid synthesis, glycolysis, and pentose phosphate pathway. In addition, hepatocytes-secreted EVs influenced lipolysis and insulin sensitivity in recipient 3T3-L1 adipocytes. Untargeted metabolomic analysis detected alterations in different adipocyte metabolic pathways in cells treated with hepatic EVs. In summary, our work showed that steatosis has a significant impact in the amount and composition of EVs secreted by hepatocytes. Moreover, our data point to the involvement of hepatic-EVs in the development of pathologies associated with MetS.

Defective glucose and lipid metabolism in rheumatoid arthritis is determined by chronic inflammation in metabolic tissues.

BACKGROUND: Rheumatoid arthritis (RA) patients are at increased risk of insulin resistance (IR); however, the specific mechanisms mediating this association are currently unknown. OBJECTIVE: To investigate whether the inflammatory activity associated with RA accounts for the observed defective glucose metabolism and lipid metabolism in these patients. METHODS: We followed two main strategies: (i) extensive metabolic profiling of a RA cohort of 100 patients and 50 healthy control subjects and (ii) mechanistic studies carried out in both a collagen-induced arthritis mouse model and 3T3-L1 adipocytes treated with conditioned serum from RA patients. RESULTS: Following the exclusion of obese and diabetic subjects, data from RA patients demonstrated a strong link between the degree of systemic inflammation and the development of IR. These results were strengthened by the observation that induction of arthritis in mice resulted in a global inflammatory state characterized by defective carbohydrate and lipid metabolism in different tissues. Adipose tissue was most susceptible to the RA-induced metabolic alterations. These metabolic effects were confirmed in adipocytes treated with serum from RA patients. CONCLUSIONS: Our results show that the metabolic disturbances associated with RA depend on the degree of inflammation and identify inflammation of adipose tissue as the initial target leading to IR and the associated molecular disorders of carbohydrate and lipid homeostasis. Thus, we anticipate that therapeutic strategies based on tighter control of inflammation and flares could provide promising approaches to normalize and/or prevent metabolic alterations associated with RA.

Sphingolipids and glycerophospholipids - The "ying and yang" of lipotoxicity in metabolic diseases.

Sphingolipids in general and ceramides in particular, contribute to pathophysiological mechanisms by modifying signalling and metabolic pathways. Here, we present the available evidence for a bidirectional homeostatic crosstalk between sphingolipids and glycerophospholipids, whose dysregulation contributes to lipotoxicity induced metabolic stress. The initial evidence for this crosstalk originates from simulated models designed to investigate the biophysical properties of sphingolipids in plasma membrane representations. In this review, we reinterpret some of the original findings and conceptualise them as a sort of "ying/yang" interaction model of opposed/complementary forces, which is consistent with the current knowledge of lipid homeostasis and pathophysiology. We also propose that the dysregulation of the balance between sphingolipids and glycerophospholipids results in a lipotoxic insult relevant in the pathophysiology of common metabolic diseases, typically characterised by their increased ceramide/sphingosine pools.

Mild cold effects on hunger, food intake, satiety and skin temperature in humans.

BACKGROUND: Mild cold exposure increases energy expenditure and can influence energy balance, but at the same time it does not increase appetite and energy intake. OBJECTIVE: To quantify dermal insulative cold response, we assessed thermal comfort and skin temperatures changes by infrared thermography. METHODS: We exposed healthy volunteers to either a single episode of environmental mild cold or thermoneutrality. We measured hunger sensation and actual free food intake. After a thermoneutral overnight stay, five males and five females were exposed to either 18°C (mild cold) or 24°C (thermoneutrality) for 2.5 h. Metabolic rate, vital signs, skin temperature, blood biochemistry, cold and hunger scores were measured at baseline and for every 30 min during the temperature intervention. This was followed by an ad libitum meal to obtain the actual desired energy intake after cold exposure. RESULTS: We could replicate the cold-induced increase in REE. But no differences were detected in hunger, food intake, or satiety after mild cold exposure compared with thermoneutrality. After long-term cold exposure, high cold sensation scores were reported, which were negatively correlated with thermogenesis. Skin temperature in the sternal area was tightly correlated with the increase in energy expenditure. CONCLUSIONS: It is concluded that short-term mild cold exposure increases energy expenditure without changes in food intake. Mild cold exposure resulted in significant thermal discomfort, which was negatively correlated with the increase in energy expenditure. Moreover, there is a great between-subject variability in cold response. These data provide further insights on cold exposure as an anti-obesity measure.

Prostaglandin profiling reveals a role for haematopoietic prostaglandin D synthase in adipose tissue macrophage polarisation in mice and humans.

BACKGROUND/OBJECTIVES: Obesity has been associated with both changes in adipose tissue lipid metabolism and inflammation. A key class of lipid-derived signalling molecules involved in inflammation are the prostaglandins. In this study, we aimed to determine how obesity affects the levels of prostaglandins within white adipose tissue (WAT) and determine which cells within adipose tissue produce them. To avoid the effects of cellular stress on prostaglandin levels, we developed a multivariate statistical approach in which metabolite concentrations and transcriptomic data were integrated, allowing the assignment of metabolites to cell types. SUBJECTS/METHODS: Eicosanoids were measured by liquid chromatography-tandem mass spectrometry and mRNA levels using real-time PCR. Eicosanoid levels and transcriptomic data were combined using principal component analysis and hierarchical clustering in order to associate metabolites with cell types. Samples were obtained from C57Bl/6 mice aged 16 weeks. We studied the ob/ob genetically obese mouse model and diet-induced obesity model. We extended our results in mice to a cohort of morbidly obese humans undergoing bariatric surgery. RESULTS: Using our modelling approach, we determined that prostglandin D2 (PGD2) in adipose tissue was predominantly produced in macrophages by the haematopoietic isoform of prostaglandin D synthase (H-Pgds). Analysis of sub-fractionated WAT confirmed that H-Pgds was expressed in adipose tissue macrophages (ATMs). Furthermore, H-Pgds expression in ATMs isolated from lean and obese mice was consistent with it affecting macrophage polarisation. Functionally, we demonstrated that H-PGDS-produced PGD2 polarised macrophages toward an M2, anti-inflammatory state. In line with a potential anti-inflammatory role, we found that H-PGDS expression in ATMs was positively correlated with both peripheral insulin and adipose tissue insulin sensitivity in humans. CONCLUSIONS: In this study, we have developed a method to determine the cellular source of metabolites within an organ and used it to identify a new role for PGD2 in the control of ATM polarisation.

Dihydroceramide desaturase 1, the gatekeeper of ceramide induced lipotoxicity.

The pathogenic relevance of sphingolipid metabolism is increasingly being recognised. Here we elaborate on a new player within the sphingolipid field: the degs1 enzyme, a recently discovered enzyme that catalyses the final step in the de novo biosynthesis of ceramides controlling the step from dihydroceramides to ceramides. Here, we describe its function and dysregulation by factors such as oxidative stress, hypoxia and inflammation and provide evidence indicating that dihydroceramides constitute a biologically active molecule from the sphingolipid family with certain differential characteristics with respect to its delta-4 unsaturated counterparts, the ceramides. Finally we present pathophysiological scenarios characterised by specific increases in dihydroceramide that challenge the concept that "all ceramides species are the same". This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.

Central obesity and altered peripheral adipose tissue gene expression characterize the NAFLD patient with insulin resistance: Role of nutrition and insulin challenge.

OBJECTIVE: Insulin resistance (IR) and white adipose tissue (WAT) dysfunction frequently are associated with nonalcoholic fatty liver disease (NAFLD); however, the pathogenic mechanisms contributing to their clustering are not well defined. The aim of this study was to define some nutritional, anthropometric, metabolic, and genetic mechanisms contributing to their clustering. METHODS: Forty-five (20 men, 25 women) patients (age 45.7 ± 11.1 y) with recent diagnosis of NAFLD were grouped according to IR state. Energy balance was assessed using a food questionnaire and indirect calorimetry, and body composition with anthropometry and dual-energy x-ray absorptiometry. Biochemical and hormonal parameters combined with adipose tissue gene expression were determined. Microarray analysis of gene expression was performed in a subset of WAT samples from IR patients (n = 9), in the fasted state, after specific test meals (monounsaturated fatty acid [MUFA], saturated fat [SAT], and carbohydrate-rich) and after being challenged with insulin. RESULTS: IR patients exhibited higher trunk fat to leg fat ratio (P < 0.05) and had a higher ratio of SAT/MUFA fat intake (P < 0.05) than insulin-sensitive (IS) individuals. Deposition of fat in the trunk but not in the leg was directly related to liver enzyme levels (P < 0.05). IR patients also had lower adiponectin serum levels and leptin (LEP) mRNA expression in WAT compared with IS patients (P < 0.01 and P < 0.05, respectively). Microarray analysis after insulin challenge confirmed that insulin treatment induces the expression of PPARG gene and LEP and decreases GCGR gene (P < 0.05 for all) in WAT. No changes in these genes were observed in the postprandial state induced after the acute effect of specific diets. CONCLUSIONS: Patients exhibiting NAFLD and IR had preferential central fat deposition directly related to their serum alanine aminotransferase levels. These patients showed peripheral adipose tissue dysfunction and exhibited inappropriately low LEP biosynthesis that could be partially restored after anabolic conditions induced by insulin signaling.

Harnessing the beneficial properties of adipogenic microbes for improving human health.

Obesity is associated with numerous metabolic comorbidities. Weight loss is an effective measure for alleviating many of these metabolic abnormalities. However, considering the limited success of most medical weight-management approaches in producing a sustained weight loss, approaches that improve obesity-related metabolic abnormalities independent of weight loss would be extremely attractive and of practical benefit. Metabolically healthy obesity supports the notion that a better metabolic profile is possible despite obesity. Moreover, adequate expansion of adipose tissue appears to confer protection from obesity-induced metabolic comorbidities. To this end, the 10th Stock conference examined new approaches to improve metabolic comorbidities independent of weight loss. In particular, human adenovirus 36 (Ad36) and specific gut microbes were examined for their potential to influence lipid and glucose homeostasis in animals and humans. While these microbes possess some undesirable properties, research has identified attributes of adenovirus Ad36 and gut microbes that may be selectively harnessed to improve metabolic profile without the obligatory weight loss. Furthermore, identifying the host signalling pathways that these microbes recruit to improve the metabolic profile may offer new templates and targets, which may facilitate the development of novel treatment strategies for obesity-related metabolic conditions.

Prediction of weight loss and regain following dietary, lifestyle, and pharmacologic intervention.

To develop statistical models for predicting weight loss and regain, we analyzed the phenotypic responses in an outpatient study of 60 obese subjects randomized to one of three 12-week interventions, diet (-600 kcal) alone, diet with exercise, and diet with sibutramine. This was followed by 12 weeks of observation. The best of the "baseline covariates" models was one that incorporated intervention group and baseline homeostasis model assessment-estimated insulin resistance (HOMA(IR)). It predicted week 12 weight change with R(2) of 0.38 and root mean square error (√MSE) of 2.92 kg. An alternative model incorporating baseline fat mass plus change in weight and HOMA(IR) at week 4 improved the prediction (R(2), 0.67, √MSE, 2.19 kg). We could not identify a satisfactory model to predict weight regain. We conclude that prediction of weight loss over 12 weeks is significantly improved when short-term weight change is incorporated into the model. This information could be utilized to forecast the success of a weight-loss program and to motivate and contribute to innovative designing of obesity trials.

Could increased time spent in a thermal comfort zone contribute to population increases in obesity?

Domestic winter indoor temperatures in the USA, UK and other developed countries appear to be following an upwards trend. This review examines evidence of a causal link between thermal exposures and increases in obesity prevalence, focusing on acute and longer-term biological effects of time spent in thermal comfort compared with mild cold. Reduced exposure to seasonal cold may have a dual effect on energy expenditure, both minimizing the need for physiological thermogenesis and reducing thermogenic capacity. Experimental studies show a graded association between acute mild cold and human energy expenditure over the range of temperatures relevant to indoor heating trends. Meanwhile, recent studies of the role of brown adipose tissue (BAT) in human thermogenesis suggest that increased time spent in conditions of thermal comfort can lead to a loss of BAT and reduced thermogenic capacity. Pathways linking cold exposure and adiposity have not been directly tested in humans. Research in naturalistic and experimental settings is needed to establish effects of changes in thermal exposures on weight, which may raise possibilities for novel public health strategies to address obesity.

Electrical and optical spectroscopy for quantitative screening of hepatic steatosis in donor livers.

Macro-steatosis in deceased donor livers is increasingly prevalent and is associated with poor or non-function of the liver upon reperfusion. Current assessment of the extent of steatosis depends upon the macroscopic assessment of the liver by the surgeon and histological examination, if available. In this paper we demonstrate electrical and optical spectroscopy techniques which quantitatively characterize fatty infiltration in liver tissue. Optical spectroscopy showed a correlation coefficient of 0.85 in humans when referenced to clinical hematoxylin and eosin (H&E) sections in 20 human samples. With further development, an optical probe may provide a comprehensive measure of steatosis across the liver at the time of procurement.

LEM-PCR: a method for determining relative transcript isoform proportions using real-time PCR without a standard curve.

Many genes express multiple transcript isoforms generated by alternative splicing of mRNA. Using real-time PCR, it is straightforward to determine the relative expression level of each isoform independently. However, it is less trivial to determine the relative proportions of different isoforms in a cDNA sample. The relative proportions of different isoforms can be important, as a small change in a highly abundant transcript may be more relevant than a large change in a minimally expressed transcript. Currently, determining the relative proportions of isoforms requires the construction of a standard curve using recombinant plasmid DNA or genomic DNA. As recombinant or genomic DNA standards often amplify with different efficiencies to cDNA samples, they may give under- or overestimations of isoform abundances. The method described in this article uses a titration curve generated from the same cDNA samples measured in the experiment. By using samples with different levels of separate isoforms, it is possible to derive linear equations which, when solved, allow the determination of the proportion of each isoform within the samples under study.

Secreted frizzled-related protein 1 regulates adipose tissue expansion and is dysregulated in severe obesity.

AIM: The Wnt/ß-catenin signaling network offers potential targets to diagnose and uncouple obesity from its metabolic complications. In this study, we investigate the role of the Wnt antagonist, secreted frizzled-related protein 1 (SFRP1), in promoting adipogenesis in vitro and adipose tissue expansion in vivo. METHODS: We use a combination of human and murine, in vivo and in vitro models of adipogenesis, adipose tissue expansion and obesity-related metabolic syndrome to profile the involvement of SFRP1. RESULTS: SFRP1 is expressed in both murine and human mature adipocytes. The expression of SFRP1 is induced during in vitro adipogenesis, and SFRP1 is preferentially expressed in mature adipocytes in human adipose tissue. Constitutive ectopic expression of SFRP1 is proadipogenic and inhibits the Wnt/ß-catenin signaling pathway. In vivo endogenous levels of adipose SFRP1 are regulated in line with proadipogenic states. However, in longitudinal studies of high-fat-diet-fed mice, we observed a dynamic temporal but biphasic regulation of endogenous SFRP1. In agreement with this profile, we observed that SFRP1 expression in human tissues peaks in patients with mild obesity and gradually falls in morbidly obese subjects. CONCLUSIONS: Our results suggest that SFRP1 is an endogenous modulator of Wnt/ß-catenin signaling and participates in the paracrine regulation of human adipogenesis. The reduced adipose expression of SFRP1 in morbid obesity and its knock-on effect to prevent further adipose tissue expansion may contribute to the development of metabolic complications in these individuals.

Regulation of insulin secretion, glucokinase gene transcription and beta cell proliferation by adipocyte-derived Wnt signalling molecules.

AIMS/HYPOTHESIS: Adipocytes secrete signalling molecules that elicit responses from target cells, including pancreatic beta cells. Wnt signalling molecules have recently been identified as novel adipocyte-derived factors. They also regulate insulin secretion in pancreatic beta cells and the cell cycle. The aim of this study was to investigate the effect of adipocyte-derived Wnt signalling molecules on insulin secretion and beta cell proliferation. METHODS: Human adipocytes were isolated to generate fat cell-conditioned medium (FCCM). Ins-1 cells were stimulated with FCCM and transiently transfected with reporter genes. Proliferation assays using [3H]thymidine incorporation were carried out in Ins-1 cells and primary islet cells. Insulin secretion from primary islets was assessed by radioimmunoassay. Gene expression in primary islets was assessed by Taqman PCR. RESULTS: Treatment with human FCCM increased the transcription of a T cell-specific transcription factor reporter gene (TOPFLASH) in Ins-1 cells (241%, p < 0.05). FCCM induced the proliferation of Ins-1 cells (1.8 fold, p < 0.05) and primary mouse islet cells (1.6 fold, p < 0.05). Antagonizing Wnt signalling with secreted Frizzled-related protein 1 (FRP-1) inhibited the proliferative effect induced by Wnt3a and FCCM on Ins-1 cells by 49 and 41%, respectively. In addition, FCCM led to a twofold (p < 0.05) induction of cyclin D1 promoter activity in Ins-1 cells. Furthermore, FCCM stimulated insulin secretion (204% of controls, p > 0.05) in primary mouse islets, and this stimulation was inhibited by sFRP-1. At a molecular level, canonical Wnt signalling induced glucokinase gene transcription in a peroxisome proliferator-activated receptor gamma-dependent fashion, thereby defining the glucokinase gene as a novel Wnt target gene. CONCLUSIONS/INTERPRETATION: Taken together, these data show that adipocyte-derived Wnt signalling molecules induce beta cell proliferation and insulin secretion in vitro, suggesting a novel mechanism linking obesity to hyperinsulinaemia.