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L'inflammation du tissu adipeux, le stress oxydatif ou encore les modifications du microbiote intestinal semblent constituer des facteurs essentiels dans l'initiation, l'aggravation, voire même la "chronicisation" du syndrome métabolique et de l'insulinorésistance. La lectine Reg3a (également appelée HIP/PAP) est un composant de l'immunité innée qui déploie des activités anti-bactérienne, anti-oxydante et anti-inflammatoire. Dans ces conditions, Reg3a pourrait jouer un rôle clé en tant que modulateur du dialogue moléculaire entre le foie, le tissu adipeux et le tube digestif. Via son expression dans le foie de souris transgéniques, ou bien son administration par voie sous-cutanée, nous montrons que la protéine humaine Reg3a présente des effets bénéfiques sur le phénotype des animaux, en contribuant à leur capacité à mieux gérer les troubles du métabolisme d’origine à la fois glucidique et lipidique liés au vieillissement, à un régime alimentaire hyperlipidique ou une obésité et une insulinorésistance génétiquement acquises. Elle permet une diminution de la proportion de masse grasse et une meilleure régulation de la glycémie. Les mécanismes moléculaires sous-jacents restent à déterminer mais pourraient d’une part impliquer le régulateur énergétique AMPK, d’autre part être en lien avec le phénomène de brunissement du tissu adipeux. / Inflammation of adipose tissue, oxidative stress or modifications of intestinal microbiota seem to stand among crucial factors contributing to the initiation, worsening or even "chronification" of metabolic syndrome and insulin resistance. The lectin Reg3a (also called HIP/PAP) is a component of innate immunity with antibacterial, antioxidant and anti-inflammatory activities. In these conditions, Reg3a could play a key role as a modulator of the molecular dialogue between the liver, the adipose tissue and the gut. Through its expression in the liver of transgenic mice or its subcutaneous administration, we show that the human protein Reg3a has a positive impact on animals’ phenotype and contribute to their capacity to better manage both glucose and lipid metabolic disorders related to ageing, a high fat diet or genetically acquired obesity and insulin resistance. Notably it leads to a decrease in the proportion of fat and allows a better control of glycaemia. The associated molecular mechanisms remain to be determined but they could involve the energy sensor AMPK and the phenomenon of adipose tissue browning.

Weight loss has been shown to significantly improve Adipose tissue (AT) function, however changes in AT gene expression profiles particularly in visceral AT (VAT) have not been systematically studied. Here, we tested the hypothesis that extensive weight loss in response to bariatric surgery (BS) causes AT gene expression changes, which may affect energy and lipid metabolism, inflammation and secretory function of AT. We assessed gene expression changes by whole genome expression chips in AT samples obtained from six morbidly obese individuals, who underwent a two step BS strategy with sleeve gastrectomy as initial and a Roux-en-Y gastric bypass as second step surgery after 12 ± 2 months. Global gene expression differences in VAT and subcutaneous (S)AT were analyzed through the use of genome-scale metabolic model (GEM) for adipocytes. Significantly altered gene expressions were PCR-validated in 16 individuals, which also underwent a two-step surgery intervention. We found increased expression of cell death-inducing DFFA-like effector a (CIDEA), involved in formation of lipid droplets in both fat depots in response to significant weight loss. We observed that expression of the genes associated with metabolic reactions involved in NAD+, glutathione and branched chain amino acid metabolism are significantly increased in AT depots after surgery-induced weight loss.

Recently, a genome-wide analysis identified DNA methylation of the HIF3A (hypoxia-inducible factor 3A) as strongest correlate of BMI. Here we tested the hypothesis that HIF3A mRNA expression and CpG-sites methylation in adipose tissue (AT) and genetic variants in HIF3A are related to parameters of AT distribution and function. In paired samples of subcutaneous AT (SAT) and visceral AT (VAT) from 603 individuals, we measured HIF3A mRNA expression and analyzed its correlation with obesity and related traits. In subgroups of individuals, we investigated the effects on HIF3A genetic variants on its AT expression (N = 603) and methylation of CpG-sites (N = 87). HIF3A expression was significantly higher in SAT compared to VAT and correlated with obesity and parameters of AT dysfunction (including CRP and leucocytes count). HIF3A methylation at cg22891070 was significantly higher in VAT compared to SAT and correlated with BMI, abdominal SAT and VAT area. Rs8102595 showed a nominal significant association with AT HIF3A methylation levels as well as with obesity and fat distribution. HIF3A expression and methylation in AT are fat depot specific, related to obesity and AT dysfunction. Our data support the hypothesis that HIF pathways may play an important role in the development of AT dysfunction in obesity.

Alterations in composition and function of human gut microbiota can affect physiological processes and are known to be associated with many diseases including inflammatory bowel disease, hypertension, asthma and colon cancer. Complex interactions between gut microbiota, environmental toxins, nutrients and host genetics may result in an increased permeability of the gut, which is closely linked to the presence of adverse metabolic conditions. As a consequence, translocation of bacterial DNA into the blood circulation increases in patients with obesity. Obesity is a growing health problem worldwide and often paired with severe secondary complications, such as type 2 diabetes or cardiovascular problems. A main feature of disease progression is a chronic low-grade inflammation of adipose tissue which contributes to the development and aggravation of insulin resistance and many of the underlying mechanisms are still unknown. Although data from mice studies suggest that the presence of bacterial components in adipose tissue can support these processes, human studies on this subject are lacking. In my thesis entitled ``Bacterial translocation to adipose tissue in metabolic diseas'' I provide evidence supporting the initial hypothesis, namely that bacterial DNA is present in adipose tissue, even after stringent controlling for contaminants. To this end, I established a wet lab routine protocol to eliminate contamination as well as a bioinformatics pipeline accounting for contamination by subtracting negative controls. Briefly, this included the use of lab ware and reagents UV-treated for at least 90 minutes, the use of breath protection, extra-long gloves and single-use lab coats as well as working under a sterile laminar flood hood in a clean lab free of any PCR products. The bioinformatics pipeline employed commonly used 16S rRNA gene analysis tools including qiime2, phyloseq and DESeq2 as well as decontam, a novel tool to extract negative controls. Observed quantity of bacterial DNA was in the range of 1 to 10 pg/µg total isolated genomic DNA, which is equivalent to about 0.01 to 0.7% of bacterial cells per human cell. The highest quantity was present in subcutaneous adipose tissue, followed by mesenteric adipose tissue. Bacterial amount correlated with adipose tissue macrophages and PPARG expression in omental and with IL1B and TNF expression in subcutaneous adipose tissue. Mesenteric adipose tissue showed the highest diversity of the observed genera. The most commonly observed phyla in all tissues were Proteobacteria and Firmicutes, which is in line with previously published data on blood bacterial DNA. Still, many genera were predominantly found in specific tissues, e.g. Enterobacter in subcutaneous and Acinetobacter in omental adipose tissue. I further showed that the distribution of observed features could partially be explained by markers of insulin resistance (HOMA-IR, HbA1c) and inflammation (IL-6, TNFa, macrophages) and that certain genera, such as Rhodoferax or Lactobacillus are associated with type 2 diabetes status. In first functional approaches I demonstrated that concentrations of bacterial DNA in the observed range are sufficient to stimulate an inflammatory response in immortalized subcutaneous adipocytes derived from a healthy donor. The effect was most prominent after four hours of treatment and increased in a dose-dependent matter. One of the aims in the present study was to determine levels of gut leakage by measuring zonulin, the most commonly used biomarker for intestinal permeability in humans, and analyze possible associations with adipose tissue bacterial signature. As there are few well-conducted studies on circulating zonulin levels in patients with metabolic diseases, I first performed a correlation study in the available and metabolically well-characterized Sorbs cohort. Circulating zonulin correlated significantly positive with BMI, fasting glucose, triglycerides and cholesterol and negatively with HOMA-IS, high density lipoprotein and circulating adiponectin levels. Albeit these strong correlations with markers of glucose and lipid metabolism supported previously reported findings, the results pointed to some inconsistencies. As zonulin is reported to be pre-haptoglobin 2 (preHP2), and about 15% percent of a typical western European population are homozygous for haptoglobin 1, they should not express zonulin at all. I confirmed in the Sorbs cohort previously reported distributions of haptoglobin genotypes and showed that the target of the only commercially available zonulin ELISA kit was not related to haptoglobin genotype, therefore presumably not measuring zonulin/ preHP2. Subsequently, I identified properdin as a possible target by employing mass spectrometry approaches. Properdin is structurally related to haptoglobin, as both proteins belong to the mannose-associated serine protease family, however further experiments are needed to validate a possible functional resemblance. In regard to bacterial translocation two adipose tissue depots were of notable interest due to their close proximity to the gastrointestinal tract. Mesenteric adipose tissue is located around the small intestine and the adipose tissue of the appendices epiploicae, small chunks of fat also called epiploic adipose tissue, are directly attached to the colon. After a thorough literature research I could also assert that both adipose tissues were rarely analyzed in the context of obesity. Therefore they were extensively investigated by measuring gene expression of adipo(cyto)kines, circulating inflammatory markers and analyzing adipocyte size and adipose tissue macrophages. Furthermore, a ``multiomics'' characterization was conducted and by analyzing transcriptome and methylome data I could identify epiploic adipose tissue as a tissue of interest in regard to type 2 diabetes and insulin resistance, which was further confirmed by untargeted proteomics data. Contrary to initial assumptions, I observed only a slight increase in translocation of bacterial DNA and no increased inflammation, as measured by tissue specific TNF and IL6 expression as well as adipose tissue macrophage infiltration. However, both transcriptome and proteome profiles allowed a clear discrimination of patients with and without insulin resistance which was most distinct in epiploic adipose tissue. Compared to other fat depots, epiploic adipose tissue exhibited a discriminable metabolic profile whereas mesenteric adipose tissue was more similar to omental-visceral adipose tissue. Most strikingly, epiploic adipose tissue showed a strong increase in leptin expression and, in general, the upregulation of various metabolic pathways involved in sugar, amino acid or sphingolipid metabolism. In accord with the leaky gut hypothesis high expression of lipopolysaccharide binding protein and various pathways involved in chemokine signaling were observed. In summary, I did not observe an increase in bacterial DNA or adipose tissue macrophages, but demonstrated elevated inflammatory signals such as increased chemokine or IL-8 signaling which are linked with an overall increase of metabolic processes and an increased expression of various adipokines. Epiploic adipose tissue might have a watch dog function by being the first adipose tissue sensing and forwarding certain (microbial) stimuli from the large intestine to the host. In the last part of my thesis I addressed a possible role of the HLA genomic region on the development of type 2 diabetes. The influence of HLA genetics on type 2 diabetes has been under debate for several decades, since HLA was recognized to largely contribute to type 1 diabetes heritability. However, studies remained inconclusive due to lacking cohorts with sample sizes providing sufficient statistical power for association analyses. More recently, animal studies suggested MHC class II proteins as crucial factors mediating adipose tissue inflammation and insulin resistance. The sample size of the leaky gut cohort was insufficient to determine any correlation between HLA class II genotypes and the presence or type of bacteria in adipose tissue due to the high variability in the observed genomic region. Yet, I had access to three large population-based cohorts which allowed me to analyze associations between HLA class II alleles and type 2 diabetes. Therefore HLA genotypes of the LIFE-Adult (N=4649), LIFE-Heart (N=4815) and Sorbs (N=949) cohort were imputed from SNP genotyping data and analyzed for association with type 2 diabetes. In a meta-analysis including all three cohorts, I identified a protective effect for the well-established type 1 diabetes protective haplotype DRB1*15:01~DQA1*01:02~DQB1*06:02 and confirmed DRB1*07:01~DQA1*02:01~DQB1*03:03 as a risk haplotype in non-insulin treated diabetes. These results suggest that the genetic foundation of both type 1 and 2 diabetes shares common elements involving the HLA class II locus. In conclusion, to the best of my knowledge, I provide in my work the first contaminant-aware identification of bacterial DNA in human adipose tissue and highlight the importance of analyzing novel adipose tissue depots by showing that fat of the appendices epiploicae, previously only considered to have a cushioning function, is metabolically active and possibly involved in the development of insulin resistance.

Despite the availability of effective therapy of hypercholesterolemia and hypertension, cardiovascular mortality continues to be very high in the Western world. Inflammatory changes occurring in the arterial wall as well as in the adipose tissue play a major role in the development of atherosclerosis. Macrophages are involved in the process of atherogenesis as early as atherosclerosis begins to develop, when, still as monocytes, they migrate and adhere to the arterial wall as a result of endothelial activation and stimulation by pro-inflammatory substances. Adipose tissue has long been recognized as an important endocrine organ, with part of adipose tissue made up by a large amount of macrophages capable of producing a large number of pro-inflammatory cytokines, which contribute to the development of low-grade chronic inflammation important in the development of atherosclerosis. In samples of subcutaneous, visceral and perivascular adipose tissue (SAT, VAT, and PVAT, respectively) obtained from healthy subjects (living kidney donors, LKD), we analyzed macrophages and their polarization, gene expression of pro-inflammatory cytokines and the effect of substances released by VAT on the level of monocyte adhesion to the endothelium. In some analyses, we included samples of SAT, VAT and PVAT obtained...

For many years, adipose tissue (AT) was thought to be a tissue primarily responsible for cushioning and insulating organs. However, significant advances in knowledge have shown that AT is necessary for maintaining an optimal metabolic balance through paracrine and endocrine mechanisms. Because AT dysfunction is related with illnesses such as obesity and diabetes, it is vital to understand the mechanisms behind these pathologies to restore metabolic health. Beige AT is a unique form of fat that generates heat through uncoupling protein 1 (UCP1), has a dense neurovascular network, and is associated with enhanced metabolic health. Hence, particular emphasis has been made on unraveling the processes behind thermogenic activation and maintenance, as increasing thermogenic activity offers considerable potential for treating metabolic disorders. Activation of beige AT is dependent on norepinephrine release from sympathetic neurons upon physiological cues such as cold exposure. Studies have revealed a major role of monoamine oxidase a (MAOA)-mediated norepinephrine clearance in the maintenance of thermogenic AT. However, major limitations are still present with regards to the mechanisms of neurotransmitter clearance and their role in thermogenic regulation. The initial objective of this thesis is to evaluate the effect of human white and thermogenic adipocytes on the formation of a neurovascular network in order to maintain thermogenesis and whether MAOA plays a direct role in thermogenic control. We demonstrate that implanted human thermogenic adipocytes generate a more vascularized and innervated AT than non-thermogenic adipocytes. Additional findings revealed that MAOA is expressed in human adipocytes and that inhibiting MAOA promotes thermogenesis. The second objective of this thesis is to determine if hAdipoGel (hAG) - a decellularized AT matrix – enhances mesenchymal stem cell (MSC) proliferation and differentiation, as well as human adipocyte engraftment in vivo. We show that MSC can proliferate in hAG and differentiate effectively into white and thermogenic adipocytes. Additionally, when white adipocytes are implanted with hAG, they differentiate into a fully functioning fat graft capable of integrating with the host. Understanding the thermogenic processes of human AT, in combination with the use of a suitable decellularized matrix, can aid in the development of therapeutic treatments that boost thermogenic activity and hence metabolic health.

Vaspin expression is increased in white adipose tissue (WAT) of diet-induced obese mice and rats and is supposed to compensate HFD-induced inflammatory processes and insulin resistance in adipose tissue by counteracting pro-inflammatory gene expression in obesity. Multiple studies have also demonstrated strong anti-inflammatory effects in vascular and skin cells. Here, we used vaspin treated 3T3-L1 murine adipocytes as well as 3T3-L1 cells with stable vaspin expression to investigate the effect of exogenous and endogenous vaspin on inflammatory processes and insulin signaling in adipocytes. Our stably transfected cells secreted significant amounts of vaspin which was in the physiological range of ∼0.5 ng/ml in cell supernatants. Adipocyte differentiation was not affected by vaspin as expression of adipogenic marker genes as well as lipid accumulation after full differentiation was similar to control cells. We found that IL-1β induced expression and secretion of pro-inflammatory cytokines, such as IL-6, MCP1 and TNFα was significantly blunted in vaspin expressing 3T3-L1 cells. Treatment of 3T3-L1 cells with exogenous vaspin resulted in reduced cytokine-induced activation of the intracellular and pro-inflammatory NFκB signaling cascades (IKKα/β, IκB and NFκB). Moreover, endogenous vaspin positively affected insulin signaling by increasing insulin-stimulated phosphorylation of the key mediator protein kinase B (AKT). Together, we demonstrate anti-inflammatory effects of vaspin in 3T3-L1 adipocytes as well as increased insulin signaling by endogenous expression or exogenous treatment. The results provide evidence for potent anti-inflammatory action of vaspin not only in vascular cells but also in adipose tissue.

Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society. C1q/TNF-related protein 3 (CTRP3) is a relatively novel adipose tissue-derived cytokine (adipokine) which has been linked to improved glucose regulation and insulin sensitivity. However, the relationship between circulating CTRP3 levels and diabetes is controversial. CTRP3 can circulate in different oligomeric complexes: trimeric, hexameric, and high molecular weight (HMW) oligomeric complexes. However, the concentration of the different oligomeric complexes in human disease states has not been previously investigated. Therefore, the purpose of this study was to compare the levels of different oligomeric complexes of CTRP3 between type 2 diabetic and nondiabetic individuals. Additionally, the association between the oligomeric complexes and other serum factors was examined. CTRP3 primarily circulates in the HMW complex (>50%) and the hexametric multimer, with no CTRP3 detected in the trimeric complex or as a monomer. Further, no differences were observed in total, hexameric, or HMW CTRP3 levels regardless of diabetic status. Surprisingly, HMW CTRP3 was found to be positively correlated with circulating triglyceride levels. Combined, these data suggest that CTRP3 is associated with triglyceride regulation, not diabetic status. These data may explain some of the discrepancies in the literature as elevated triglyceride levels are often detected in patients with obesity and type 2 diabetes.

Obesity results from expansion of white adipose tissue. The inability of white adipose tissue to adequately store lipids leads to ectopic deposition of lipids in non-adipose tissue that can lead to systemic insulin resistance. It is well known that insulin resistance correlates with inflammation of adipose tissue in obese animals and humans. Decreasing inflammation in the adipose tissue has been proven as a therapeutic strategy for improvement of insulin sensitivity in vivo. Numerous factors secreted by immune cells, including macrophages, have been suggested as regulating adipose tissue insulin sensitivity. In the first part of my thesis, I describe the role of one such factor, CD40 in adipose tissue inflammation. The CD40-CD40L dyad acts as co-stimulation in the interaction of antigen-presenting cells, such as macrophages and dendritic cells, with effector cells, such as T cells, in adaptive immunity. We found that CD40 knockout mice were smaller but surprisingly more insulin resistant and glucose intolerant compared to wild-type mice when fed a high fat diet. Consistent with their metabolic phenotype, knockout mice displayed increased adipose tissue inflammation with infiltration of immune cells including macrophages and T cells. Consistent with increased inflammation, CD40 knockout adipose tissue displayed decreased lipid storage. Deficiency of CD40 also led to increased lipid deposition in liver, which may be due to increased lipid release into circulation from the adipose tissue as well as increased lipid synthesis in the liver. CD40 knockout mice had increased hepatic insulin resistance and increased gluconeogensis despite decreased hepatic inflammation. These findings suggest that CD40 is a novel regulator of adipose tissue inflammation in diet-induced obesity. In the second part of this thesis we examined perivascular adipose tissue and brown adipose tissue for the presence of inflammation. In contrast to visceral adipose tissue, macrophage infiltration was absent in perivascular and brown adipose tissue as defined by reduced F480+ cells by flow cytometry and immunohistochemistry. We also found that perivascular adipose tissue was similar to brown adipose tissue as shown by gross morphology and gene expression pattern. Inflammatory gene expression was not increased in brown or perivascular adipose tissue in obese mice as determined by microarray gene expression analysis. These findings suggest that perivascular adipose tissue is more similar to brown adipose tissue than white adipose tissue and that both perivascular and brown adipose tissue are resistant to inflammation. We conclude that, (1) CD40 protects against adipose tissue inflammation in diet-induced obesity, (2) the CD40 knockout mouse is an interesting model of hepatic steatosis with decreased inflammation and (3) perivascular adipose tissue is almost identical to brown adipose tissue in obese mice and that both are resistant to inflammation.