Mechanism and significance of adipose inflammatory recruitment

Adipose tissue has significant roles in whole body energy homeostasis, systemic insulin sensitivity, and lipid metabolism. Increased food intake, physical inactivity, and genetic predisposition lead to over-expansion of adipose tissues. Under constant energy surplus, adipocytes become hypertrophic and adipose tissues undergo hyperplasia. These tissue modifications lead to recruitment of preadipocytes and preadipocyte progenitors (mesenchymal stem cells) into adipogenic lineage, thereby increases the lipid storage capacity of adipose tissues. This keeps circulating blood glucose and fatty acids below toxic levels; however, adipocytes have a saturation point where they cannot store more lipids; when adipocytes are completely engorged with lipids, they start expressing stress signals to recruit inflammation into the tissue. While the mechanisms involved in recruitment of adipose inflammation remain largely unknown, some findings point to “extensive adiposity” as the responsible factor. This thesis focuses on persistent adipose inflammation and its relationship with metabolic comorbidities such as insulin resistance, type 2 diabetes, and particularly, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). A theme of this research is that adipose tissue is not inert, but as closely linked functional “mini endocrine organs” distributed throughout the body. The current literature regarding structural and functional differences in different adipose tissues is reviewed with a focus on morphometric changes (under energy surplus), and systemic effects of adipose inflammation and dysfunction (development of insulin resistance, NAFLD, etc.). The contributions of muscle activity in bodily energy homeostasis and the close interaction between muscle, adipose, liver and insulin metabolism are discussed, and the implications of “adipose failure” for lipid partitioning into the liver, a key pathway to pathogenesis of NAFLD/NASH is highlighted. The low yield of cellular material and the excessive lipid contamination make it harder to work with adipose compared to liver. The candidate developed experimental protocols for this study, which were optimised, including adipose morphometric analysis, that were used to describe the development and progression of adipose tissue inflammation and fatty liver disease in a mouse model of NASH. We examined the effects of feeding an atherogenic diet (23% fat, 45% carbohydrate, 0.19% cholesterol) on adipose morphometry and the recruitment of inflammatory cells. We studied Alms1 mutant foz/foz mice, which have a profound disturbance of hypothalamic appetite regulation. foz/foz mice fed an atherogenic diet developed adipocyte hypertrophy, adipose inflammation, hyperglycemia, and evidence of NASH. Wildtype (WT) mice fed the same diet developed milder metabolic phenotype compared to foz/foz mice. The phenotypic switch towards a proinflammatory phenotype in enlarged adipocytes, this increasing cellular stress causes recruitment of macrophage crown-like structures (CLSs) into adipose tissue and a higher rate of adipocyte injury/necrosis. Toll-like receptors (TLRs) are innate immune system receptors activated by danger-associated molecular patterns (DAMPs). Necrotic cellular debris contains DAMPs which can stimulate TLR9 signalling. Activation of TLR9, particularly in macrophages, exacerbates adipose inflammation. Other works support a role for TLR4 in adipose inflammation, but the roles of other pattern recognizing receptors are less clear. In this study, we used Tlr9-/- mouse to investigate the contribution of TLR9 signalling, if any, to adipose inflammatory recruitment. Increased calorie intake with atherogenic feeding for 24 weeks, led to inflammation in adipose tissue. TLR9 deletion abolished this effect. Correspondingly, NASH prevalence was much less in Tlr9-/- mice. The farnesoid X receptor (FXR) agonist obeticholic acid (6-ECDCA) improves steatosis in patients with NASH, but protective mechanisms remain unresolved. We therefore investigated the effects of 6-ECDCA (1mg/kg/day) on glucose metabolism, multiple adipose compartments and liver in atherogenic diet-fed foz/foz and WT mice. 6-ECDCA reduced body weight, liver mass and hepatic lipid partitioning with striking improvement of glucose tolerance in WT but not foz/foz mice, in which it had no effect on liver histology. 6-ECDCA limited expansion of adipose tissues in atherogenic diet-fed WT but not foz/foz mice. In addition, 6-ECDCA treatment altered macrophage polarization towards a more anti-inflammatory phenotype in WT mouse adipose compartments but not foz/foz mice. To conclude, 6-ECDCA improves glucose metabolism, adiposity and adipose inflammation in animals with a milder metabolic phenotype. Conversely, 6-ECDCA fails to improve adipose inflammation or hepatic lipid partitioning in profoundly obese mice, and there is no reversal of NASH. These results help explain why 6-ECDCA treatment against human NASH improves steatosis but fails to reverse NASH pathology. Physical inactivity contributes to adverse effects of overnutrition. We studied the effects of an exercise intervention on adipose and liver pathology. From weaning, mice were provided with an in-cage exercise wheel, in which they were calculated to run over 4 km/day. In this study, voluntary exercise protected mice against the metabolic effects of high calorie intake and atherogenic dietary feeding by reducing adipose inflammation and maintaining systemic insulin sensitivity, the latter principally with its effects on muscle. Improvements in fatty liver pathology appear to be one of the benefits conferred by exercise. In the final chapter, the most important findings of earlier experiments are discussed in relation to and how they extend the knowledge in the field. Dysfunction of adipose tissue related to stress and inflammation is of pathologic importance. Limited lipid storage capacity and pro-inflammatory factors released from adipose depots can disrupt normal functioning of other organs. Circulating lipids increase when adipose is inflamed and leading to adverse effects on other tissues, primarily the liver. Meanwhile, inflammation in adipose tissue becomes persistent. Obesity-associated NAFLD/NASH, type 2 diabetes and metabolic syndrome are closely linked to persistent adipose inflammation

Characterization Of The Adipogenic Differentiation Potentials Of Bone Marrow And Adipose Tissue-derived Mesenchymal Stem Cells And Dermal Fibroblast

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2011Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2011Özellikle son on yılda yapılan çalışmalar, adipoz dokunun inert bir doku olmaktan çok, aktif bir endokrin organ olduğunu göstermektedir. Bu gelişmeler ise bilimcileri adipoz hücreleri olan adipositlerin kökenlerini araştırmaya yöneltmiştir. Bu bağlamda yapılan en önemli keşif, adipoz dokuda yerleşik olarak bulunan mezenkimal kök hücrelerin bulunmasıdır. Ancak bu hücrelerin kısıtlı çoğalma kapasiteleri sonradan anlaşılmıştır, dolayısıyla adipoz dokuyu dışarıdan besleyen, adipoz dokuda yerleşik olmayan ata hücrelerin bulunduğu hücresel kaynaklar fikri ortaya çıkmıştır. Belirtilen açıklamaya göre bu yüksek lisans tezinin birincil önceliği, kemik iliğinden (BM72 & BM78) ve adipoz dokudan (LA47) elde edilen mezenkimal kök hücrelerin ve dermal fibroblast (DF24) hücrelerinin adipojenik farklılaşma kapasitelerinin anlaşılmasıdır. Bu amaç için 3 tane marker gen (proliferator-activated receptor-γ (PPAR-γ), perilipin, and adiponectin (ADPQ)) ekspresyon dereceleri açısından karşılaştırmalı qRT-PCR tekniği ile analiz edilmişlerdir. Adipojenik uyarıma bağlı olarak mezenkimal kök hücrelerde, marker genlerin ekspresyonları yüksek oranda artmış, fibroblastlarda ise tam etkinleşmemiştir. Şaşırtıcı olan sonuç ise mezenkimal kök hücrelerin gen ekspresyon kinetiklerinin karşılaştırılmalarıyla bulunmuştur. Buna göre, ekspresyondaki değişiklikler bu hücre soylarının yeni çevrelere adaptasyon geliştirdikleri veya doğalarında farklı özellikler taşıdıklarını göstermektedir. Bu yüzden, yapılan buluş bu kök hücrelerin farklı adanmışlık seviyelerine sahip oldukları anlamına gelebilir ki, bu ise adipositlere farklılaşan hücrelerin ve onların atalarının ayırımı açısından anlamlı olabilir. Fibroblast hücreler, adipojenik farklılaşma kapasitelerinin daha iyi anlaşılması için, gen ekspresyon çalışmalarına ek olarak immünofloresans boyama ile analiz edilmişlerdir. Bu hücrelerin yağ parçacık yapılarına ve hücre şekillerine dair anlamlı bulgular elde edilmiş olsa da, fibroblastların bu farklılaşma türüne başlayıp devam ettiklerini net olarak söylememize olanak sağlayacak bulgulara rastlanılamıştır. Diğer bağ doku üyeleri gibi, adipoz dokuda da ekstrasellüler matriks yapısal bir iskele görevi görür. Bu ise dokudaki sinyalizasyon olayları açısından gereklidir. Tezin bu son aşamasında, farklı türdeki ekstrasellüler matriks proteinleri, adipojenik farklılaşma üzerindeki etkileri açısından analiz edilmişlerdir. Bu bağlamda, kollajen tip I’in ve fibronektinin adipojenik farklılaşmayı arttırıcı ve koruyucu etkilerine rastlanılırken, kollajen tip IV ve laminin nötral bir etki göstermişlerdir.Research of especially the past decade has pointed adipose tissue as a highly active endocrine organ rather than being an inert tissue. This also promoted investigations on the origins of adipocytes. In this context, the most important finding was the discovery of the adipose tissue-resident mesenchymal stem cells. However, it has been lately understood that these cells have a limited proliferative capacity, thus the idea occured that there should be other sources, feeding the tissue from non-adipose tissue resident progenitor cells. According to this statement, the principal aim of this master’s thesis was to understand the adipogenic differentiation potentials of two bone marrow (BM72 & BM78) and one adipose tissue (LA47) -derived mesenchymal stem cell strains and a dermal fibroblast cell strain (DF24). For this purpose the relative expression levels of the adipogenic marker genes proliferator-activated receptor-γ (PPAR-γ), perilipin, and adiponectin (ADPQ) were analyzed with qRT-PCR. Upon adipogenic induction, the expression profiles of marker genes were largely intensified in all MSCs from bone marrow or fat tissue, while expression was only rudimentary in dermal fibroblasts. The surprising finding at this part was gained by the comparison of the gene expression kinetics of the MSCs. Accordingly, the differences in expression between these cell strains mirrored an initial adaptation to the different environments or distinct inherent traits. This may point to a difference in the commitment stages of MSCs from bone marrow and adipose tissue, also allowing the discrimination of early progenitors and their progeny, the pre-adipocytes. For better understanding of the adipogenic differentiation potential of DF24 cells, they were analyzed by immunofluorescence staining in parallel to gene expression assays. Although the observed oil droplet formation or cell shape changes were indicative of adipogenesis, there was no definite proof to say that dermal fibroblasts initiate and proceed in true adipogenic differentiation. As other connective tissues, adipose tissue is structured by a sscaffold of extracellular matrix (ECM) which exerts signalling functions, too. At the last part of the thesis, different types of ECM proteins were examined for their influences on adipogenesis. Herein, promoting and mainteaning effects on adipogenic differentiation were found for collagen type I and fibronectin whereas collagen type IV and laminin proteins did not reveal any measurable effect.Yüksek LisansM.Sc

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

Background & Aims: NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods: We fed foz/foz and wild-type mice an atherogenic diet for 16 weeks, gavaged MCC950 or vehicle until 24 weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6 weeks and determined the effects on liver fibrosis. Results: In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24 weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion: MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH.Supported by NIH Project grants R2 AA023574 and U01 AA022489 (to AEF), Australian NHMRC project grants 1084136 and 1044288 (to GCF), and 1086786 (to MAC and AABR), and Deutsche Forschungsgemeinschaft WR 173/3-1 (to AW). Matthew Cooper is an NHMRC Principle Research Fellow (1059354

Exercise-induced ‘browning’ of adipose tissues

Global rates of obesity continue to rise and are necessarily the consequence of a long-term imbalance between energy intake and energy expenditure. This is the result of an expansion of adipose tissue due to both the hypertrophy of existing adipocytes and hyperplasia of adipocyte precursors. Exercise elicits numerous physiological benefits on adipose tissue, which are likely to contribute to the associated cardiometabolic benefits. More recently it has been demonstrated that exercise, through a range of mechanisms, induces a phenotypic switch in adipose tissue from energy storing white adipocytes to thermogenic beige adipocytes. This has generated the hypothesis that the process of adipocyte ‘browning’ may partially underlie the improved cardiometabolic health in physically active populations. Interestingly, ‘browning’ also occurs in response to various stressors and could represent an adaptive response. In the context of exercise, it is not clear whether the appearance of beige adipocytes is metabolically beneficial or whether they occur as a transient adaptive process to exercise-induced stresses. The present review discusses the various mechanisms (e.g. fatty acid oxidation during exercise, decreased thermal insulation, stressors and angiogenesis) by which the exercise-induced ‘browning’ process may occur

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

Background & Aims NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. Methods We fed foz/foz and wild-type mice an atherogenic diet for 16\ua0weeks, gavaged MCC950 or vehicle until 24\ua0weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6\ua0weeks and determined the effects on liver fibrosis. Results In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24\ua0weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. Conclusion MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. Lay summary Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis

Seladelpar (MBX-8025), a selective PPAR-δ agonist, in patients with primary biliary cholangitis with an inadequate response to ursodeoxycholic acid: a double-blind, randomised, placebo-controlled, phase 2, proof-of-concept study.

BACKGROUND: Many patients with primary biliary cholangitis have an inadequate response to first-line therapy with ursodeoxycholic acid. Seladelpar is a potent, selective agonist for the peroxisome proliferator-activated receptor-delta (PPAR-δ), which is implicated in bile acid homoeostasis. This first-in-class study evaluated the anti-cholestatic effects and safety of seladelpar in patients with an inadequate response to ursodeoxycholic acid. METHODS: The study was a 12-week, double-blind, placebo-controlled, phase 2 trial of patients with alkaline phosphatase of at least 1·67 times the upper limit of normal (ULN) despite treatment with ursodeoxycholic acid. Patients, recruited at 29 sites in North America and Europe, were randomly assigned to placebo, seladelpar 50 mg/day, or seladelpar 200 mg/day while ursodeoxycholic acid was continued. Randomisation was done centrally (1:1:1) by a computerised system using an interactive voice-web response system with a block size of three. Randomisation was stratified by region (North America and Europe). The primary outcome was the percentage change from baseline in alkaline phosphatase over 12 weeks, analysed in the modified intention-to-treat (ITT) population (any randomised patient who received at least one dose of medication and had at least one post-baseline alkaline phosphatase evaluation). This study is registered with ClinicalTrials.gov (NCT02609048) and the EU Clinical Trials Registry (EudraCT2015-002698-39). FINDINGS: Between Nov 4, 2015, and May 26, 2016, 70 patients were screened at 29 sites in North America and Europe. During recruitment, three patients treated with seladelpar developed fully reversible, asymptomatic grade 3 alanine aminotransferase increases (one on 50 mg, two on 200 mg), ranging from just over five to 20 times the ULN; as a result, the study was terminated after 41 patients were randomly assigned. The modified ITT population consisted of 12 patients in the placebo group, 13 in the seladelpar 50 mg group, and 10 in the seladelpar 200 mg group. Mean changes from baseline in alkaline phosphatase were -2% (SD 16) in the placebo group, -53% (14) in the seladelpar 50 mg group, and -63% (8) in the seladelpar 200 mg group. Changes in both seladelpar groups versus placebo were significant (p<0·0001 for both groups vs placebo), with no significant difference between the two seladelpar groups (p=0·1729). All five patients who received seladelpar for 12 weeks had normal alkaline phosphatase values at the end of treatment, based on a central laboratory ULN for alkaline phosphatase of 116 U/L. The most frequently reported adverse events were pruritus (16%; one patient on placebo, four on seladelpar 50 mg, and one on seladelpar 200 mg), nausea (13%; one patient on placebo, three on seladelpar 50 mg, and one on seladelpar 200 mg), diarrhoea (10%; two patients on placebo, one on seladelpar 50 mg, and one on seladelpar 200 mg), dyspepsia (8%; two patients on seladelpar 50 mg and one on seladelpar 200 mg), muscle spasms (8%; three patients on seladelpar 200 mg), myalgia (8%; one patient on placebo and two on seladelpar 200 mg), and dizziness (8%; one patient on placebo and two on seladelpar 50 mg). INTERPRETATION: Seladelpar normalised alkaline phosphatase levels in patients who completed 12 weeks of treatment. However, treatment was associated with grade 3 increases in aminotransferases and the study was stopped early. The effects of seladelpar should be explored at lower doses. FUNDING: CymaBay Therapeutics

‘Browning’ the cardiac and peri-vascular adipose tissues to modulate cardiovascular risk

Excess visceral adiposity, in particular that located adjacent to the heart and coronary arteries is associated with increased cardiovascular risk. In the pathophysiological state, dysfunctional adipose tissue secretes an array of factors modulating vascular function and driving atherogenesis. Conversely, brown and beige adipose tissues utilise glucose and lipids to generate heat and are associated with improved cardiometabolic health. The cardiac and thoracic perivascular adipose tissues are now understood to be composed of brown adipose tissue in the healthy state and undergo a brown-to-white transition i.e. during obesity which may be a driving factor of cardiovascular disease. In this review we discuss the risks of excess cardiac and vascular adiposity and potential mechanisms by which restoring the brown phenotype i.e. “re-browning” could potentially be achieved in clinically relevant populations