Korišćenje in vitro model sistema za proučavanje osnovnih bioloških procesa kod riba

With the increasing replacement of fish meal and fish oil with new ingredients in aquaculture diets, imbalances in amino acids, fatty acids, vitamins and minerals can occur. The metabolic and regulatory processes underlying these nutrition-induced imbalances in fish are still not fully understood. At the cellular level, essential dietary compounds and micro-nutrients have been shown to influence lineage determination, differentiation and proliferation of certain cell types, and hence the development of tissue structures and organogenesis. An improved understanding of cellular and molecular events occurring during development in teleosts will enable us to better characterize and define particular requirements, customize feed components, and thus enable development of sustainable feeds, minimize the occurrence of disorders as well as maintain the continuous growth of the fish. In vitro techniques have a great potential in experiments involving mechanism based hypothesis testing, where there is a significant need for a complete understanding of basic biological processes. Cell cultures provide means to study single-factor effects and the combinations thereof in detail, and further, to investigate the role of particular nutrients and their specific gene interactions, which are not possible when working at the organism level. In addition, whole organisms are complex and vary individually, depending on age, sex, health status, type of meal, genetics etc., which makes it difficult to accurately simulate nutritional processes. In vitro experiments offer the unique opportunity to develop standardized methods to study quality of novel and fortified feed products by studying the cellular and molecular effect of different types of food products, ranging from proteins to n-3 fatty acids (FAs) and from fat-soluble vitamins to minerals and trace elements. The system may also be used for studies on the development of functional feeds such as probiotics, prebiotics, bioactive peptides, lipase inhibitors, fat and cholesterol binders and antioxidants. Results from selected in vitro trials, showing how different nutrients may influence development of fat, bone and muscle cells and lipid metabolism in liver cells, will be presented.Zamena ribljeg brašna i ulja sa novim, alternativnim izvorima proteina i masti u hrani za ribe može dovesti do narušavanja odnosa amino kiselina, masnih kiselina i minerala kod ovih organizama. Promene u metaboličkim i regulatornim procesima koje mogu da nastanu loše balansiranom ishranom još uvek nisu dobro proučene kod riba. Esencijalne hranljive materije i mikronutrijenti mogu da utiču na razvoj pojedinih ćelijskih linija, njihovu proliferaciju, a samim tim i na pravilan razvoj tkiva i organa. Bolje razumevanje ćelijskih i molekularnih procesa, koji se dešavaju tokom razvića košljoriba će nam omogućiti da bolje razumemo i definišemo određene zahteve za ishranom na svakom stupnju razvoja riba, prilagodimo komponente riblje hrane, a samim tim i omogućimo normalan razvoj i rast riba, kao i da sprečimo pojavu različitih poremećaja. Korišćenje in vitro sistema predstavlja veliki potencijal za testiranje novih komponenata hrane i daje nam mogućnost za razumevanje osnovnih bioloških procesa kod riba. Prednost korišćenja ćelijskih kultura je što nam one omogućavaju da proučavamo uticaj pojedinačnih faktora, ali i kombinaciju dva ili više različitih faktora i njihov uticaj na gene, što nije moguće istraživati na nivou organizma. Pored toga proučavanje organizma kao celine je jako složeno, jer zavisi od pola, zdravstvenog stanja jedinke, ishrane, genetike, itd., te je na ovaj način lakše simulirati procese ishrane. In vitro eksperimenti nude jedinstvenu priliku da razvijemo standardizovane metode za proučavanje kvaliteta i efekata novih hranljivih materija: od proteina, preko masnih kiselina i minerala, do elemenata, koji se u hrani nalaze u tragovima. Ovi sistemi mogu se koristiti i za proučavanje novih, funkcionalnih hraniva, kao što su: probiotici, prebiotici, bioaktivni peptidi, masti, inhibitori lipaze, antioksidansi. Biće prikazani rezultati odabranih in vitro ispitivanja uticaja različitih hraniva na metabolizam masnog tkiva, kostiju i mišića kao i na sam metabolizam masti

Role of long non-coding RNAs in adipose tissue metabolism and associated pathologies

The incidence of obesity and its related disorders has increased dramatically in recent years and has become a pandemic. Adipose tissue is a crucial regulator of these diseases due to its endocrine capacity. Thus, understanding adipose tissue metabolism is essential to finding new effective therapeutic approaches. The 'omic' revolution has identified new concepts about the complexity of the signaling pathways involved in the pathophysiology of adipose tissue-associated disorders. Specifically, advances in transcriptomics have allowed its application in clinical practice and primary or secondary prevention. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of adipose tissue since they can modulate gene expression at the epigenetic, transcriptional, and post-transcriptional levels. They interact with DNA, RNA, protein complexes, other non-coding RNAs, and microRNAs to regulate a wide range of physiological and pathological processes. Here, we review the emerging field of lncRNAs, including how they regulate adipose tissue biology, and discuss circulating lncRNAs, which may represent a turning point in the diagnosis and treatment of adipose tissue-associated disorders. We also highlight potential biomarkers of obesity and diabetes that could be considered as therapeutic targets. Keywords: Adipose tissue; Biomarkers; Diabetes; Obesity; Therapeutics; lncRNA

Regional fat depot masses are influenced by protein-coding gene variants

Waist-to-hip ratio (WHR) is a prominent cardiometabolic risk factor that increases cardio-metabolic disease risk independently of BMI and for which multiple genetic loci have been identified. However, WHR is a relatively crude proxy for fat distribution and it does not capture all variation in fat distribution. We here present a study of the role of coding genetic variants on fat mass in 6 distinct regions of the body, based on dual-energy X-ray absorptiometry imaging on more than 17k participants. We find that the missense variant CCDC92(S70C), previously associated with WHR, is associated specifically increased leg fat mass and reduced visceral but not subcutaneous central fat. The minor allele-carrying transcript of CCDC92 is constitutively more highly expressed in adipose tissue samples. In addition, we identify two coding variants in SPATA20 and UQCC1 that are associated with arm fat mass. SPATA20(K422R) is a low-frequency variant with a large effect on arm fat only, and UQCC1(R51Q) is a common variant reaching significance for arm but showing similar trends in other subcutaneous fat depots. Our findings support the notion that different fat compartments are regulated by distinct genetic factors.Peer reviewe

Possible mediators of metabolic endotoxemia in women with obesity and women with obesity-diabetes in The Gambia.

AIMS/HYPOTHESIS: Translocation of bacterial debris from the gut causes metabolic endotoxemia (ME) that results in insulin resistance, and may be on the causal pathway to obesity-related type 2 diabetes. To guide interventions against ME we tested two hypothesised mechanisms for lipopolysaccharide (LPS) ingress: a leaky gut and chylomicron-associated transfer following a high-fat meal. METHODS: In lean women (n = 48; fat mass index (FMI) 9.6 kg/m2), women with obesity (n = 62; FMI 23.6 kg/m2) and women with obesity-diabetes (n = 38; FMI 24.9 kg/m2) we used the lactulose-mannitol dual-sugar permeability test (LM ratio) to assess gut integrity. Markers of ME (LPS, EndoCAb IgG and IgM, IL-6, CD14 and lipoprotein binding protein) were assessed at baseline, 2 h and 5 h after a standardised 49 g fat-containing mixed meal. mRNA expression of markers of inflammation, macrophage activation and lipid metabolism were measured in peri-umbilical adipose tissue (AT) biopsies. RESULTS: The LM ratio did not differ between groups. LPS levels were 57% higher in the obesity-diabetes group (P < 0.001), but, contrary to the chylomicron transfer hypothesis, levels significantly declined following the high-fat challenge. EndoCAb IgM was markedly lower in women with obesity and women with obesity-diabetes. mRNA levels of inflammatory markers in adipose tissue were consistent with the prior concept that fat soluble LPS in AT attracts and activates macrophages. CONCLUSIONS/INTERPRETATION: Raised levels of LPS and IL-6 in women with obesity-diabetes and evidence of macrophage activation in adipose tissue support the concept of metabolic endotoxemia-mediated inflammation, but we found no evidence for abnormal gut permeability or chylomicron-associated post-prandial translocation of LPS. Instead, the markedly lower EndoCAb IgM levels indicate a failure in sequestration and detoxification

Implantation of CPT1AM-expressing adipocytes reduces obesity and glucose intolerance in mice

Obesity and its associated metabolic comorbidities are a rising global health and social issue, with novel therapeutic approaches urgently needed. Adipose tissue plays a key role in the regulation of energy balance and adipose tissue-derived mesenchymal stem cells (AT-MSCs) have gained great interest in cell therapy. Carnitine palmitoyltransferase 1A (CPT1A) is the gatekeeper enzyme for mitochondrial fatty acid oxidation. Here, we aimed to generate adipocytes expressing a constitutively active CPT1A form (CPT1AM) that can improve the obese phenotype in mice after their implantation. AT-MSCs were differentiated into mature adipocytes, subjected to lentivirus-mediated expression of CPT1AM or the GFP control, and subcutaneously implanted into mice fed a high-fat diet (HFD). CPT1AM-implanted mice showed lower body weight, hepatic steatosis and serum insulin and cholesterol levels alongside improved glucose tolerance. HFD-induced increases in adipose tissue hypertrophy, fibrosis, inflammation, endoplasmic reticulum stress and apoptosis were reduced in CPT1AM-implanted mice. In addition, the expression of mitochondrial respiratory chain complexes was enhanced in the adipose tissue of CPT1AM-implanted mice. Our results demonstrate that implantation of CPT1AM-expressing AT-MSC-derived adipocytes into HFD-fed mice improves the obese metabolic phenotype, supporting the future clinical use of this ex vivo gene therapy approach

Fettvevets utvikling og funksjoner i atlantisk laks

The trend in salmon aquaculture is to use feed with high lipid content. In current feeds, energy from lipids can comprise as much as 50% of the total energy, compared to 10% approximately 20 years ago. This change was undertaken in order to increase growth and reduce expensive and scarce marine proteins in the feed [1]. However, these lipid-rich diets lead to high lipid deposition in salmonid fishes both in the fillet and around the internal organs [2]. High levels of visceral adipose tissue result in production losses when the salmon is gutted at harvest [3]. The relationship between excess lipid deposition in viscera and health is not well known for fish, but the occurrence of high mortality in slaughter-sized salmonids due to poor heart health and stress has been observed by Tørud and Hillestad [4]. The overall aim of the work described in this thesis was to gain more insight into Atlantic salmon visceral adipose tissue development and functions as well as the effects of n-3 highly unsaturated fatty acids (HUFAs) on adipocytes by combining in vivo fish trials with an in vitro cell culture model. Article I: describes an in vitro method for studying the differentiation of preadipocytes isolated from Atlantic salmon visceral adipose tissue. Isolated preadipocytes differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. After one week in culture, preadipocytes reach confluence. At this stage, the differentiation process is triggered with hormones. Several adipogenic gene markers were measured in order to follow the differentiation process. The expression of the adipogenic gene markers; peroxisome proliferated activated receptor (PPAR) α, lipoprotein lipase, microsomal triglyceride transfer protein, fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during the maturation of adipocytes. In this article, we further describe a novel alternatively spliced form of PPAR γ (PPAR γ short). The expression of PPAR γ short increased during differentiation, while the expression of PPAR γ long was down regulated. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are known to lower the triacylglycerol (TAG) accumulation in human adipocytes both in vivo and in vitro. We show that this is also the situation in fish, both EPA and DHA significantly lower TAG accumulation and increase fatty acid (FA) -oxidation in salmon adipocytes compared to OA. Article II: describes how increasing dietary levels of n-3 HUFAs affects lipid storage and mitochondrial β-oxidation in Atlantic salmon white adipose tissue in vivo. Further, it investigates how EPA and DHA influence the susceptibility to oxidative stress. Increased dietary levels of n-3 HUFAs resulted in lower fat percentage in white adipose tissue; in agreement with the in vitro observations in Article I. Mitochondrial FA β-oxidation activity was higher in the fish oil group than it was in the rapeseed oil group. The relative levels of EPA and DHA in phospholipids from white adipose tissue and mitochondrial membranes increased with the increasing dietary levels of these HUFAs. Together with reduced cytochrome c oxidase activity and increased superoxide dismutase activity in the high HUFA groups, these data show an increased incidence of oxidative stress resulting in non-functional mitochondria with no detectable mitochondrial FA β-oxidation activity. The increased activity of caspase 3, in the high n-3 HUFA groups, further indicated some degree of apoptosis occurring in white adipose tissue of these groups. Decreased fat cell number due to apoptosis, may be one factor explaining the lower TAGs percentage found in the high HUFA groups. Article III: describes development-associated changes in gene expression during determination and terminal differentiation of Atlantic salmon adipose-derived stromo-vascular fraction (aSVF). The determination phase was characterised with cellular heterogeneity. After confluence, however, the cellular heterogeneity decreased as evidenced by the down-regulation of markers of osteo/chondrogenic, myogenic, immune and vasculature cell lineages. Genes involved in nucleotide metabolism and DNA replication, essential processes for cellular division, implied attenuation of proliferation after day 9, in agreement with the number of cells staining positive for proliferating cell nuclear antigen. The terminal differentiation phase was characterised by high lipid accumulation and decreased recruitment of new adipocytes. This was accompanied with increased expressions of several genes involved in lipid and glucose metabolism, including markers of the adipocyte lineage. The gene expression of glyceraldehyde 3-phosphate dehydrogenase and transaldolase, suggested interplay between glycolysis and pentose phosphate pathways in order to secure the production of the glycerol backbone for TAG synthesis. The coordinated-expression of several genes in different antioxidant producing pathways, including the glutathione-based system, pointed to the importance of maintaining a reduced intracellular environment in cells after confluence in order to be able to safely store large amounts of lipids. Signs of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occurred at the later stages of adipocyte differentiation, in parallel with increased lipid droplet formation and increased gene expression of the secretory proteins adipsin and visfatin. The UPR markers, Xbox binding protein 1 and activating transcription factor 6, were induced together with genes involved in ubiquitin-proteasome and lysosomal proteolysis. Notably, changes in expression of a panel of genes belonging to different immune pathways were observed throughout adipogenesis. Article IV: describes how HUFAs may influence oxidative stress responses in salmon adipocytes in vitro. Terminally differentiating adipocytes were cultivated on HUFA-rich media and treated with two agents that affected their oxidative status: buthionine sulfoximine depleted stores of the intracellular antioxidant glutathione and exacerbated oxidative stress, while α-tochopherol protected cells from oxidative stress. Lipid accumulation and the expression of adipogenic gene markers were lower in cells with high HUFA levels and no antioxidant supplemented than in cells added the antioxidant α-tocopherol. Depletion of glutathione with buthionine sulfoximine was associated with the highest activity of superoxide dismutase and the highest levels of reactive oxygen species (ROS) as measured by increased level of thiobarbituric acid reactive substances. α-tocopherol supplementation mediated a reduction in the oxidative stress response in the glutathione-depleted cells, independent of glutathione peroxidases and superoxide dismutase. α-tocopherol seem to have a strong pro-adipogenic effect, while oxidative stress induced by HUFAs and buthionine sulfoximine have anti-adipogenic effects. In addition, α-tocopherol had anti-apoptotic and anti-inflammatory effects and induced the expression of activating transcription factor 6, a marker of ER-stress. The high expression of transcription factor 6 in the α-tocopherol groups may be explained by the higher lipid level found in these groups, since high lipid level is known to induce ER stress.Norges forskningsrå

Lipid Deposition and Mobilisation in Atlantic Salmon Adipocytes

publishedVersio

Gene expression in Atlantic salmon skin in response to infection with the parasitic copepod <it>Lepeophtheirus salmonis</it>, cortisol implant, and their combination

<p>Abstract</p> <p>Background</p> <p>The salmon louse is an ectoparasitic copepod that causes major economic losses in the aquaculture industry of Atlantic salmon. This host displays a high level of susceptibility to lice which can be accounted for by several factors including stress. In addition, the parasite itself acts as a potent stressor of the host, and outcomes of infection can depend on biotic and abiotic factors that stimulate production of cortisol. Consequently, examination of responses to infection with this parasite, in addition to stress hormone regulation in Atlantic salmon, is vital for better understanding of the host pathogen interaction.</p> <p>Results</p> <p>Atlantic salmon post smolts were organised into four experimental groups: lice + cortisol, lice + placebo, no lice + cortisol, no lice + placebo. Infection levels were equal in both treatments upon termination of the experiment. Gene expression changes in skin were assessed with 21 k oligonucleotide microarray and qPCR at the chalimus stage 18 days post infection at 9°C. The transcriptomic effects of hormone treatment were significantly greater than lice-infection induced changes. Cortisol stimulated expression of genes involved in metabolism of steroids and amino acids, chaperones, responses to oxidative stress and eicosanoid metabolism and suppressed genes related to antigen presentation, B and T cells, antiviral and inflammatory responses. Cortisol and lice equally down-regulated a large panel of motor proteins that can be important for wound contraction. Cortisol also suppressed multiple genes involved in wound healing, parts of which were activated by the parasite. Down-regulation of collagens and other structural proteins was in parallel with the induction of proteinases that degrade extracellular matrix (MMP9 and MMP13). Cortisol reduced expression of genes encoding proteins involved in formation of various tissue structures, regulators of cell differentiation and growth factors.</p> <p>Conclusions</p> <p>These results suggest that cortisol-induced stress does not affect the level of infection of Atlantic salmon with the parasite, however, it may retard repair of skin. The cortisol induced changes are in close concordance with the existing concept of wound healing cascade.</p

Precursor cells from Atlantic salmon (Salmo salar) visceral fat holds the plasticity to differentiate into the osteogenic lineage

In order to study the potential plasticity of Atlantic salmon (Salmo salar) precursor cells (aSPCs) from the adipogenic mesenchyme cell lineage to differentiate to the osteogenic lineage, aSPCs were isolated and cultivated under either osteogenic or adipogenic promoting conditions. The results strengthen the hypothesis that aSPCs most likely are predestined to the adipogenic lineage, but they also hold the flexibility to turn into other lineages given the right stimuli. This assumption is supported by the fact that the transcription factor pparγ , important for regulation of adiopogenesis, was silent in aSPCs grown in osteogenic media, while runx2, important for osteogenic differentiation, was not expressed in aSPCs cultivated in adipogenic media. After 2 weeks in osteogenic promoting conditions the cells started to deposit extracellular matrix and after 4 weeks, the cells started mineralizing secreted matrix. Microarray analyses revealed large-scale transcriptome responses to osteogenic medium after 2 days, changes remained stable at day 15 and decreased by magnitude at day 30. Induction was observed in many genes involved in osteogenic differentiation, growth factors, regulators of development, transporters and production of extracellular matrix. Transcriptome profile in differentiating adipocytes was markedly different from differentiating osteoblasts with far fewer genes changing activity. The number of regulated genes slowly increased at the mature stage, when adipocytes increased in size and accumulated lipids. This is the first report on in vitro differentiation of aSPCs from Atlantic salmon to mineralizing osteogenic cells. This cell model system provides a new valuable tool for studying osteoblastogenesis in fish