Increased Adipocyte Differentiation Research Articles

1505-P: CD44 Knockdown in Human Adipocytes—Effect on Adipocyte Proliferation and Differentiation

Introduction: eGWAS implicates the immune-cell receptor CD44 as the top candidate gene in the molecular pathogenesis of T2D1. We have shown that in human adipose tissue, CD44 expression is associated with inflammatory gene expression, M1 macrophage phenotype, and systemic insulin resistance2. Here we sought to determine whether CD44 directly impairs adipocyte function via siRNA knockdown in human preadipocytes with quantification of proliferation, differentiation, and gene expression. Methods: Abdominal subcutaneous adipose tissue was harvested from five healthy nondiabetic obese individuals during bariatric surgery. Preadipocytes were isolated via collagenase digestion and seeded at 1×10^4 cells/well and treated with siRNA-CD44 or mock condition (control) using siRNA Maxi Transfection Reagents (Invitrogen). Cell proliferation was assayed by MTS (Promega) on Days 1, 2, 3 post transfection. Differentiation was induced at confluence (as described3), and cells were harvested at days 3, 6, 12 after confluence for gene expression and at day 12 for oil red O staining (total lipid uptake) and immunofluorescence microscopy (% differentiation = bodipy/dapi*100). Results: CD44 knockdown decreased preadipocyte proliferation and increased adipocyte differentiation and lipid uptake. Conclusion: CD44 may contribute to human insulin resistance by impairing adipocyte differentiation and fat storage. Disclosure H.Chen: None. N.R.Saini: None. E.M.Ayhan: None. T.Mclaughlin: Board Member; January, Inc., Research Support; Merck & Co., Inc., Stock/Shareholder; Eiger Bio. Funding National Institutes of Health (1R21AI159024-01A1)

Hydrogen Sulfide Promotes Adipocyte Differentiation, Hyperplasia, and Hypertrophy

Hydrogen sulfide (H2S) is endogenously produced in adipocytes and fat tissues and stimulates adipogenesis. The integrated pathogenic effects of H2S on the development of obesity and the underlying mechanisms, however, have been unclear. Here, we find that a decreased endogenous H2S level lowered lipid accumulation in mouse adipocytes. Exogenous H2S treatment significantly increased the adipogenesis of primary mouse preadipocytes after six days of adipogenic induction. In the early phase of adipogenesis, H2S increased cell proliferation and prepared cells to go through hyperplasia. After H2S treatment for ten days, preadipocytes exhibited significantly greater cell surface area and diameter, indicating cell hypertrophy. Although it stimulated lipid accumulation and adipogenesis, H2S had no effect on lipolysis. With nutrition overload and high glucose/insulin incubation, H2S further stimulated glucose consumption and deteriorated adipocyte hypertrophy. H2S upregulated hyperplasia genes (CCAAT/enhancer-binding protein (C/EBPβ), cell division cycle 25 (Cdc25), minichromosome maintenance 3 (Mcm3), and cell division cycle (Cdc45)) and cyclin-dependent kinase 2 protein (Cdk2), which regulates cell proliferation. H2S also upregulated the insulin receptor β (Irβ)-activated mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) pathways, leading to adipogenesis. In conclusion, H2S increases adipocyte differentiation, hypertrophy, and hyperplasia, implying that it plays a pathogenic role in obesity disorder.

Adipocyte commitment of 3T3-L1 cells is required to support human adenovirus 36 productive replication concurrent with altered lipid and glucose metabolism.

Human adenovirus 36 (HAdV-D36) can cause obesity in animal models, induces an adipogenic effect and increased adipocyte differentiation in cell culture. HAdV-D36 infection alters gene expression and the metabolism of the infected cells resulting in increased glucose internalization and triglyceride accumulation. Although HAdV-D36 prevalence correlates with obesity in humans, whether human preadipocytes may be targeted in vivo has not been determined and metabolic reprogramming of preadipocytes has not been explored in the context of the viral replication cycle. HAdV-D36 infection of the mouse fibroblasts, 3T3-L1 cells, which can differentiate into adipocytes, promotes proliferation and differentiation, but replication of the virus in these cells is abortive as indicated by short-lived transient expression of viral mRNA and a progressive loss of viral DNA. Therefore, we have evaluated whether a productive viral replication cycle can be established in the 3T3-L1 preadipocyte model under conditions that drive the cell differentiation process. For this purpose, viral mRNA levels and viral DNA replication were measured by RT-qPCR and qPCR, respectively, and viral progeny production was determined by plaque assay. The lipogenic effect of infection was evaluated with Oil Red O (ORO) staining, and expression of genes that control lipid and glucose metabolism was measured by RT-qPCR. In the context of a viral productive cycle, HAdV-D36 modulated the expression of the adipogenic genes, C/EBPα, C/EBPβ and PPARγ, as well as intracellular lipid accumulation, and the infection was accompanied by altered expression of glucolytic genes. The results show that only adipocyte-committed 3T3-L1 cells are permissive for the expression of early and late viral mRNAs, as well as viral DNA replication and progeny production, supporting productive HAdV-D36 viral replication, indicating that a greater effect on adipogenesis occurs in adipocytes that support productive viral replication.

Bisphenol A and 2,3,7,8-tetrachlorodibenzo-p-dioxin at non-cytotoxic doses alter the differentiation potential and cell function of rat adipose-stem cells.

The possibility of chemical contamination is an important issue to consider when designing a cell therapy strategy. Both bisphenol A (BPA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are among the most environmentally relevant endocrine disrupting chemicals (EDCs, compounds with a high affinity for adipose tissue) recently studied. Adipose-derived stem cells (ASCs) are mesenchymal stromal cells (MSCs) obtained from adipose tissue widely used in regenerative medicine to prevent and treat diseases in several tissues and organs. Although the experimental use of tissue-engineered constructs requires careful analysis for approval and implantation, there has been a recent increase in the number of approved clinical trials for this promising strategy. This study aimed to evaluate cell viability, apoptosis, DNA damage, and the adipogenic or osteogenic differentiation potential of rat adipose-derived stem cells (rASCs) exposed to previously established non-cytotoxic doses of BPA and TCDD in vitro. Results demonstrated that 10μM of BPA and 10nM of TCDD were able to significantly reduce cell viability, while all exposure levels resulted in DNA damage, although did not increase the apoptosis rate. According to the analysis of adipogenic differentiation, 1μM of BPA induced the significant formation of oil droplets, suggesting an increased adipocyte differentiation, while both 10μM of BPA and 10nM of TCDD decreased adipocyte differentiation. Osteogenic differentiation did not differ among the treatments. As such, BPA and TCDD in the concentrations tested can modify important processes in rASCs such as cell viability, adipogenic differentiation, and DNA damage. Together, these findings prove that EDCs play an important role as contaminants, putatively interfering in cell differentiation and thus impairing the therapeutic use of ASCs.

MicroRNA-133a Regulates the Viability and Differentiation Fate of Bone Marrow Mesenchymal Stem Cells via MAPK/ERK Signaling Pathway by Targeting FGFR1.

Dysfunction of bone marrow mesenchymal stem cells (BMSCs) is recognized critical in bone deteriorations of osteoporosis. However, the specific mechanisms that determine the fate of BMSCs remain elusive. MicroRNA-133a (miR-133a), a highly conserved microRNA, was investigated under both in vitro and in vivo conditions. In the in vitro study, cell proliferation, cell apoptosis, and osteoblast/adipocyte differentiation of BMSCs as a result of overexpression or knockdown of miR-133a was investigated. In the in vivo study, the ovariectomy (OVX) model was applied on mice, with further treatment of the models with BMSC-specific miR-133a antagomir through femur intramedullary injection. Microcomputed tomography scanning and histological analysis of the proximal and middle femur were performed to evaluate the morphological changes. The results revealed that overexpression of miR-133a suppressed cell proliferation, cell viability, and osteoblast differentiation of BMSCs, but increased adipocyte differentiation. We also found that FGFR1, an important upstream regulator of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signal pathway, was a major target of miR-133a. We also recorded that BMSC-specific knockdown of miR-133a attenuates bone loss in OVX mice. Our study suggested that miR-133a played an important role in maintaining the viability and balance between osteoblast and adipocyte differentiation of BMSCs through the MAPK/ERK signaling pathway by targeting FGFR1.

Hexane Extract of Chloranthus japonicus Increases Adipocyte Differentiation by Acting on Wnt/β-Catenin Signaling Pathway.

Chloranthus japonicus has been heavily investigated for the treatment of various diseases. This paper attempts to show that Chloranthus japonicus can modulate adipocyte differentiation of preadipocytes. To establish this, we investigated the effects of Chloranthus japonicus extract in peroxisome proliferator-activated receptor γ (PPARγ) expression, adipogenesis, and the underlying molecular mechanisms in C3H10T1/2 and 3T3-L1 cells. Our data showed that Chloranthus japonicus methanol extract increased lipid accumulation and promoted adipocyte differentiation. Further studies on the fractionation with various solvents led to the identification of Chloranthus japonicus hexane extract (CJHE) as the most potent inducer of adipocyte differentiation. CJHE consistently increased lipid accumulation and adipocyte marker expression including Pparγ and it acted during the early stages of adipocyte differentiation. Mechanistic studies revealed that CJHE and a Wnt inhibitor similarly stimulated adipogenesis and were active in Wnt-selective reporter assays. The effects of CJHE were inhibited by Wnt3a protein treatment and were significantly blunted in β-catenin-silenced cells, further suggesting that CJHE acted on Wnt pathways to promote adipogenesis. We also showed that Chloranthus japonicus extracts generated from different plant parts similarly promoted adipocyte differentiation. These results identified Chloranthus japonicus as a pro-adipogenic natural product and suggest its potential use in metabolic syndrome.

(+)-Catechin & Proanthocyanidin Fraction of Uncaria gambir Roxb. Improve Adipocytes Differentiation & Glucose Uptake of 3T3-L1 Cells Via Sirtuin-1, Peroxisome Proliferator-Activated Receptor γ (PPAR γ), Glucose Transporter Type 4 (GLUT-4) Expressions.

Purpose: To improve adipocytes differentiation & glucose uptake activity of 3T3-L1 cells through sirtuin-1, peroxisome proliferator-activated receptor γ (PPAR γ), glucose transporter type 4 (GLUT-4) of (+)-catechin & proanthocyanidin fraction Uncaria gambir Roxb. Methods: Adipocytes differentiation activity of (+)-Catechin of Uncaria gambir Roxb. was determined by oil red O staining method & glucose uptake activity was determined by measuring 2-deoxyglucose uptake on 3T3-L1 cells. The ability of (+) - catechin as an activator of sirtuin-1 was assessed by administration of (+) - catechin with the presence of a specific inhibitor of sirtuin-1, nicotinamide. Metformin 1 mM & 5 mM were used as positive control. Sirtuin-1, PPAR γ & GLUT-4 expressions were determined by RT-PCR. Results: (+)-Catechin & proanthocyanidin fraction of Uncaria gambir Roxb. were found to increase adipocyte differentiation & glucose uptake by increasing activity of sirtuin-1 as well as metformin (P ≤0.05). PPAR γ, GLUT-4 and sirtuin-1 expressions were known to be responsible for this activities. Conclusion: These results indicate that (+)–catechin & proanthocyanidin fraction of Uncaria gambir Roxb. could be utilized as a renewable bioresource to develop potential antidiabetic and antiobesity agents.

Epidermal Mineralocorticoid Receptor Inactivation Affects the Homeostasis of All Skin Layers in Chronologically Aged Mice

The increased production of endogenous glucocorticoids (GCs) in the skin of the elderly population contributes to age-related defects strikingly similar to those occurring after pharmacologic treatments with GCs. GCs act through the ligand-dependent transcription factors GC receptor (GR) and mineralocorticoid receptor (MR). We reported that epidermal MR plays nonredundant roles relative to GR in adult mouse skin homeostasis; however, its relative contribution to natural skin aging has not been previously investigated. A 13-month-old MR epidermal knockout (MREKO) mice showed differential features of aging relative to controls (CO) in all skin compartments. MREKO mice were resistant to age-induced epidermal atrophy but showed reduced dermal thickness, with decreased collagen deposition and decreased SMAD2 and 3 activity. Importantly, the dermal white adipose tissue (dWAT) was 2.5-fold enlarged in 13-month MREKO versus CO, featuring adipocyte hyperplasia and hypertrophy at least in part through early increases in Pparg. These changes correlated with compartment-specific alterations in GC signaling. In addition, conditioned medium from MREKO keratinocytes increased adipocyte differentiation, indicating paracrine regulation of adipogenesis through mechanisms that include activation of β-catenin signaling. These findings highlight the importance of epidermal MR in regulating cross-talk among skin compartments in naturally aged skin through GC and β-catenin signaling pathways.

The Effect of Methanolic Valeriana officinalis Root Extract on Adipocyte Differentiation and Adiponectin Production in 3T3-L1 Adipocytes.

Adipose tissue is an endocrine organ and its endocrine function is closely associated with type 2 diabetes mellitus. Valeriana officinalis (Valerian) exerts some physiological effects; however, its influence on adipocytes remains unclear. We investigated the effect of methanolic Valerian root extract (Vale) on 3T3-L1 adipocytes. Vale (1, 10, and 100μg/mL) dose-dependently promoted adipocyte differentiation with increasing lipid accumulation. In addition, Vale significantly increased the mRNA levels in genes associated with adipocyte differentiation, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α , and adipocyte protein 2, in dose-dependent manner. Vale also significantly enhanced mRNA and protein levels in adiponectin. A PPARγ antagonist assay and a PPARγ binding assay revealed that Vale-induced increased adipocyte differentiation and adiponectin production were partly associated with direct binding to PPARγ. Valerenic acid, a characteristic component in Valerian, also demonstrated the ability to induce adipocyte differentiation and adiponectin secretion, suggesting that it is one of the functional components in Vale.

MIR221HG Is a Novel Long Noncoding RNA that Inhibits Bovine Adipocyte Differentiation.

Adipogenesis is a complicated but precisely orchestrated process mediated by a series of transcription factors. Our previous study has identified a novel long noncoding RNA (lncRNA) that was differentially expressed during bovine adipocyte differentiation. Because this lncRNA overlaps with miR-221 in the genome, it was named miR-221 host gene (MIR221HG). The purpose of this study was to clone the full length of MIR221HG, detect its subcellular localization, and determine the effects of MIR221HG on bovine adipocyte differentiation. The 5′ rapid amplification of cDNA ends (RACE) and 3′ RACE analyses demonstrated that MIR221HG is a transcript of 1064 nucleotides, is located on the bovine X chromosome, and contains a single exon. Bioinformatics analyses suggested that MIR221HG is an lncRNA and the promoter of MIR221HG includes the binding consensus sequences of the forkhead box C1 (FOXC1) and krüppel-like factor5 (KLF5). The semi-quantitative PCR and quantitative real-time PCR (qRT-PCR) of nuclear and cytoplasmic fractions revealed that MIR221HG mainly resides in the nucleus. Inhibition of MIR221HG significantly increased adipocyte differentiation, as indicated by a dramatic increment in the number of mature adipocytes and in the expression of the respective adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and fatty acid binding protein 4 (FABP4). Our results provide a basis for elucidating the mechanism by which MIR221HG regulates adipocyte differentiation.

The Effect of Chronic Inflammation and Oxidative and Endoplasmic Reticulum Stress in the Course of Metabolic Syndrome and Its Therapy.

Metabolic syndrome (MetS) is highly associated with a modern lifestyle. The prevalence of MetS has reached epidemic proportion and is still rising. The main cause of MetS and finally type 2 diabetes occurrence is excessive nutrient intake, lack of physical activity, and inflammatory cytokines secretion. These factors lead to redistribution of body fat and oxidative and endoplasmic reticulum (ER) stress occurrence, resulting in insulin resistance, increase adipocyte differentiation, and much elevated levels of proinflammatory cytokines. Cellular therapies, especially mesenchymal stem cell (MSC) transplantation, seem to be promising in the MetS and type 2 diabetes treatments, due to their immunomodulatory effect and multipotent capacity; adipose-derived stem cells (ASCs) play a crucial role in MSC-based cellular therapies. In this review, we focused on etiopathology of MetS, especially on the crosstalk between chronic inflammation, oxidative stress, and ER stress and their effect on MetS-related disease occurrence, as well as future perspectives of cellular therapies. We also provide an overview of therapeutic approaches that target endoplasmic reticulum and oxidative stress.

The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPAR\u03b3

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor that regulates adipocyte differentiation and glucose homeostasis. The transcriptional activity of PPARγ is regulated not only by ligands but also by post-translational modifications (PTMs). In this study, we demonstrate that a novel E3 ligase of PPARγ, tripartite motif-containing 25 (TRIM25), directly induced the ubiquitination of PPARγ, leading to its proteasome-dependent degradation. During adipocyte differentiation, both TRIM25 mRNA and protein expression significantly decreased and negatively correlated with the expression of PPARγ. The stable expression of TRIM25 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 cells. In contrast, the specific knockdown of TRIM25 increased PPARγ protein levels and stimulated adipocyte differentiation. Furthermore, TRIM25-knockout mouse embryonic fibroblasts (MEFs) exhibited an increased adipocyte differentiation capability compared with wild-type MEFs. Taken together, these data indicate that TRIM25 is a novel E3 ubiquitin ligase of PPARγ and that TRIM25 is a novel target for PPARγ-associated metabolic diseases.

Abstract 312: Cardiomyocyte-secreted Signaling Factors Modulate Adipocyte Differentiation And Function

Heart failure (HF) alters cardiomyocyte metabolism by altering energy substrate utilization and mitochondrial function, which has potential implications for global metabolic health. Recent studies demonstrated that cardiomyocyte-released signaling factors communicate with peripheral organ systems to alter metabolic function and that signaling pathways activated by HF control the release of these factors. However, the identity of these factors is still under investigation. Conditioned media (CM) collected from neonatal rat ventricular myocytes (NRVMs) was used to treat 3T3-L1 pre-adipocytes to 1) identify signaling molecules released by cardiomyocytes that could regulate global metabolic function and 2) determine how HF signaling pathways alter their release. NRVM-CM slowed adipocyte differentiation as measured by lipid accumulation using the fluorescent probe BODIPY 493/503 (57±8% decrease in normalized fluorescence compared to control media) and reduced the expression of adipocyte-related proteins. NRVM CM treatment also increased basal mitochondrial oxygen consumption (78 ± 3 vs 62±4 pmol/min) and maximal respiration (270±12 vs 220 ± 16 pmol/min) in mature adipocytes. CM protein fractionation revealed that released-factor activity was retained in >100kDa fractions and this activity was abolished by trypsin digestion, suggesting a novel, high-MW cardiomyocyte-released signaling protein that targets adipocytes. To determine if NRVM-released factor activity is altered by HF signaling, the ability of G-protein kinase 2 (GRK2, upregulated by HF) signaling activity to alter NRVM-released factor activity was investigated. GRK2 overexpression in NRVMs increased the activity of this released-factor as measured by further decreased 3T3-L1 differentiation (lipid accumulation measured by BODIPY fluorescence was 70±8% compared to CM from GFP-overexpressing NRVMs), while overexpression of the GRK2 inhibitor βARKct increased adipocyte differentiation (140±13% compared to control NRVM-CM). These results suggest a novel, high-MW cardiomyocyte-released signaling protein under the control of HF signaling pathways communicates with adipocytes to alter adipose tissue function and potentially alter global metabolic function.

IKKß and Wnt/ß-Catenin Signaling Interaction Regulates Adipogenesis and Osteogenesis in Mesenchymal Stem Cells of Nonhuman Primates and Humans

IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been implicated in the pathogenesis of obesity-associated metabolic disorders. We recently demonstrated that IKKβ also functions in adipose progenitors to regulate adipogenesis and adipose tissue development in mice. Deficiency of IKKβ in adipose progenitors decreased high-fat (HF)-elicited adipogenesis and protected mice from diet-induced adiposity and insulin resistance. Here we report that IKKβ can regulate both adipogenesis and osteogenesis in mesenchymal stem cells (MSCs) of non-human primates (Macaca fascicularis) and humans. Activation or overexpression of IKKβ increased adipocyte differentiation but inhibited osteoblast differentiation of MSCs isolated from adipose tissue and bone marrow of macaques and humans. By contrast, IKKβ loss of function decreased adipogenesis and increased osteogenesis in macaque and human MSCs. Mechanistically, IKKβ can directly interact with Wnt/β-catenin signaling and phosphorylate β-catenin at serine-33, -37 and -45, leading to its ubiquitination and degradation. Inhibition of canonical Wnt/β-catenin signaling then contributed to the increased adipogenesis and decreased osteogenesis in MSCs. Our findings demonstrate IKKβ as a key molecule that regulates MSC differentiation, and provide a connection between IKKβ and Wnt/β-catenin signaling which may lead to new insights into the pathogenesis of obesity and osteoporosis. Disclosure Y. Sui: None. S. Park: None. P.A. Kern: None. R. Temel: Research Support; Self; Ionis Pharmaceuticals, Inc., Regulus Therapeutics Inc.. Stock/Shareholder; Self; Merck & Co., Inc., Ionis Pharmaceuticals, Inc., Resverlogix Corp.. C. Zhou: None.

3,3'-Diindolylmethane Enhances Glucose Uptake Through Activation of Insulin Signaling in 3T3-L1 Adipocytes.

Indole-3-carbinol (I3C), a naturally occurring compound found in cruciferous vegetables, and its metabolite 3,3'-diindolylmethane (DIM) reduce body mass and serum glucose levels in high-fat-diet-induced obese mice. This study aimed to determine whether I3C or DIM could increase glucose uptake via enhanced insulin sensitivity in 3T3-L1 adipocytes, as well as the mechanism involved. 3T3-L1 preadipocytes were differentiated by using a mixture of adipogenic inducers, including a suboptimal concentration of insulin. DIM, but not I3C, increased adipocyte differentiation through upregulation of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α. DIM also enhanced glucose uptake by increasing expression of glucose transporter 4 in adipocytes. This was associated with DIM-enhanced phosphorylation of the signaling intermediates Akt, insulin receptor substrate-1, and insulin receptor early in differentiation. Our findings suggest that DIM may improve insulin sensitivity through the activation of the insulin signaling pathway, leading to enhanced glucose uptake.

Low temperature exposure induces browning of bone marrow stem cell derived adipocytes in vitro

Brown and beige adipocytes are characterised as expressing the unique mitochondrial uncoupling protein (UCP)1 for which the primary stimulus in vivo is cold exposure. The extent to which cold-induced UCP1 activation can also be achieved in vitro, and therefore perform a comparable cellular function, is unknown. We report an in vitro model to induce adipocyte browning using bone marrow (BM) derived mesenchymal stem cells (MSC), which relies on differentiation at 32 °C instead of 37 °C. The low temperature promoted browning in adipogenic cultures, with increased adipocyte differentiation and upregulation of adipogenic and thermogenic factors, especially UCP1. Cells exhibited enhanced uncoupled respiration and metabolic adaptation. Cold-exposed differentiated cells showed a marked translocation of leptin to adipocyte nuclei, suggesting a previously unknown role for leptin in the browning process. These results indicate that BM-MSC can be driven to forming beige-like adipocytes in vitro by exposure to a reduced temperature. This in vitro model will provide a powerful tool to elucidate the precise role of leptin and related hormones in hitherto functions in the browning process.

Improved Growth Patterns in Cystic Fibrosis Mice after Loss of Histone Deacetylase 6

Growth failure in cystic fibrosis (CF) patients has been well-documented and shown to correlate with poorer disease outcomes. This observation is also true in CF animal models, including mouse, pig, rat, and ferret. The etiology underlying growth deficits is unknown, and our previous work demonstrated reduced tubulin acetylation in CF cell models and tissue that is correctable by inhibition of histone deacetylase-6 (HDAC6). Here, we hypothesize that loss of HDAC6 will improve growth phenotype in a CF mouse model. Hdac6 knockout mice were crossed with F508del (CF) mice to generate F508del/Hdac6 (CF/HDA) mice. Growth, fat deposits, survival, and bioelectric measurements were analyzed. CF/HDA mice displayed improvements in length and weight with no correction of CFTR function. Mechanistically, Igf1 levels likely account for increased length and improvements in fertility. Weight gain is attributed to increased fat deposits potentially mediated by increased adipocyte differentiation. CF-related growth deficits can be improved via inhibition of HDAC6, further implicating it as a potential therapeutic target for CF.

Altered Adipogenesis in Zebrafish Larvae Following High Fat Diet and Chemical Exposure Is Visualised by Stimulated Raman Scattering Microscopy.

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on adipogenesis in the zebrafish (Danio rerio). We used label-free Stimulated Raman Scattering (SRS) microscopy for the first time to image zebrafish adipogenesis at 15 days post fertilization (dpf) and compared standard feed conditions (StF) to a high fat diet (HFD) or high glucose diet (HGD). We also exposed zebrafish embryos to a non-toxic concentration of tributyltin (TBT, 1 nM) or Tris(1,3-dichloroisopropyl)phosphate (TDCiPP, 0.5 µM) from 0–6 dpf and reared larvae to 15 dpf under StF. Potential molecular mechanisms of altered adipogenesis were examined by qPCR. Diet-dependent modulation of adipogenesis was observed, with HFD resulting in a threefold increase in larvae with adipocytes, compared to StF and HGD. Developmental exposure to TBT but not TDCiPP significantly increased adipocyte differentiation. The expression of adipogenic genes such as pparda, lxr and lepa was altered in response to HFD or chemicals. This study shows that SRS microscopy can be successfully applied to zebrafish to visualize and quantify adipogenesis, and is a powerful approach for identifying obesogenic chemicals in vivo.

Increased adipocyte differentiation may be mediated by extracellular calcium levels through effects on calreticulin and peroxisome proliferator activated receptor gamma expression in intramuscular stromal vascular cells isolated from Hanwoo beef cattle

Marbling is the intramuscular deposition of adipose tissue and is said to be directly associated with beef quality. Nutritional manipulations are a common practice among beef cattle producers in order to achieve better quality meat. The objective of the current study was to investigate the effects of increasing extracellular calcium on the differentiation of intramuscular stromal vascular cells (IM SVCs) isolated from Hanwoo beef cattle. Primary cell isolates of SVCs were differentiated for 14 days while exposed to increasing concentrations (1.8, 3.6, 7.2, 10.8 mM) of calcium in the media. The correlation between adipogenesis and calcium concentrations was determined through SVC differentiation monitored by RT-PCR, Western blot analysis for PPARγ, C/EBPα, calreticulin, FABP4 and GLUT4 expression. In addition, expression of both phospholipase C gamma (PLC-γ) and protein kinase C (PKC) were investigated since both have possible links to intracellular calcium increase and the expression of several adipogenic genes including FABP4 and GLUT4. Results of the current study provide evidence that stromal vascular cells exposed to the lower concentrations of extracellular calcium have higher rates of adipogenesis possibly due to a decrease in the expression of calreticulin, a known inhibitor of PPARγ expression, and PKC activation followed by PLC-γ activation, leading to the expression of adipogenic genes. Data derived from this study shows that in vitro, decreasing calcium levels present in the microenvironment of fat precursor cells lead to a higher percentage of adipogenesis.

Functional characterisation of bone marrow-derived mesenchymal stromal cells from COPD patients.

Autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) are evaluated for clinical use in chronic obstructive pulmonary disease (COPD) patients, but it is unclear whether COPD affects BM-MSCs.To investigate this, BM-MSCs from nine COPD patients and nine non-COPD age-matched controls were compared with regard to immunophenotype, growth and differentiation potential, and migration capacity. Other functional assays included the response to pro-inflammatory stimuli and inducers of the nuclear factor (erythroid derived 2)-like 2 antioxidant response element (Nrf2-ARE) pathway, and effects on NCI-H292 airway epithelial cells.No significant differences were observed in terms of morphology, proliferation and migration, except for increased adipocyte differentiation potential in the COPD group. Both groups were comparable regarding mRNA expression of growth factors and inflammatory mediators, and in their potential to induce mRNA expression of epidermal growth factor receptor ligands in NCI-H292 airway epithelial cells. MSCs from COPD patients secreted more interleukin-6 in response to pro-inflammatory stimuli. Activation of the Nrf2-ARE pathway resulted in a comparable induction of mRNA expression of four target genes, but the expression of the NAD(P)H:quinone oxidoreductase 1 gene NQO1 was lower in MSCs from COPD patients.The observation that MSCs from COPD patients are phenotypically and functionally comparable to those from non-COPD controls implies that autologous MSCs can be considered for use in the setting of clinical trials as a treatment for COPD.