Adipocyte Staining Research Articles

Libanoridin Isolated from Corydalis heterocarpa Inhibits Adipogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow adiposity is a complication in osteoporotic patients. It is a result of the imbalance between adipogenic and osteogenic differentiation of bone marrow cells. Phytochemicals can alleviate osteoporotic complications by hindering bone loss and decreasing bone marrow adiposity. Corydalis heterocarpa is a biennial halophyte with reported bioactivities, and it is a source of different coumarin derivatives. Libanoridin is a coumarin isolated from C. heterocarpa, and the effect of libanoridin on adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) was evaluated in the present study. Cells were induced to undergo adipogenesis, and their intracellular lipid accumulation and expression of adipogenic markers were observed under libanoridin treatment. Results showed that 10 μM libanoridin-treated adipocytes accumulated 44.94% less lipid compared to untreated adipocytes. In addition, mRNA levels of PPARγ, C/EBPα, and SREBP1c were dose-dependently suppressed with libanoridin treatment, whereas only protein levels of PPARγ were decreased in the presence of libanoridin. Fluorescence staining of adipocytes also revealed that cells treated with 10 μM libanoridin expressed less PPARγ compared to untreated adipocytes. Protein levels of perilipin and leptin, markers of mature adipocytes, were also suppressed in adipocytes treated with 10 μM libanoridin. Analysis of MAPK phosphorylation levels showed that treatment with libanoridin inhibited the activation of p38 and JNK MAPKs observed by decreased levels of phosphorylated p38 and JNK protein. It was suggested that libanoridin inhibited adipogenic differentiation of hBM-MSCs via suppressing MAPK-mediated PPARγ signaling. Future studies revealing the anti-adipogenic effects of libanoridin in vivo and elucidating its action mechanism will pave the way for libanoridin to be utilized as a nutraceutical with anti-osteoporotic properties.

Exosomes Derived From Human Adipose-Derived Stem Cells Inhibit Lipogenesis Involving Hedgehog Signaling Pathway

Since obesity impairs wound closure and adipose-derived exosomes (ADEs) regulate wound healing in clinical applications, we hypothesized that ADEs may inhibit adipogenesis of adipose-derived stem cells (ADSCs) to reduce the adverse effects of obesity on wound healing. Hedgehog (Hh) signaling has been previously shown to inhibit adipogenesis in ADSCs. The present study aimed to determine the role of ADEs in the adipogenesis of ADSCs and the Hh signaling pathway. ADSCs collected from human adipose tissues were co-cultured with ADEs and treated with an adipogenic inducer. qRT-PCR showed that ADEs could inhibit adipogenic differentiation of ADSCs and activate Hh signaling. The differences in the mRNA expression profiles of genes related to Hh signaling between the groups that were exposed to either high fat or low fat indicated that increased Hh signaling activation is necessary but not sufficient to inhibit adipogenic differentiation in the ADSC differentiation process. The Hh signaling pathway can be activated effectively by ADEs, especially during high-fat exposure after treatment with ADEs. Oil Red O staining of adipocytes suggested that ADEs inhibited not only adipogenic differentiation, but also lipogenesis in ADSCs. Overall, targeted activation of Hh signaling by ADEs reduced lipid accumulation in ADSCs and may be explored for clinical applications.

Effects of bisphenol A at the safe reference dose on abdominal fat deposition in aged hens

Bisphenol A (BPA) is an endocrine-disrupting chemical. Its influence on lipid homeostasis remains to be proven. In this study, the obese model of laying hens were induced using high-fat diet (HFD) to determine the lipid metabolism interference of BPA, especially its influence on estrogen receptors (ERs) and oxidative damage, at the dose of tolerable daily intake (TDI, 50 μg/kg body weight [BW]/day) and no observable adverse effect level (NOAEL, 5000 μg/kg BW/day). The results demonstrated that the TDI dose of BPA interacted with ERα more effectively than the NOAEL dose of BPA. The TDI dose of BPA increased the expression of ERα (esr1), which further changed the expression of lipid metabolism-related genes, such as cpt-1, lpl, creb1, and apov1. Furthermore, the abdominal fat rate, hematoxylin-eosin staining of adipocytes, and the average area of the hens were reduced. Therefore, the TDI dose of BPA played an estrogen-compensating role and weakened the effect of HFD on obesity in aged hens. By contrast, BPA at NOAEL dose exhibited great oxidative stress, which remarkably inhibited the activities of antioxidant-related enzymes (total superoxide dismutase and glutathione peroxidase) and promoted the excessive accumulation of lipid peroxidation products (malondialdehyde). Moreover, the increase in oxidative stress corresponded well with the increase in the expression of fat-forming genes (srebp-1, fas, acc, and ppar γ). That is, BPA at NOAEL may accelerate the process of fat formation.

In vivo and in vitro effects of chronical exposure to nonylphenol on lipid metabolism

BackgroundThe incidence of obesity has soared over the last several decades. There is mounting evidence suggesting that the increased presence of environmental endocrine disruptors (EEDs), including nonylphenol (NP), plays an important role in the incidence of lipid metabolism disorders. The aim of this work was to determine whether chronical exposure to NP could induce obesity and lipid metabolism disorders, both in vivo in Sprague–Dawley rats, and in vitro in 3T3-L1 preadipocytes. Forty rats (n = 10 per group) were gavaged with NP in corn oil at dose levels of 0.02 μg/kg/day (low dose, L), 0.2 μg/kg/day (middle dose, M), and 2.00 μg/kg/day (high dose, H) or corn oil alone (vehicle control, C) for 180 days. In vitro study, 3T3-L1 preadipocytes were exposed to NP at concentrations of 0, 40 pM, 40 nM, or 40 μM for 12 days.ResultsIn vivo, the fat weight (F = 103.605, P < 0.001) and fat coefficient (F = 169.807, P < 0.001) of NP-exposed rats were higher than those of control group rats. The serum levels of TC (F = 3.798, P < 0.05), LDL-C (F= 4.946,P < 0.05), and TG (F = 14.117,P < 0.05) in the H group were higher than those in the control group. Protein concentrations of CEBPα (F = 189.104, P < 0.001), FAS (F = 51.011, P < 0.001), PPARγ (F = 114.306,P < 0.001), and SREBP1 (F = 30.432,P < 0.001) in serum in the NP group were higher than those in the control group. The concentration of NP in adipose tissues of rats increased with an increase in NP exposure dose in a dose–response manner (F = 561.353,P < 0.001). The numbers of adipocytes in the M and H groups decreased, and the volume of a single cell increased with cells’ membranes ruptured. With the increase in NP exposure dose, the number of adipocytes per microscope decreased gradually (F = 85.873, P < 0.001). The expression levels of PPARγ (F = 169.936, P < 0.001) and FAS (F = 295.249, P < 0.001) proteins in the H group were higher than those in the control group. CEBPα (F = 101.086, P < 0.001) mRNA expression was up-regulated in the M and H groups; and FAS (F = 439.600, P < 0.001), PPARγ (F = 10.540, P < 0.001), and SREBP1 (F = 123.499, P < 0.001) mRNA expression in NP-exposed groups were significantly higher than those in the control group. In vitro, compared with the control group, the Oil Red Staining of adipocytes in the NP groups was darker, the fat cells were more densely distributed, and some of them fused into large lipid droplets. Expressions of CEBPα (F = 539.103, P < 0.001), FAS (F = 715.740, P < 0.001), PPARγ(F = 114.783, P < 0.001), and SREBP1 (F = 139.600, P < 0.001) proteins in 3T3-L1 preadipocytes were higher in group exposed to 40 μM NP than those in the control group.ConclusionsThe results of this in vivo and in vitro experiment were consistent, and both have demonstrated that NP exposure interfered with the expression of proteins and/or mRNAs of lipid metabolism-related regulators (CEBPα, FAS, SREBP1, PPARγ), promoted the proliferation and differentiation of adipocytes and intracellular accumulation of lipids, and eventually lead to blood lipid disorders and obesity in rats.

Optimizing Collagenase, Fetal Bovine Serum, and Insulin to Isolate, Proliferate, and Differentiate Rat Preadipocyte in vitro

BackgroundThe aim of this study is to develop a method for optimal in vitro proliferation and differentiation of rat adipocyte. MethodsPreadipocyte were isolated from omentum of Rattus norvegicus Wistar using Collagenase type I dan II (Sigma). Cells were cultured in M199 culture containing either 0%, 8% or 10% Fetal Bovine Serum (FBS). Insulin were added into the media once the cells attached to the culture plate Proliferating and differentiated cells were counted and analysed by using Oil Red O dan Hematoxylen (HE) staining. Results This study demonstrated that adipocyte could be isolated using Collagenase type I but not type II. The addition of 10% FBSsignificantly increased the number of preadipocyte and differentiation of adipocyte more than those of 8% FBS and without FBS. The timing of FBS addition was best performed on day 8 using 10% FBS. Specific adipocyte staining using Oil Red O revealed thatthere were core lipids in mature adipocyte. ConclusionsCollagenase tipe I could be used to isolate preadipocyte cells. Supplementation of culture media with 8-10% FBS could enhance the in vitro proliferation of preadipocyte. Standard media M199 containing 10% FBS and insulin may provide an environment to differentiate preadipocyte specifically into adipocyte in vitro.

Effect of different combination of maternal and postnatal diet on adipose tissue morphology in male rat offspring

Purpose Adipose tissue expansion can occur through several different ways and, under certain conditions, can be connected with chronic inflammation. TNF-α is one of the important cytokines involved in this process. Prolonged inflammation in obesity can lead to obesity-related insulin resistance and tissue dysfunction. The aim of our study was to investigate how different combination of maternal and postnatal diet affects offspring adipose tissue morphology and adipose tissue TNF-α expression. Methods Ten female Sprague Dawley rats, 9 weeks old, were randomly divided into two groups and fed either standard laboratory chow or food rich in saturated fatty acids during 6 weeks and then mated with the same male rat. After birth and lactation male rat offspring from both groups were divided into four subgroups depending on the diet they were fed until 22 weeks old. Samples of white adipose tissue were taken from the subcutaneous, epididymal, and perirenal fat pad. On tissue sections, histomorphometric analysis was conducted using CellProfiler program v 2.1.1, and immunohistochemical staining for TNF-α was performed. Results Greater mean surface area of subcutaneous and epididymal adipocytes was found in groups of male rat offspring with altered diet. In perirenal adipose tissue, the highest number of adipocytes was measured in the group where both mother and offspring were fed a high-fat diet. Adipocyte staining intensity for TNF-α did not differ significantly between the groups. Conclusions Together with our previously published data, our results lead to the conclusion that alteration of postnatal diet can lead to TNF-α and adipocyte morphology changes.

Biological activities of the legume crops Melilotus officinalis and Lespedeza capitata for skin care and pharmaceutical applications

The search for natural principles is attracting much interest in the field of skin care. Fabaceae are major agricultural crops and potential sources of bioactive compounds with possible applications in human health and skin care. This study concerns the biological activities of the legume crops Melilotus officinalis (L.) Pall. and Lespedeza capitata Michx. for their potential use in skin care applications. The effects of plant ethanolic extracts at doses ranging from 0.25 to 50μg/mL (from 1 to 5000μg/mL in cell viability assays) were evaluated using in vitro tests on HaCaT human keratinocytes, 46BR.1N fibroblasts, and adipocyte cell cultures, and on matrix-degrading enzymes. MTT assay revealed weak effects on cell viability (IC50>1000μg/mL) and significant increase of fibroblast growth rate with both extracts. Similar induction of cell motility by the two extracts was observed on keratinocytes, while on fibroblasts M. officinalis induced a stronger effect with respect to L. capitata. Cell-free enzymatic assays showed stronger collagenase inhibition by L. capitata, while an ELISA assay revealed more efficient stimulation of fibroblast collagen production by M. officinalis. Oil-Red-O adipocyte staining showed more pronounced lipolytic effect of M. officinalis with respect to L. capitata. Both extracts showed the ability of stimulating skin cells in order to promote tissue regeneration, prevent skin aging, and reduce fat deposition. In most cases, different patterns of activation/inhibition were observed. Data indicate that these legume crops could be profitably exploited in skin care applications, possibly in combined formulations for the development of antiaging and anticellulite products.

Expression of NGF, BDNF and their receptors in subcutaneous adipose tissue of lactating cows

Currently, there are no reports of neurotrophins in adipose tissue of cows. The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their high-affinity tyrosine kinase receptors TrkA and TrkB, was investigated by immunohistochemical method in the subcutaneous adipose tissue of cow at mid-lactation. Results revealed the localization of NGF and BDNF along the plasma membrane and cytoplasm of adipocytes. Neurotrophin receptors TrkA and TrkB showed moderate and strong positive staining in adipocytes, respectively. The expression of NGF, BDNF, TRKB — but not of TRKA — was also confirmed at transcriptional level by RT-PCR analyses.Considering the involvement of BDNF on fat metabolism and of NGF on activation of the sympathetic response in human and rodents, these neurotrophins could be related to lipogenesis and lipolysis occurring during lactation in cows. The local production of these neurotrophins supports their potential paracrine function for the regulation of adipocyte activity and deserve further investigations.

399 ISOLATION AND CHARACTERIZATION OF BOVINE MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE

Stem cells have been widely used because of their multi-differentiation ability. Compared with embryonic stem cells, mesenchymal stem cells (MSC) are more easily sourced and cultured, besides being easily obtained from adult individuals. In this regard, bovine MSC is of great interest because of its wide application in basic and applied research, e.g. in somatic cell nuclear transfer (SCNT). Success rates of SCNT are expected to be enhanced with the use of MSC as donor cells because they exhibit a more undifferentiated condition when compared with the most widely used cell type, fibroblast. There are few reports on MSC referring to this species. Therefore, our aim was to isolate and culture adipose MSC from cattle. Mesenchymal stem cells were induced to differentiate into adipocytes and osteocytes to prove their multi-differentiation ability. A small piece of adipose tissue was sourced from the base of the tail of a cow and extensively washed in cold saline solution containing 5% antibiotic. Recovered tissue was minced and digested in 0.001% collagenase 1 and incubated for 3 h. Collagenase was inactivated in alpha minimal essential medium (MEM) supplemented with 15% FCS. The treated tissue was centrifuged and the pellet plated on plastic dishes in alpha MEM supplemented with 15% FCS and 1% antibiotic. Culture medium was replaced every 2 days, and cells were plated on new dishes before reaching 75% confluence. To test their resistance to grow after thawing, cells were frozen after each passage, thawed, and cultured. Cell growth was accompanied through several weeks, and cells were tested for differentiation ability after 3 passages, when adipocyte and osteocyte differentiation was accomplished using a standard protocol. Briefly, adipocyte differentiation was inducted in DMEM containing 2% FCS, isobutyl-methylxanthine, dexamethasone, insulin, and indomethacin. Osteocyte differentiation was carried out in alpha MEM without FCS containing ascorbate-2-phosphate, dexamethasone, and beta-glycerophosphate.To confirm differentiation, a tissue-specific staining was carried out using Oil Red for adipocyte staining and Alizarin Red for osteocyte staining. The cells adhered to plastic dishes shortly after plating, presented fibroblast-like morphology, and showed an exponential growth curve during the first 6 passages. The cells were capable of growing after thawing similarly to unthawed cells. After 1 week under differentiation protocols, standard morphological changes were observed in the cells. Mesenchymal stem cells subjected to differentiation into adipocytes showed an increase in their size, developed lipid-like vesicles, and stained positive for Oil Red. An increase in the cell size was also observed in the cells subjected to differentiation into osteocyte. Moreover, these cells stained positive for Alizarin Red. Altogether these results provide evidence that these cells are multi-potent MSC. Therefore, we conclude that the current methodology was efficient in isolating bovine MSC from adipose tissue, and the cells resemble those isolated from other mammals. Bovine MSC constitute a significant source of nuclear donor cells for future experiments in SCNT. Financial support was provided by FAPESPA and Cnpq.

Cardiotrophin-1 is expressed in adipose tissue and upregulated in the metabolic syndrome

Adipose tissue is a target for cardiotrophin-1 (CT-1), a cytokine member of the IL-6 family of cytokines that is involved in cardiac growth and dysfunction. However, it is unknown whether adipocytes are a source of CT-1 and whether CT-1 is overexpressed in diseases characterized by increased fat depots [i.e., the metabolic syndrome (MS)]. Thus this work aimed 1) to test whether adipose tissue expresses CT-1 and whether CT-1 expression can be modulated and 2) to compare serum CT-1 levels in subjects with and without MS diagnosed by National Cholesterol Education Program Adult Treatment Panel III criteria. Gene and protein expression of CT-1 was determined by real-time RT-PCR, ELISA, and Western blotting. CT-1 expression progressively increased, along with differentiation time from preadipocyte to mature adipocyte in 3T3-L1 cells. CT-1 expression was enhanced by glucose in a dose-dependent manner in these cells. mRNA and protein CT-1 expression was also demonstrated in human adipose biopsies. Immunostaining showed positive staining in adipocytes. Finally, increased CT-1 serum levels were observed in patients with MS compared with control subjects (127 +/- 9 vs. 106 +/- 4 ng/ml, P < 0.05). Circulating levels of CT-1 were associated with glucose levels (r = 0.2, P < 0.05). Taken together, our data suggest that adipose tissue can be recognized as a source of CT-1, which could account for the high circulating levels of CT-1 in patients with MS.

Salivary gland epithelial cells: A new source of the immunoregulatory hormone adiponectin

Adiponectin is an adipocytokine that displays insulin-sensitizing and immunoregulatory properties. Adipocyte development in association with fibrosis is frequently detected in primary Sjögren's syndrome lesions, connoting a healing process. The aim of this study was to examine the expression of adiponectin in minor salivary gland biopsy specimens obtained from patients with primary SS and controls. The expression of adiponectin in minor salivary gland biopsy specimens and in long-term-cultured non-neoplastic salivary gland epithelial cell (SGEC) lines obtained from patients with primary SS and control subjects was examined, using immunohistochemistry and immunoblotting, respectively. The expression of adiponectin, adiponectin receptor 1 (AdipoR1), and AdipoR2 messenger RNA (mRNA) by SGECs was investigated by reverse transcription-polymerase chain reaction. Immunohistochemical analysis for adiponectin revealed positive staining of adipocytes from primary SS lesions as well as ductal epithelial cells from both patients with primary SS and controls. All of the SGEC lines tested were shown to express adiponectin, AdipoR1, and AdipoR2 mRNA, whereas adiponectin protein expression was detected by immunoblotting in SGECs from patients with primary SS but not in those from controls. The analysis of concentrated culture supernatants also revealed increased adiponectin expression by SGECs from patients with SS compared with controls. Our findings provide novel evidence that adiponectin is produced by SGECs. The high constitutive expression of adiponectin by SGECs from patients with primary SS is likely attributable to aberrant activation of these cells. Although the significance of adiponectin expression remains unknown, it is possible that adiponectin functions in an autocrine manner, as suggested by concurrent expression of the relevant receptors.

Strategies for vascular targeting in tumors.

Strategies for vascular targeting in tumors.

Isolation and tissue profiles of a large panel of phage antibodies binding to the human adipocyte cell surface

Phage display is a powerful technique for the rapid selection and isolation of antibodies to any given target antigen. We have applied this technology to isolate over 100 different human antibodies that bind to antigens expressed in situ on the human adipocyte cell surface. This is a diverse panel of antibodies, as indicated by the V-region sequences. The binding profile of each anti-adipocyte antibody has been characterised using phage antibody immunocytochemistry against a panel of normal human tissues. Although there was some variation in the intensity of the adipocyte staining, each antibody consistently recognised adipocytes, where present, irrespective of the tissue source. In addition, all of the antibodies recognised at least one other cell type other than the adipocyte cell surface. In total, over 50 different tissue-binding profiles were recorded, with the most frequently recognised tissues identified as capillaries or smooth muscle. Extensive tissue binding profiles were generated for some antibodies using a panel of 37 different human tissues. This identified anti-adipocyte antibodies with unexpected profiles, such as FAT.13, which binds only to adipocytes and capillaries in the entire tissue panel. We believe this is the most extensive survey ever undertaken of the human adipocyte cell surface. Moreover, similar methodology could be used to derive complete tissue-binding profiles of antibodies against cell-surface antigens of any cell type. Indeed, by screening antibodies on both normal and diseased tissues, it may be possible to identify antigenic associations between different cell types and the pathologies of many diseases.

Wortmannin, a Specific Phosphatidylinositol 3-Kinase Inhibitor, Inhibits Adipocytic Differentiation of 3T3-L1 Cells

The effect of wortmannin, which inhibits phosphatidylinositol 3-kinase (PI 3-kinase), on adipocytic differentiation of 3T3-L1 cells was examined. The extent of differentiation was evaluated by the staining of adipocytes with Oil Red O and measurement of glycerol 3-phosphate dehydrogenase (GPDH) activity. Wortmannin, at over 100 nM, significantly inhibited adipogenesis of cells treated with isobutyl-methylxanthine, dexamethasone, and insulin with an IC50 value about 50 nM. These results suggest that PI 3-kinase plays a role in adipocytic differentiation of 3T3-L1 cells.

Vascular and cellular development in fetal adipose tissue: Lectin binding studies and immunocytochemistry for laminin and type IV collagen

A cytochemical study of vascular and cellular development in fetal adipose tissue was conducted utilizing 10 plant lectins (fluorescein isothiocyanate (FITC)-labeled), antibodies against laminin, types II and IV collagen, and a probe for actin. Throughout fetal development (50–110 days) blood vessels were stained by galactose binding lectins and stained for actin, type IV collagen, and laminin. Adipocyte reactivity for laminin was strong throughout development, whereas adipocyte staining for type IV collagen and several lectins increased from weak to moderate between 70 and 110 days of fetal life. In general, staining intensity for lectins was greater for blood vessels than for adipocytes at every age, and staining for lectins and type IV collagen was detected much earlier on blood vessels than on adipocytes. However, the ontogeny and intensity of laminin staining were similar for developing adipocytes and vasculature. Adipocyte staining by several lectins was dependent on location within the tissue, whereas blood vessel lectin staining was not location-dependent. Neuraminidase pretreatment abolished the variation in cellular lectin staining due to location (within the tissue) but did not alter age-related changes in cellular staining. This study indicates that the differentiation of the extracellular matrix of blood vessels and adipocytes is clearly distinct in regard to glycoconjugate composition and temporal pattern of glycoconjugate and type IV collagen deposition.