Adipose-like Cells Research Articles

The SR proteins SF2 and RBP1 regulate triglyceride storage in the fat body of Drosophila

In Western societies where food is abundant, these excess nutrients are stored as fats mainly in adipose tissue. Fats are stored in structures known as lipid droplets, and a genome-wide screen performed in Drosophila cells has identified several genes that are important for the formation of these droplets. One group of genes found during this screen included those that regulate mRNA splicing. Previous work from our lab has identified some splicing factors that play a role in regulating fat storage; however, the full complement of splicing proteins that regulate lipid metabolism is still unknown. In this study, the levels of a number of serine-arginine (SR) domain containing splicing factors (RSF1, RBP1, RBP1-like, SF2 and Srp-54) were decreased using RNAi in the adult fat body to assess their role in the control of Drosophila metabolism. Decreasing SF2 and RBP1 showed increased triglycerides, while inducing RNAi towards RSF1, RBP1-Like and Srp-54 had no effect on triglycerides. Interestingly, the increased triglyceride phenotype in the SF2-RNAi flies was due to an increase in the amount of fat stored per cell while the RBP1-RNAi flies have more fat cells. In addition, the splicing of the β-oxidation enzyme, CPT1, was altered in the SF2-RNAi flies potentially promoting the increased triglycerides in these animals. Together, this study identifies novel splicing factors responsible for the regulation of lipid storage in the Drosophila fat body and contributes to our understanding of the mechanisms, which influence the regulation of fat storage in adipose-like cells.

Pathology of Melanotic Schwannoma.

Melanotic schwannoma (MS) is a nerve sheath tumor with a uniform composition of variably melanin-producing Schwann cells and metastatic potential. The MS is an uncommon neoplasm, accounting for less than 1% of all nerve sheath tumors, with a predilection for spinal nerve involvement. Microscopically, the tumors are characterized by spindle and epithelioid cells arranged in interlacing fascicles, with marked accumulation of melanin in neoplastic cells and associated melanophages. The MSs are frequently associated with Carney complex, showing features of psammoma bodies and adipose-like cells. Strict criteria of malignancy in MS are not well developed, although a combination of worrisome histologic features (large, vesicular nuclei, with macronucleoli, brisk mitotic activity, and necrosis) raises concern for aggressive behavior. To review the current status of the MS literature, discussing putative etiology, histopathology, current genetics, and differential diagnoses, including overlap with other pigmented tumors. Search of PubMed (National Center for Biotechnology Information, Bethesda, Maryland) and the authors' own experiences. The occurrence of MS at several unusual anatomic sites and its spectrum of morphologic patterns can result in significant diagnostic difficulty, and correct diagnosis is particularly important because of its high tendency to recur locally and to metastasize, which highlights the importance of diagnostic recognition, ancillary molecular genetic testing, and close clinical follow-up of patients with MS.

Third ventricle liponeurocytoma: one case report and review of literature

Objective To study clinicopathological features, diagnosis, differential diagnosis and prognosis of central nervous system (CNS) liponeurocytoma outside the cerebellum. Methods One case of the third ventricle liponeurocytoma was reported focusing on the following aspects: clinical manifestations, histopathological features and immunophenotypes, and the relevant literatures were reviewed. Results A 52-year-old female presented headache, and her head CT scan showed that there was a low-density lesion area at the former of the brain midline. The tumor was detected in the third ventricle during surgery. The size of tumor was 5.80 cm × 4.00 cm × 3.80 cm. Under optical microscopy, the tumor was biphasic in appearance, which consisted isomorphic small neuronal cells and mature adipose cells and adipose-like cells. Mature osseous tissue and intensive areas of calcification could also be seen. There was no necrosis or mitosis. By using immunohistochemical staining, the tumor cells were diffusely positive for S-100 protein (S-100) and β-catenin. A part of tumor cells were positive for neuronal nuclear antigen (NeuN), synaptophysin (Syn), murine double minute 2 (MDM2) and P53. Besides, the tumor cells were negative for glial fibrillary acidic protein (GFAP), oligodendrocyte transcription factor-2 (Oligo-2), isocitrate dehydrogenase 1 (IDH1), desmin (Des), CD68 and myelin basic protein (MBP). Ki-67 labeling index was about 1%. Conclusions Central nervous system liponeurocytoma is a rare tumor, which is predominantly located in the cerebellar hemispheres, and those located out of cerebellum are much more seldom. Definite diagnosis could be made by typical histopathological characteristics and immunohistochemical expressions. Central nervous system liponeurocytoma has a very well prognosis. Most patients can survive over 5 years, and the longest sugvival is more than 18 years. doi: 10.3969/j.issn.1672-6731.2014.09.012

Transient Brown Adipocyte-Like Cells Derive from Peripheral Nerve Progenitors in Response to Bone Morphogenetic Protein 2

Perineurial-associated brown adipocyte-like cells were rapidly generated during bone morphogenetic protein 2 (BMP2)-induced sciatic nerve remodeling in the mouse. Two days after intramuscular injection of transduced mouse fibroblast cells expressing BMP2 into wild-type mice, there was replication of beta-3 adrenergic receptor(+) (ADRB3(+)) cells within the sciatic nerve perineurium. Fluorescence-activated cell sorting and analysis of cells isolated from these nerves confirmed ADRB3(+) cell expansion and their expression of the neural migration marker HNK1. Similar analysis performed 4 days after BMP2 delivery revealed a significant decrease in ADRB3(+) cells from isolated sciatic nerves, with their concurrent appearance within the adjacent soft tissue, suggesting migration away from the nerve. These soft tissue-derived cells also expressed the brown adipose marker uncoupling protein 1 (UCP1). Quantification of ADRB3-specific RNA in total hind limb tissue revealed a 3-fold increase 2 days after delivery of BMP2, followed by a 70-fold increase in UCP1-specific RNA after 3 days. Expression levels then rapidly returned to baseline by 4 days. Interestingly, these ADRB3(+) UCP1(+) cells also expressed the neural guidance factor reelin. Reelin(+) cells demonstrated distinct patterns within the injected muscle, concentrated toward the area of BMP2 release. Blocking mast cell degranulation-induced nerve remodeling resulted in the complete abrogation of UCP1-specific RNA and protein expression within the hind limbs following BMP2 injection. The data collectively suggest that local BMP2 administration initiates a cascade of events leading to the expansion, migration, and differentiation of progenitors from the peripheral nerve perineurium to brown adipose-like cells in the mouse, a necessary prerequisite for associated nerve remodeling.

Effect of porcine placenta steroid extract on myogenic satellite cell proliferation, transdifferentiation, and lipid accumulation

Intramuscular long-chain fatty acids (LCFAs) play an important role in energy production and initiation of mitochondrial oxidation of lipids. Herein, we report a natural porcine placenta steroid extract (PPSE) that stimulates transdifferentiation and lipid accumulation in bovine myogenic satellite cells (MSCs). The steroids hormones in PPSE were analyzed using enzyme-linked immunosorbant assay and presence of LCFA was established using gas chromatography. At 70% confluent growth, cells were treated with PPSE, LCFAs, transdifferentiation cocktail and commercially available steroid hormones. The working concentrations of all chemicals were manipulated similar to PPSE. The cells were observed for morphological changes and subjected to quantitative analysis of lipid deposition on Days 2, 4, and 6 of treatment. PPSE-treated MSCs exclusively transformed into lipid-accumulated adipose-like cells (ALCs). However, myotubes or adipocytes were formed in cells treated with other chemicals. Expression of different genes was studied to ascertain the molecular mechanism involved in ALC formation. CD36, fatty acid binding protein 4, and peroxisome proliferator-activated receptor-gamma were up-regulated. The expression of CD36 was established through immunocyto-chemical analysis. A viability assay was used to confirm the effect of PPSE on proliferation of MSCs. Hence, a natural steroid extract from porcine was found as a nontoxic mixture, which induces lipid accumulation and transdifferentiation of MSCs to ALCs. From the gene expression studies, it was established that the extract works almost in homogenous manner with other lipid inducers.

Peroxisome Proliferator-activated Receptor β/δ Induces Myogenesis by Modulating Myostatin Activity

Classically, peroxisome proliferator-activated receptor β/δ (PPARβ/δ) function was thought to be restricted to enhancing adipocyte differentiation and development of adipose-like cells from other lineages. However, recent studies have revealed a critical role for PPARβ/δ during skeletal muscle growth and regeneration. Although PPARβ/δ has been implicated in regulating myogenesis, little is presently known about the role and, for that matter, the mechanism(s) of action of PPARβ/δ in regulating postnatal myogenesis. Here we report for the first time, using a PPARβ/δ-specific ligand (L165041) and the PPARβ/δ-null mouse model, that PPARβ/δ enhances postnatal myogenesis through increasing both myoblast proliferation and differentiation. In addition, we have identified Gasp-1 (growth and differentiation factor-associated serum protein-1) as a novel downstream target of PPARβ/δ in skeletal muscle. In agreement, reduced Gasp-1 expression was detected in PPARβ/δ-null mice muscle tissue. We further report that a functional PPAR-responsive element within the 1.5-kb proximal Gasp-1 promoter region is critical for PPARβ/δ regulation of Gasp-1. Gasp-1 has been reported to bind to and inhibit the activity of myostatin; consistent with this, we found that enhanced secretion of Gasp-1, increased Gasp-1 myostatin interaction and significantly reduced myostatin activity upon L165041-mediated activation of PPARβ/δ. Moreover, we analyzed the ability of hGASP-1 to regulate myogenesis independently of PPARβ/δ activation. The results revealed that hGASP-1 protein treatment enhances myoblast proliferation and differentiation, whereas silencing of hGASP-1 results in defective myogenesis. Taken together these data revealed that PPARβ/δ is a positive regulator of skeletal muscle myogenesis, which functions through negatively modulating myostatin activity via a mechanism involving Gasp-1.

Osteogenic and Adipogenic Induction Potential of Human Periodontal Cells

Human periodontium contains different cell types that have various potential roles in hard and soft tissue regeneration. However, there is limited knowledge about how these diverse cell populations contribute to the regenerative process. In this study, we investigated the surface marker difference between different periodontal cells (alveolar osteoblasts [AOs], periodontal ligament fibroblasts [PDLFs], and gingival fibroblasts [GFs]) and their differentiation potential toward osteogenic and adipogenic phenotypes. Periodontal cells (AOs, PDLFs, and GFs) from 14 subjects were isolated. The surface antigen expression pattern of cells was analyzed by cell flow cytometry, and the molecular and histologic characterizations under osteogenic and adipogenic inductions were monitored by reverse transcription-polymerase chain reaction, Western blot, and immunocytohistology. The cell phenotypes of AOs were verified by the high expressions of CD29 and CD49a, whereas PDLFs showed distinctively low levels of CD63 and CD73. Under adipogenic induction, limited AOs formed cube-shaped adipose-like cells, whereas PDLFs formed spindle-shaped adipose-like cells. All three cell types expressed baseline osteo-related genes. AOs demonstrated the highest osteogenic ability followed by PDLFs and GFs. Cells in alveolar bone and periodontal ligament contain osteogenic and adipogenic progenitors. These observations indicate a possible application for periodontium cells in hard or soft tissue regeneration.

Satellite Cells Isolated from Adult Hanwoo Muscle Can Proliferate and Differentiate into Myoblasts and Adipose-like Cells

Satellite Cells Isolated from Adult Hanwoo Muscle Can Proliferate and Differentiate into Myoblasts and Adipose-like Cells

Fourth ventricle neurocytoma with lipomatous and ependymal differentiation

We report the case of a 4-year-old girl with a fourth ventricle tumor diagnosed as a cerebellar liponeurocytoma which recurred, showing ependymal differentiation, 14 months after surgery. Magnetic resonance imaging at initial presentation revealed a large mass in the fourth ventricle, and histology showed a neoplasm characterized by a combination of well-differentiated neurocytes and cells resembling adipocytes. The tumor recurrence was histologically identical to the original tumor in some regions, but with fewer adipose-like cells, while other areas presented an endocrine architecture with oligo-like or pleiomorphic cells, and rosette-like arrangements of tumoral cells were seen around the thin vessels, with features similar to cellular ependymoma. The cells in the liponeurocytoma areas expressed synaptophysin, chromogranin A, and epithelial membrane antigen. Glial fibrillary acidic protein was expressed in some dispersed tumoral cells, in lipidized tumoral cells, and in reactive astrocytes. Cytokeratin was focally expressed in the ependymal region of the recurrence. The immunophenotype of our case, with glial, ependymal, and neuronal or neuroendocrine markers, suggest a neurocytoma with lipomatous and ependymal differentiation. This tumor resembled those derived from circumventricular organs. Its localization in the area postrema region led us to hypothesize that it may be derived from this circumventricular organ.

Roles of peroxisome proliferator-activated receptors delta and gamma in myoblast transdifferentiation

Dietary long chain fatty acids and thiazolidinediones act as potent activators of adipogenesis in established preadipose cell lines. High concentrations of thiazolidinediones have also been shown to induce terminal differentiation of non-preadipose cells, such as fibroblasts and myoblasts, into adipose-like cells. This transdifferentiation was observed in both rodent and human myoblasts. In this report, we show that PPARδ mediates some of the effects exerted by long chain fatty acids on myogenesis and adipogenesis. Activation of PPARδ by long chain fatty acids impairs the expression of the determination factor MyoD1 and α-actin, abolishes the development of multinucleated myotubes, and in parallel induces the expression of PPARγ gene, a master regulator of adipogenesis. Ectopic expression of PPARδ in C2C12 myoblasts potentiated the fatty acid-induced expression of adipogenic markers, while expression of a dominant negative PPARδ mutant exerted opposite effects. Furthermore, a sequential activation of first PPARδ with long chain fatty acids and then PPARγ with thiazolidinediones is required for adipogenesis in C2C12 myoblasts. This study demonstrates that PPARδ, at least in part, is responsible for the dual effects of long chain fatty acids as inhibitors of myogenesis and inducers of transdifferentiation into preadipose-like cells.

Thiazolidinediones and Fatty Acids Convert Myogenic Cells into Adipose-like Cells

Fatty acids and thiazolidinediones act as potent activators of the adipose differentiation program in established preadipose cell lines. In this report, the effects of these agents on the differentiation pathway of myoblasts have been investigated. Exposure of C2C12N myoblasts (a subclone of the C2C12 cell line) to thiazolidinediones or fatty acids prevents the expression of myogenin, alpha-actin, and creatine kinase, thus abolishing the formation of multinucleated myotubes. These treatments lead in parallel to the expression of a typical adipose differentiation program including acquisition of adipocyte morphology and activation of adipose-related genes. A similar transition toward the adipose differentiation pathway also occurs in mouse muscle satellite cells maintained in primary culture. Thiazolidinediones exert their adipogenic effects only in non-terminally differentiated myoblasts; myotubes are insensitive to the compounds. Continuous exposure to inducers after growth arrest is not required to maintain the adipose phenotype, but proliferation of adipose-like C2C12N cells leads to a complete reversion toward undifferentiated cells able to undergo either myogenic or adipogenic differentiation depending on the composition of culture medium. These results indicate that adipogenic inducers, such as thiazolidinediones or fatty acids, specifically convert the differentiation pathway of myoblasts into that of adipoblasts.

Late expression of alpha 2-adrenergic-mediated antilipolysis during differentiation of hamster preadipocytes.

In order to study the ontogenesis of the beta- and alpha 2-adrenergic control of lipolysis during the adipose conversion process, a model based on preadipocytes isolated from the stromal-vascular fraction of hamster adipose tissue was developed. When cultured in an ITT (insulin, transferrin, triiodothyronine) medium supplemented with 2% fetal calf serum, adipose precursors differentiated into adipose-like cells. On 8-day-post-confluent differentiating preadipocytes, the rank order of potency of activation of lipolysis by various beta-adrenergic agonists (BRL37344 greater than norepinephrine = isoproterenol greater than epinephrine greater than fenoterol) was equivalent to that determined in mature adipocytes isolated from adult hamster adipose tissue. On 8-day-post-confluent differentiating preadipocytes, phenylisopropyladenosine (A1-adenosine agonist) and prostaglandin E1 evoked a strong antilipolytic response whereas that evoked by UK 14304 and clonidine (alpha 2-adrenergic agonists) remained undetectable at this step of differentiation. The activity of UK 14304 and clonidine only appeared on 20- to 25-day-post-confluent differentiating preadipocytes. They induced dose-dependent antilipolysis with a maximal effect reaching 80-85% inhibition of adenosine deaminase-stimulated lipolysis. Their action was blocked by increased concentrations of different alpha 2-adrenergic antagonists with the following order of potency, RX 821002 greater than phentolamine much greater than yohimbine. This order of potency was similar to that determined on mature adipocytes isolated from adult hamsters. Both the density of the alpha 2-adrenoceptors, identified with the selective alpha 2-adrenergic radioligand [3H]RX-821002 (19 +/- 1 vs. 30 +/- 1 fmol/mg protein: P less than 0.01) and the amount of Gi proteins identified by pertussis toxin-catalyzed ADP-ribosylation (31 +/- 4 vs. 43 +/- 4% of the amount defined in mature fat cells from adult hamsters: P less than 0.05) were significantly increased between 8 days and 20-25 days after confluence, explaining the late emergence of the alpha 2-adrenergic control of lipolysis during preadipocyte differentiation. In conclusion, the late emergence of the alpha 2-adrenergic control of lipolysis, which is also supported by previous data obtained in vivo that demonstrated the age and/or the fat cell size dependence of alpha 2-adrenoreceptor expression in mature adipocytes, allows the alpha 2-adrenoceptor to be considered as a marker of adipocyte hypertrophy.

Fetuin modulates growth and differentiation of Ob17 preadipose cells in serum-free hormone-supplemented medium

A serum-free hormone-supplemented medium able to support the growth of rodent adipose precursor cells has been used to characterize additional components from serum required for the differentiation of preadipose Ob17 cells into adipose-like cells. Fetuin is shown to behave as a growth-promoting agent for these cells. In addition to growth hormone, triiodothyronine and a low-molecular weight component(s) also purified from serum, fetuin is required for the full expression of the differentiation program. Other serum proteins as well as other mitogenic factors are unable to substitute for fetuin. A possible role of fetuin in the development of adipose tissue is discussed.

Characterization of ouabain-resistant mutants of the preadipocyte Ob17 clonal line Adipose conversion in vitro and in vivo

After exposure to a mutagenic drug (ethylmethanesulfonate), mutant clones of Ob17 preadipose cells resistant to the cytotoxic action of ouabain have been isolated. Their ability to grow in serum-supplemented medium containing ⩾3 mM ouabain is consistent with the decreased ouabain sensitivity of K + transport observed in one of these clones (Ob17-OR11). Ouabain-resistant mutants retain their ability to convert into adipose-like cells in vitro with a frequency similar to that of original Ob17 cells. Long-term maintenance of Ob17-OR11 cells in ouabain-enriched medium leads to a higher frequency of adipose conversion, which is linked to a rather specific and limited growth-promoting effect of the drug. Undifferentiated Ob17-OR11 cells, when injected subcutaneously into athymic mouse, give rise in vivo to a fat pad containing ouabain-resistant mature adipocytes. Therefore, Ob17-OR11 clone should be a useful tool to distinguish in vivo between intrinsic and extrinsic factors involved in the differentiation of adipose precursor cells.

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium.

Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described.

Modulation of lipid-synthesizing enzymes by insulin in differentiated ob17 adipose-like cells.

ob17 cells convert into adipose-like cells when maintained in the presence of physiological concentrations of insulin and tri-iodothyronine. After this conversion, insulin removal from differentiated ob17 cells gives within 24-48 h a large decrease in fatty acid synthetase, glycerol 3-phosphate dehydrogenase and acid:CoA ligase activities, as well as in the rate of fatty acid synthesis determined by [14C]acetate incorporation into lipids. All parameters are restored by insulin addition to initial values within 24-48 h. Dose-response curves of insulin on the restoration of glycerol 3-phosphate dehydrogenase activity and of fatty acid synthesis give half-maximally effective concentrations close to 1 nM, in agreement with the affinity for insulin of the insulin receptors previously characterized in these cells. Immunotitration experiments indicate that the changes in the specific activity of fatty acid synthetase are due to parallel changes in the cellular enzyme content. Therefore the ob17 cell line should be a useful model to study the long-term effects of insulin on the modulation of lipid synthesis in adipose cells.

Lipogenic and mitogenic effects of insulin during conversion of Ob17 cells to adipose-like cells

Chronic exposure to insulin of confluent cells of a preadipocyte clonal line (Ob 17) leads to an acceleration of their development into adipose cells. The short-term effects of insulin have been examined by the stimulation of [ 14C]α-aminoisobutyrate uptake and the long-term effects by the increase in the activity levels of several lipogenic enzymes and in the intracellular triacylglycerol content. These metabolic effects of insulin occur within a physiological range of concentrations (EC 50 ∼- 1 nM). As compared to insulin, dose-response curves obtained with proinsulin on these parameters show at least a 10-fold decrease in sensitivity of the cells. In contrast, the growth-promoting effects of both insulin and proinsulin occur at supraphysiological concentrations (EC 50 > 300 nM). This mitogenic response is likely mediated through binding to receptors of insulin-like growth factors. Our data demonstrate that long-term effects of insulin on lipid synthesis can be dissociated from its effect on cell growth. Therefore the Ob17 cell line should be a useful model to study the role of insulin in the regulation of lipid synthesis in adipose cells.

60] Growth and cytodifferentiation of 3T3-L1 preadipocytes into Adipocytes

This chapter describes the growth and cytodifferentiation of 3T3-L1 preadipocytes into adipocytes. The 3T3-L1 preadipose cell line was isolated from the original uncloned stock (3T3M) of Swiss mouse fibroblasts. This preadipose cell line exhibits fibroblast-like properties during growth. However, when confluent cultures are maintained in a resting state for 2–3 weeks, adipose-like cells develop as clusters throughout the monolayer. The cultured adipocytes have many properties that are similar to those of normal adipocytes. 3T3-L1 cells are plated at 20,000 cells per 60-mm culture dish and fed three times per week with DMEM-10% FCS. It is found that, when plated in this manner, the cells grow to confluence in 5–7 days, reaching a cell number of 50 to 55 × 103 cells/ cm 2 . The adipose conversion does not occur until after the cells reach confluence and enter a resting state. The most striking morphological feature of the adipose conversion is the appearance of triglyceride droplets in the cell cytoplasm. The altered metabolism that leads to the accumulation of triglyceride in the cultured adipocyte results from the development of hormonal responses and enzymatic activities characteristic of mature adipocytes.

Synthesis of collagen in ob 17 preadipocyte cell line

This chapter describes the isolation of cell lines from the adipocyte fraction of epididymal fat pads of C57 BL/6J genetically obese and nonobese mice. Cells of these fibroblast-like established lines are able to differentiate into adipose-like cells, having both morphological and biochemical properties characteristic of adipocytes. Fibroblast-like cells of established cell lines and cell strains, which convert into adipocyte-like cells in culture, have provided unique opportunities for studying the process of adipose conversion. Some of the advantages of this approach are (1) the ease of conducting long-term experiments in vitro, because the viability of adipocytes isolated from adipose tissue does not exceed a few hours, and to do so with no interference of other cell types as the case in explants of adipose tissue; (2) the possibility to control the cell environment and show the direct effects of growth factors and hormones; and (3) the possibility to study morphological and biochemical events occurring during adipose conversion at both cellular and molecular levels.