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Ionic liquids (ILs) have been widely used in biomedical and pharmaceutical fields as solvents or permeation enhancers. Recently, more and more researchers focused on optimizing the physicochemical properties of active pharmaceutical ingredient (API) by ILs technology. Converting APIs into ILs (API-ILs) has shown great potential for drug delivery by eliminating polymorphism, tailoring solubility, improving thermal stability, increasing dissolution, controlling drug release, modulating the surfactant properties, enhancing permeability of APIs and modulating cytotoxicity on tumor cells. In addition, API-ILs are also used in various formulations as active ingredients, such as solutions, emulsions, even tablets or nanoparticles. This paper aims to review current status of API-ILs, including the rational and design, preparation and characterization, the improvement on the physicochemical characteristics of APIs, the compatibility of API-ILs with various formulations, and the future prospects of API-ILs in biomedical and pharmaceutical fields.

MF59, an oil-in-water nanoemulsion, has been used in licensed seasonal influenza vaccines for many years. Administration of such vaccines by injection is associated with pain and safety issues. Here, we evaluated the potential of administering MF59 via a transcutaneous route with antigen loading (either encapsulated into or mixed with MF59) to intact or microneedle-pretreated skin. In addition to commercial MF59, we also prepared a nanoemulsion to encapsulate hydrophilic antigens by mimicking the formulation and preparation technique of MF59. The nanoemulsion was prepared using a water-in-oil-in-water emulsion method, and was similar to MF59 in composition, particle size, and morphology. Compared with the intact skin group, the microneedle-pretreated group showed significant enhanced antigen penetration. In vivo transcutaneous immunization analysis showed that the MF59-adjuvant influenza vaccine elicited approximately 3–5 times higher hemagglutination inhibition titers than the influenza solution alone in BALB/c mice after microneedle pretreatment. The intact skin group showed negative immune results at the same dose, suggesting that microneedle pretreatment was critical for efficient delivery of antigens, to obtain strong immune responses. Furthermore, the loading method (encapsulation or mixing with the vehicle) did not affect the dermal penetration or transcutaneous immunization of antigens on microneedle-pretreated skin. Moreover, in vitro cellular assays showed that MF59 facilitated the maturation of dendritic cells and enhanced antigen uptake by antigen-presenting cells. In conclusion, the combination of microneedle pretreatment and mixing of MF59 with antigen provides a simple approach to enhance transcutaneous immunization.

Subcutaneous adipose tissue is a rich source of stromal vascular fraction (SVF) and adipose-derived stromal/stem cells (ASCs) that are inherently multipotent and exhibit regenerative properties. In current practice, lipoaspirate specimens harvested from liposuction surgeries are routinely discarded as a biohazard waste due to a lack of simple, cost effective, and validated cryopreservation protocols. The aim of this study is to develop a xenoprotein-free cryoprotective agent cocktail that will allow for short-term (up to 6 months) preservation of lipoaspirate tissues suitable for fat grafting and/or stromal/stem cell isolation when stored at achievable temperatures (−20 °C or −80 °C). Lipoaspirates donated by three consenting healthy donors undergoing elective cosmetic liposuction surgeries were suspended in five freezing media (FM1: 10% DMSO and 35% BSA; FM2: 2% DMSO and 43% BSA; FM3: 10% DMSO and 35% lipoaspirate saline; FM4: 2% DMSO and 6% HSA; and FM5: 40% lipoaspirate saline and 10% PVP) all suspended in 1X DMEM/F12 and frozen using commercially available freezers (−20 °C or −80 °C) and stored at least for a 1 month. After 1 month of freezing storage, SVF cells and ASCs were isolated from the frozen-thawed lipoaspirates by digestion with collagenase type I. Cell viability was evaluated by fluorescence microscopy after staining with acridine orange and ethidium bromide. The SVF isolated from lipoaspirates frozen at −80 °C retained comparable cell viability with the tested freezing media (FM2, FM3, FM4) comparable with the conventional DMSO and animal serum media (FM1), whereas the FM5 media resulted in lower viability. In contrast, tissues frozen and stored at −20 °C did not yield live SVF cells after thawing and collagenase digestion. The surface marker expression (CD90, CD29, CD34, CD146, CD31, and CD45) of ASCs from frozen lipoaspirates at −80 °C in different cryoprotectant media were also evaluated and no significant differences were found between the groups. The adipogenic and osteogenic differentiation potential were studied by histochemical staining and gene expression by qRT-PCR. Oil Red O staining for adipogenesis revealed that the CPA media FM1, FM4 and FM5 displayed robust differentiation. Alizarin Red S staining for osteogenesis revealed that FM1 and FM4 media displayed superior differentiation in comparison to other tested media. Measurement of adipogenic and osteogenic gene expression by qRT-PCR provided similar outcomes and indicated that FM4 CPA media comparable with FM1 for adipogenesis and osteogenesis.

The aim of this study is to evaluate the enhanced ability of various choline based ionic liquids (ILs) for dermal delivery of hyaluronic acid (HA). Eight ILs were synthesized via neutralization reactions, which were characterized using Fourier transform infrared spectrometer and proton nuclear magnetic resonance. The enhanced capacity of ILs varied with organic acids. The IL formed by choline and citric acid ([Ch][Cit]) exerted the strongest ability to promote HA penetrating deep into the skin among all ILs. The enhanced ability of [Ch][Cit] were still maintained and even improved after diluted by water although it could be disassociated partially according to electricity. The moisture and transepidermal water loss (TEWL) were employed to evaluate the effect of attenuating skin dehydration. As expected, the IL of choline and malic acid ([Ch][Mala]) and [Ch][Cit] increased moisture and decreased TEWL significantly by improving the penetration of HA into skin. Meanwhile, no irritation was observed on skin of nude mice after treatment by the three ILs which showed optimal enhanced effect. In conclusion, ILs could be high potential biocompatible carriers for facilitating HA dermal delivery to resist skin dehydration for medical or cosmetic uses.

Essential oils (EO) are concentrated hydrophobic liquids containing volatile aromatic compounds obtained from plants, which have properties as withdrawn antibiotic growth promoters. The objective of this study was to explore the effects of EO on growth performance, digestibility, immunity and intestinal health in broilers. A total of 500 1-day-old Arbor Acre broilers were randomly put into five groups with 10 replicate cages containing 10 birds each. Birds in the 5 groups were fed a basal diet (CON), and basal diet with 50, 100, 200 or 400 mg/kg EO (EO0.5, EO1, EO2 and EO4) for 42 d respectively. Birds were euthanized at 21d and 42 d, blood and tissue samples were collected. In the study, the digestibility of DM, GE and EE in groups with EO supplementation were significantly increased compared with CON group (P < 0.05). However, only EO2 and EO4 significantly increased the digestibility of CP compared with CON group (P < 0.05). In contrast to CON group, EO0.5 and EO1 in jejunum at 21 d, and EO1 in jejunum at 42 d markedly increased the activity of sucrase (P < 0.05). In addition, the level of SOD of EO2 and EO4 in serum at 21 d was significantly increased compared with CON group (P < 0.05). What's more, the concentration of intestinal mucosa SIgA in jejunum and ileum at 21 d of groups with EO supplementation was significantly increased compared with CON group (P < 0.05). Moreover, V/C in jejunum at 21 d of groups with EO supplementation, CD in jejunum at 42 d was also significantly increased to compare with CON group (P < 0.05). Furthermore, the expression levels of critical genes associated with nutrient transportation (i.e., GLUT2, SGLT1, SLC38A, SLC79A and SLC27A4) and barrier function (TJP1) were quadratically and linearly up-regulated in jejunum and ileum with EO supplementation (P < 0.05). These results suggest that EO has a positive impact on growth, immunity and intestinal health in broilers, and 200 mg/kg of EO was recommended in broiler diet.

The aim of this work was to develop biocompatible ionic liquids (ILs) for enhancement of dermal delivery of hydrophilic macromolecules. The ILs were prepared from malic acid and choline, which are commonly used as food additives and generally regarded as safe. The choline malate IL (CM-IL) was formed via ionic interactions, which has been validated by Fourier transform infrared spectroscopy and nuclear magnetic resonance. Analysis by differential scanning calorimetry confirmed a melting point of −65.2 °C for CM-IL. In skin penetration study, CM-IL was proved to be able to deliver a model hydrophilic macromolecule dextran into deep skin. The amount of dextran delivered to epidermis and dermis by CM-IL is approximately two folds that of by dextran solution. Enhanced in vivo skin penetration by CM-IL was proved as well. The fluorescence of FITC-dextran could be observed permeating pervasively throughout the skin from 4 h to 24 h, while in the control groups it was mainly concentrated in stratum cornea (SC) and hair follicles. In addition, CM-IL did not shown any irritation to mice skin within 7 days of successive treatment and any toxicity to human epidermal cells (HEK-A) within 24 h. In conclusion, CM-IL could be potentially used as enhancers or vehicles for dermal delivery of hydrophilic macromolecules.

Decellularized human adipose tissue has potential clinical utility as a processed biological scaffold for soft tissue cosmesis, grafting, and reconstruction. Adipose tissue decellularization has been accomplished using enzymatic-, detergent-, and/or solvent-based methods. To examine the hypothesis that distinct decellularization processes may yield scaffolds with differing compositions, the current study employed mass spectrometry to compare the proteomes of human adipose-derived matrices generated through three independent methods combining enzymatic-, detergent-, and/or solvent-based steps. In addition to protein content, bioscaffolds were evaluated for deoxyribose nucleic acid depletion, extracellular matrix composition, and physical structure using optical density, histochemical staining, and scanning electron microscopy. Mass spectrometry based proteomic analyses identified 25 proteins (having at least two peptide sequences detected) in the scaffolds generated with an enzymatic approach, 143 with the detergent approach, and 102 with the solvent approach, as compared to 155 detected in unprocessed native human fat. Immunohistochemical detection confirmed the presence of the structural proteins actin, collagen type VI, fibrillin, laminin, and vimentin. Subsequent in vivo analysis of the predominantly enzymatic- and detergent-based decellularized scaffolds following subcutaneous implantation in GFP+ transgenic mice demonstrated that the matrices generated with both approaches supported the ingrowth of host-derived adipocyte progenitors and vasculature in a time dependent manner. Together, these results determine that decellularization methods influence the protein composition of adipose tissue-derived bioscaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2481-2493, 2018.

Over the last decade and half, the optimization of cryopreservation for adipose tissue derived stromal/stem cells (ASCs) especially in determining the optimal combination of cryoprotectant type, cooling rate, and thawing rate have been extensively studied. In this study, we examined the functionality of ASCs that have been frozen-stored for more than 10 years denoted as long-term freezing, frozen within the last 3 to 7 years denoted as short-term freezing and compared their response with fresh ASCs. The mean post-thaw viability for long-term frozen group was 78% whereas for short-term frozen group 79% with no significant differences between the two groups. The flow cytometry evaluation of stromal surface markers, CD29, CD90, CD105, CD44, and CD73 indicated the expression (above 95%) in passages P1-P4 in all of the frozen-thawed ASC groups and fresh ASCs whereas the hematopoietic markers CD31, CD34, CD45, and CD146 were expressed extremely low (below 2%) within both the frozen-thawed and fresh cell groups. Quantitative real time polymerase chain reaction (qPCR) analysis revealed some differences between the osteogenic gene expression of long-term frozen group in comparison to fresh ASCs. Intriguingly, one group of cells from the short-term frozen group exhibited remarkably higher expression of osteogenic genes in comparison to fresh ASCs. The adipogenic differentiation potential remained virtually unchanged between all of the frozen-thawed groups and the fresh ASCs. Long-term cryopreservation of ASCs, in general, has a somewhat negative impact on the osteogenic potential of ASCs, especially as it relates to the decrease in osteopontin gene expression but not significantly so with respect to RUNX2 and osteonectin gene expressions. However, the adipogenic potential, post thaw viability, and immunophenotype characteristics remain relatively intact between all the groups.

Beyond their traditional use as green solvents, new applications have become available for ionic liquids (ILs) in drug delivery. Their flexible tunability enables task-specific optimization of ILs at molecular level. Thus, ILs have been exploited to improve the solubility and permeability of drugs and relieve the polymorphic problems associated with crystalline active pharmaceutical ingredients (APIs). Controlled preparation of drug nanocarriers are also achieved by using ILs either as media or as functional agents. Here, we highlight the importance and advantages of ILs in pharmaceutics and look towards the future of IL-based drug delivery.

Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a wide range of disease states; however, many expansion protocols rely on the use of fetal bovine serum (FBS) as a cell culture nutrient supplement. The current study explores the substitution of lysates from expired human platelets (HPLs) as an FBS substitute. Methods: Expired human platelets from an authorized blood center were lysed by freeze/thawing and used to examine human ASCs with respect to proliferation using hematocytometer cell counts, colony forming unit-fibroblast (CFU-F) frequency, surface immunophenotype by flow cytometry, and tri-lineage (adipocyte, chondrocyte, osteoblast) differentiation potential by histochemical staining. Results: The proliferation assays demonstrated that HPLs supported ASC proliferation in a concentration dependent manner, reaching levels that exceeded that observed in the presence of 10% FBS. The concentration of 0.75% HPLs was equivalent to 10% FBS when utilized in cell culture media with respect to proliferation, immunophenotype, and CFU-F frequency. When added to osteogenic, adipogenic, and chondrogenic differentiation media, both supplements showed appropriate differentiation by staining. Conclusion: HPLs is an effective substitute for FBS in the culture, expansion and differentiation of human ASCs suitable for pre-clinical studies; however, additional assays and analyses will be necessary to validate HPLs for clinical applications and regulatory approval.

This study aimed to exert the synergistic action of ketoconazole (KCZ) and ionic liquids (ILs) for improving antifungal effect. Various ILs were engineered and demonstrated different solubilization capacity for KCZ. Among them, the IL formed by choline and geranic acid ([Ch][Ger]) was the optimal one and able to imporve the solubility of KCZ by around 100-fold. The in vitro antifungal test revealed the [Ch][Ger] significantly inhibited the activity of T. Interdigitale and exerted the synergistic action with KCZ. Compared to Daktarin®, the [Ch][Ger] not only promoted KCZ to penetrate into deep skin layer but also improved in vivo anti-T. Interdigitale activity significantly. Besides, the [Ch][Ger] was able to strip the skin of the lesion site in a flaky manner to remove fungi more thoroughly. However, the skin can recover to be normal state after treatment and there was no evident skin irritation found in [Ch][Ger] group. The ILs may offer promising opportunities to deliver anti-fungal drugs to treat inner skin fungal infections by synergistic action.

Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Adipose tissue stromal vascular cells or primary adipocytes derived from murine adipose tissue and grown in culture are essential tools for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stromal/stem cells along with protocols for inducing adipogenesis in this cell population or isolating the adipose stromal vascular fraction cells for flow cytometric analysis.

Objectives This study explored the effects of different fiber fractions on growth performance, nutrient digestibility and intestinal development in a porcine model. Methods Blood and tissue samples were collected from thirty-two weaned pigs fed with control diet (CON) or diet containing 5 % cellulose (CEL), xylan (XYL) or β-glucan (GLU). Results This reserch showed that GLU supplementation decreased the growth performance of pigs ( P 0.05). The digestibility of gross energy, ash and crude fiber were higher in the XYL group than that in the GLU and CEL group ( P 0.05). NSPs supplementation decreased serum glucose concentration ( P 0.05). However, the serum superoxide dismutase and glutathione concentrations were elevated in GLU group ( P 0.05). GLU supplementation not only increased the ratio of villus height/crypt depth in duodenum and ileum ( P 0.05), but also elevated the activity of sucrase in the jejunum and ileum compared to CEL group ( P 0.05). Interestingly, both XYL and GLU group showed higher expression levels of genes associated with intestinal barrier functions (zonulaoccludens 1 and claudin 1) and nutrient transportation (Na + -glucose co-transporter 1 and facilitated glucose transporter 2). Moreover, pigs on XYL-containing diet showed higher abundance of Bifidobacterium than the GLU group in the ileum and cecum ( P 0.05). Conclusions This study not only indicated how the gut digestive physiology adapted to different NSP fractions, but also provided novel (to our knowledge) information that could promote our understanding of the role of dietary fibers in maintaining gut health.

This study aimed to develop biocompatible deep eutectic solvents (DESs) as carriers for improving the nasal delivery of insulin. The DES was prepared from malic acid and choline chloride broadly used in foods, drugs, or cosmetics as biocompatible additives. The DES of choline chloride and malic acid (CM-DES) demonstrated lower melting point (−59.1 °C) and higher viscosity (120,000 cP) compared with hydrogels based on sodium carboxyl methyl cellulose (CMC-Na). The conformational structure of insulin does not change in CM-DES as characterized by circular dichroism. The in vitro results showed that CM-DES dissociated gradually but did not disintegrate immediately upon contact with water. CM-DES was able to improve the hypoglycemic effect of insulin significantly at different doses compared with hydrogels or solutions of insulin, which could be ascribed to facilitated penetration of insulin across the nasal epithelia by CM-DES. The hypoglycemic effect of CM-DES loading insulin at a dose of 25 IU/kg was similar to that of subcutaneous insulin at 1 IU/kg. In addition, no evident toxicity to nasal epithelia was observed after nasal administration to rats for seven consecutive days. In conclusion, CM-DES showed promising potential in enhancing the hypoglycemic effect of insulin via the nasal route.

Until recently, the complexity of adipose tissue and its physiological role was not well appreciated. This changed with the discovery of adipokines such as leptin. The cellular composition of adipose tissue is heterogeneous and changes as a function of diabetes and disease states such as diabetes. Tissue engineers view adipose tissue as a rich source of adult stromal/stem cells isolated by collagenase digestion. In vitro and in vivo studies have documented that adipose stromal/stem cells are multipotent, with the ability to differentiate along the adipocyte, chondrocyte, osteoblast and other lineage pathways. The adipose stromal/stem cells secrete a wide range of cytokines and growth factors with potential paracrine actions. Furthermore, adipose stromal/stem cells exert immunomodulatory functions when added to mixed lymphocyte reactions, suggesting that they can be transplanted allogeneically. This review article focuses on these mechanisms of adipose stromal/stem cell action and their potential utility as cellular therapeutics.

Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase.

Adipose tissue serves as a source of adipokines and cytokines with both local and systemic actions in health and disease. In this study, we examine the hypothesis that multipotent human adipose-derived stem cells (ASCs), capable of differentiating along the adipocyte, chondrocyte, and osteoblast pathways, contribute to adipose tissue-derived cytokine secretion. Following exposure to basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), the ASCs significantly increase their secretion of hepatocyte growth factor (HGF), a cytokine implicated in hematopoiesis, vasculogenesis, and mammary epithelial duct formation. Ascorbic acid synergizes with these inductive factors, further increasing HGF levels. Following exposure to lipopolysaccharide, ASCs increase their secretion of both hematopoietic (granulocyte/monocyte, granulocyte, and macrophage colony stimulating factors, interleukin 7) and proinflammatory (interleukins 6, 8, and 11, tumor necrosis factor alpha) cytokines based on ELISA and RT-PCR. In co-cultures established with umbilical cord blood-derived CD34(+) cells, the ASCs support long-term hematopoiesis in vitro. Furthermore, in short-term 12-day co-cultures, the ASC maintain and expand the numbers of both myeloid and lymphoid progenitors. These observations are consistent with the functionality of the secreted cytokines and confirm recent reports by other laboratories concerning the hematopoietic supportive capability of ASCs. We conclude that the ASCs display cytokine secretory properties similar to those reported for bone marrow-derived mesenchymal stem cells (MSCs).

Studies of adipogenic protein induction have led to a new appreciation of the role of adipose tissue as an endocrine organ. Adipocyte-derived "adipokines" such as adiponectin, leptin, and visceral adipose tissue-derived serine protease inhibitor (vaspin) exert hormone-like activities at the systemic level. In this study, we examined the secretome of primary cultures of human subcutaneous adipose-derived stem cells as an in vitro model of adipogenesis. Conditioned media obtained from four individual female donors after culture in uninduced or adipogenic induced conditions were compared by two-dimensional gel electrophoresis and tandem mass spectrometry. Over 80 individual protein features showing > or =2-fold relative differences were examined. Approximately 50% of the identified proteins have been described previously in the secretome of murine 3T3-L1 preadipocytes or in the interstitial fluid derived from human mammary gland adipose tissue. As reported by others, we found that the secretome included proteins such as actin and lactate dehydrogenase that do not display a leader sequence or transmembrane domain and are classified as "cytoplasmic" in origin. Moreover we detected a number of established adipokines such as adiponectin and plasminogen activator inhibitor 1. Of particular interest was the presence of multiple serine protease inhibitors (serpins). In addition to plasminogen activator inhibitor 1, these included pigment epithelium-derived factor (confirmed by Western immunoblot), placental thrombin inhibitor, pregnancy zone protein, and protease C1 inhibitor. These findings, together with the recent identification of vaspin, suggest that the serpin protein family warrants further proteomics investigation with respect to the etiology of obesity and type 2 diabetes.

Adipogenesis plays a critical role in energy metabolism and is a contributing factor to the obesity epidemic. This study examined the proteome of primary cultures of human adipose-derived adult stem (ADAS) cells as an in vitro model of adipogenesis. Protein lysates obtained from four individual donors were compared before and after adipocyte differentiation by two-dimensional gel electrophoresis and tandem mass spectroscopy. Over 170 individual protein features in the undifferentiated adipose-derived adult stem cells were identified. Following adipogenesis, over 40 proteins were up-regulated by ≥2-fold, whereas 13 showed a ≥3-fold reduction. The majority of the modulated proteins belonged to the following functional categories: cytoskeleton, metabolic, redox, protein degradation, and heat shock protein/chaperones. Additional immunoblot analysis documented the induction of four individual heat shock proteins and confirmed the presence of the heat shock protein 27 phosphoserine 82 isoform, as predicted by the proteomic analysis, as well as the crystallin α phosphorylated isoforms. These findings suggest that the heat shock protein family proteome warrants further investigation with respect to the etiology of obesity and type 2 diabetes.

Complex physiological stimuli differentially regulate the tissue-specific transcription of the lipoprotein lipase (LPL) gene. A conserved DNA recognition element (-171 to -149 bp) within the promoter functions as a transcriptional enhancer when bound by the peroxisome proliferator-activated receptor-gamma2 (PPARgamma2)/retinoid X receptor alpha (RXRalpha) heterodimer, but serves as a transcriptional silencer in the presence of unidentified double and single stranded DNA-binding proteins. To address this apparent paradox, the current study examined the effect of two classes of candidate comodulatory proteins, COUP-TF (chicken ovalbumin upstream promoter transcriptional factor) and the corepressor SMRT (silencing mediator of retinoic acid receptor and thyroid receptor). The expression of COUP-TF was detected by Western and Northern blots in a preadipocyte 3T3-L1 cell model during periods corresponding to increased LPL transcription. Cotransfection of COUP-TF expression constructs in the renal epithelial 293T cell line significantly increased transcription from the LPL promoter in synergy with PPARgamma2/RXRalpha heterodimers. The COUP-TFII (ARP-1) protein specifically bound the LPL PPAR recognition element inelectromobility shift assays and interacted directly with the ligand-binding domain of PPARgamma in pull-down experiments. In contrast, cotransfection of SMRT repressed PPARgamma2/ RXRalpha-mediated LPL transcription in the absence or presence of COUP-TFII (ARP-1). The interaction between PPARgamma2 and SMRT localized to the receptor-interactive domain 2 (amino acids 1260-1495) of the SMRT protein based on cotransfection and pull-down assays. These in vitro data indicate that COUP-TF proteins and SMRT modulate PPARgamma-mediated LPL transcription in the 293T cell line.

The peroxisome proliferator activated receptors (PPARs) alpha, beta/delta, and gamma are novel nuclear hormone receptors activated by long chain fatty acids and synthetic ligands and which regulate lipid metabolism. Recent studies have detected PPARgamma mRNA in human mammary tumor cell lines. The current study examined the expression profile of PPAR mRNAs in normal and malignant rodent mammary tissues. Virgin murine mammary glands contained PPAR alpha, beta/delta, and gamma mRNAs based on northern blot analysis. The PPARgamma isoform was predominantly gamma2 based on quantitative PCR analysis. During pregnancy and lactation, the PPARalpha and gamma mRNAs decreased while the PPAR beta/delta mRNA remained relatively unchanged. NMuMG cells, an epithelial line derived from normal murine mammary gland, expressed PPAR alpha, beta/delta, and gamma mRNAs, independent of the presence or absence of compounds modifying PPAR activity. In rats, the physiologic expression pattern of PPARgamma mRNA paralleled the murine model; levels were detected in virgin but not lactating mammary glands. In addition, the PPARgamma mRNA was not detected in several histologically distinct 7,12-dimethylbenz(a)anthracene induced mammary tumors. These findings suggest that PPARs may regulate mammary epithelial and stromal cell function in response to physiologic or pathologic stimuli that profoundly alter lipid metabolism.

The peroxisome proliferator activated receptor gamma 2 (PPAR gamma 2) is a critical transcriptional regulator of adipogenesis. Lipoprotein lipase is one of the earliest genes induced following exposure of pre-adipocytes to PPAR gamma 2 ligands such as the thiazolidinediones. A unique PPAR gamma 2 DNA recognition element was mapped to the region between -171 to -149 bp of the murine LPL promoter, based on transfection analysis of deletion constructs and gel retention assays using bacterially expressed, affinity purified recombinant proteins. Circular permutation analysis determined that binding of the PPAR gamma 2/retinoic acid X receptor (RXR) heterodimer to its LPL promoter recognition element induced DNA bending at an angle of approximately 46 degrees. Parallel studies using an optimal PPAR recognition element obtained a comparable bending angle of 56 degrees. This is the first demonstration that binding of a PPAR protein to its recognition element causes a distortion of the DNA configuration. It indicates that PPAR gamma 2 utilizes a common mechanism shared by other nuclear hormone receptor proteins reported to induce bending at their DNA binding sites.

The current work examines the expression of acute phase genes in a murine-derived bone marrow stromal cell model (BMS2) which exhibits adipocyte and osteoblast characteristics and supports lymphopoiesis in vitro. Each of these physiologic processes is responsive to inflammatory events such as endotoxemia. Exposure of BMS2 cells to pro-inflammatory cytokines induced the expression of the serum amyloid A and complement factor B. During adipocyte differentiation, expression of complement C3, complement factor D (adipsin), and angiotensinogen increased in a time dependent manner. The bone metabolic steroid, 1,25 dihydroxy vitamin D3, specifically induced complement C3 expression in a time- and dose-dependent manner. Based on gel retention analysis, BMS2 nuclear extract contained proteins recognizing specific response elements from the complement C3, angiotensinogen, and complement factor B promoters. These results suggest that the bone marrow's repertoire of acute phase proteins is dependent on the stromal cell's phenotype or activation state.

A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7–10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated. © 1993 Wiley-Liss, Inc.

Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Primary adipocytes grown in culture and derived from murine adipose tissue are essential for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stem cells along with a protocol for inducing adipogenesis in this cell population.

The objective of this study was to test the hypothesis that human adipose tissue-derived adult stem cells (ASCs) can be effectively cryopreserved and stored in liquid nitrogen using a freezing medium containing a high-molecular-weight polymer, polyvinylpyrrolidone (PVP), as the cryoprotective agent (CPA) instead of dimethylsulfoxide (DMSO). To this end we investigated the postfreeze/thaw viability and apoptotic behavior of passage 1 ASCs cryopreserved in 15 different media: (i) the traditional media containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% DMSO; (ii) DMEM with 80% human serum (HS) and 10% DMSO; (iii) DMEM with various concentrations (1%, 5%, 10%, 20%, and 40%) of PVP as the sole CPA; (iv) DMEM with PVP (5%, 10%, and 20%) and HS (10%); (v) DMEM with PVP (5%, 10%, and 20%) and FCS (10%); and (vi) DMEM with PVP (10%) and FCS (40% and 80%). Approximately 1 mL (10(6) cells/mL) of passage 1 ASCs were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees C water bath (1-2 min of agitation), resuspended in culture media, and seeded in separate wells of a six-well plate for a 24-h incubation period at 37 degrees C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 10% PVP and DMEM was comparable with that obtained in freezing media with DMSO and serum (HS or FCS), that is, approximately 70% + or - 8% and approximately 83% + or - 8%, respectively. Slightly enhanced cell viability was observed with the addition of serum (either HS or FCS) to the freezing media containing PVP as the CPA. Adipogenic and osteogenic differentiation behaviors of the frozen thawed cells were also assessed using histochemical staining and optical density measurements and the expression of adipogenic-associated genes was analyzed using reverse transcription-polymerase chain reaction. Our results suggest that after thawing, ASC viability and adipogenic and osteogenic differentiation abilities can be maintained even when ASCs are frozen in the absence of serum but with 10% PVP in DMEM.

Previous studies have demonstrated that EGF and bFGF maintain the stem cell properties of proliferating human adipose-derived stromal/stem cells (hASCs) in vitro. While the expansion and cryogenic preservation of isolated hASCs are routine, these manipulations can impact their proliferative and differentiation potential. This study examined cryogenically preserved hASCs (n = 4 donors), with respect to these functions, after culture with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) at varying concentrations (0-10 ng/ml). Relative to the control, cells supplemented with EGF and bFGF significantly increased proliferation by up to three-fold over 7-8 days. Furthermore, cryopreserved hASCs expanded in the presence of EGF and bFGF displayed increased oil red O staining following adipogenic induction. This was accompanied by significantly increased levels of several adipogenesis-related mRNAs: aP2, C/EBPalpha, lipoprotein lipase (LPL), PPARgamma and PPARgamma co-activator-1 (PGC1). Adipocytes derived from EGF- and bFGF-cultured hASCs exhibited more robust functionality based on insulin-stimulated glucose uptake and atrial natriuretic peptide (ANP)-stimulated lipolysis. These findings indicate that bFGF and EGF can be used as culture supplements to optimize the proliferative capacity of cryopreserved human ASCs and their adipogenic differentiation potential.

Adipocyte function is highly regulated in response to changing oxygen levels and physiological regulation of adipocyte formation involves factors originally identified as hypoxia-responsive proteins. Inhibition of adipogenesis at low oxygen tension is associated with activation of hypoxia inducible factor-1 alpha (HIF-1alpha), a transcription factor essential for cellular responses to decreased oxygen levels whose activity is regulated by prolyl hydroxylase (PHD) enzymes. However, HIF-1alpha RNA expression has been detected during the initial stages of adipocyte formation under aerobic conditions, suggesting a physiological role for HIF-1alpha during adipogenesis under a range of oxygen levels. Here we investigated the expression of HIF-1alpha during adipogenesis using the murine 3T3-L1 adipocyte model. Our results indicate the tissue-specific form of HIF-1alpha is upregulated during adipogenesis with maximal levels obtained within the first 24 h after induction. The increase in HIF-1alpha l.1 gene expression corresponds to increased nuclear HIF-1alpha protein, which gradually declines throughout adipogenesis under aerobic conditions. Each of the three HIF prolyl hydroxylases involved in regulating HIF-1alpha stability is expressed during adipogenesis. The prolyl hydroxylase domain 1 (PHD1) isoform of the HIF prolyl hydroxylases is expressed in early adipogenesis and the PHD2 and PHD3 isoforms are expressed during late adipogenesis. Pharmacological inhibition of PHD activity during the initial stages of adipogenesis abrogates the formation of adipocytes and inhibits gene expression of each of the PHD. However, inhibition of PHD activity does not consistently regulate HIF-1alpha l.1 expression or HIF-1alpha protein levels, suggesting that hydroxylation-independent mechanisms are involved in regulating HIF-1alpha expression in adipocytes under aerobic conditions.

The adipocyte is the most abundant stromal cell phenotype in adult human bone marrow. Four hypotheses may explain their function. First, adipocytes may serve a passive role, simply occupying excess space in the bone marrow cavity. Second, they may play an active role in systemic lipid metabolism. Third, adipocytes may provide a localized energy reservoir in the bone marrow. Or fourth, marrow adipocytes may contribute directly to the promotion of hematopoiesis and influence osteogenesis. This article reviews recent findings concerning bone marrow adipocyte morphology and physiology, the transcriptional and cytokine mechanisms regulating their differentiation, and the interrelationships existing between bone marrow adipocytes, hematopoiesis, and osteogenesis. Overall, these data lend support to a "plastic" model of bone marrow stromal cell differentiation; adipocytes may share common functions with stromal stem cells, osteoblasts, and hematopoietic supportive cells.

Polyclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of retrorsine (1, 351 g/mol), monocrotaline (2, 325 g/mol), and retronecine (3, 155 g/mol) in the parts per billion (ppb) range. A set of three bifunctional linking arms (6-8) was synthesized. By N-alkylation of pyrrolizidine alkaloids (PAs) retrorsine, monocrotaline, and retronecine acetonide (9), six haptens (6.1, 6.2, 7.1, 7.2, 7.9, and 8.9) were synthesized and used to generate rabbit antisera. The resulting anti-retrorsine antiserum gave a 50% inhibition (I50) value of 0.9 +/- 0.2 ppb for retrorsine with detection limits of 0.5-10 ppb. The same ELISA system also detected isatidine (4, retrorsine N-oxide) dihydrate (403 g/mol) with an I50 of 1 ppb and senecionine (5,352 g/mol) with an I50 of 100 ppb. A second monocrotaline-based ELISA detected monocrotaline with an I50 of 36 +/- 9 ppb 2 with detection limits of 5-500 ppb and shows no cross-reactivity with 1 or 5; this ELISA demonstrates the potential for the substrate-specific detection method. A third retronecine-based ELISA detects 3 with an I50 of 3000 +/- 600 ppb (3 +/- 0.6 ppm) and detection limits of 600-10,000 ppb. None of these ELISAs cross-react with the structurally similar swainsonine (10) or lupinine (11) alkaloids. PAs were detected in extracts of Senecio vulgaris and Crotalaria retusa, but not in Lupinus spp., as a demonstration of the ELISA's usefulness.

The enzyme lipoprotein lipase is expressed in a number of cell types and plays a central role in lipid metabolism. Multiple factors regulate its expression in a tissue-specific manner. In murine macrophages, lipopolysaccharide inhibits lipoprotein lipase enzyme activity. The current work examines this process in the established J774 macrophage line and primary peritoneal macrophages from endotoxin-sensitive (C3HeB/Fej) and endotoxin-resistant (C3H/Hej) murine strains. Lipopolysaccharide inhibition of macrophage lipoprotein lipase occurred at the enzyme and mRNA levels in a time- and concentration-dependent manner. Cells from endotoxin-resistant animals maintained their expression of lipoprotein lipase following treatment with lipopolysaccharide. Results of gel retention assays showed that lipopolysaccharide treatment of the J774 macrophages altered the level of nuclear proteins recognizing and binding the lipoprotein lipase promoter DNA. Nuclear extracts from resting J774 cells contained proteins which bound specifically to the octamer motif and to the CAAT box within the lipoprotein lipase promoter. Exposure of the J774 cells to lipopolysaccharide for 16 h increased the level of protein-octamer DNA complexes. Similar responses were obtained in endotoxin-sensitive, but not endotoxin-resistant, primary macrophages following in vitro treatment with lipopolysaccharide. This finding suggests that transcriptional events may contribute to the lipopolysaccharide regulation of macrophage lipoprotein lipase expression.