Analyses and Utilization of Selectively Tuned Human Adipose-Derived Stromal/Stem Cell Exosomes.

We have developed a hollow fiber bioreactor-based production system for manufacturing large quantities of extracellular vesicles (EVs) containing exosomes from adult human adipose-derived stromal/stem cells (ASCs). By manipulating the cellular bioreactor environment, we have found that we can alter ASC EV production, secretion, and surface protein composition. The aims of this chapter are to describe the methodology for culturing and tuning of adipose ASCs in a bioreactor, along with the collection and isolation of the EVs containing exosomes demonstrating increased HSP70 content.

Biguanides Induce Acute de novo Lipogenesis in Human Primary Sebocytes.

INTRODUCTION: Acne arises during puberty, in part, due to elevated hormones and growth factors which stimulate de novo lipogenesis (DNL) in primary sebocytes to significantly increase sebum production. Oral isotretinoin is an effective acne therapy, reducing sebum production through inducing apoptosis in sebocytes. However, isotretinoin is teratogenic and has additional unwanted side effects, including an initial acne flare-up, which limits its utility. The biguanide, metformin has been found to alleviate severe acne in women with polycystic ovary syndrome (PCOS) through normalization of their insulin and androgen hormone levels. Metformin's broader effectiveness to improve acne in non-PCOS populations lacks significant clinical support. In an effort to determine whether biguanides directly affect sebogenesis, we investigated their ability to alter DNL in cell-based assays in vitro. METHODS: De novo lipogenesis was measured in human primary sebocytes using [14C]-acetate labeling. Lipid species analysis was performed by extracting newly synthesized lipids and subjecting them to thin layer chromatography. Gene expression changes in sebocytes were identified through qPCR analysis of isolated RNA. Metabolic parameters including oxygen consumption rate, lactate production and activation of adenosine monophosphate-dependent protein kinase (AMPK) were assessed in human primary sebocytes. RESULTS: Using human primary sebocytes, we found that biguanides, isotretinoin and azithromycin induced an acute dose and time-dependent increase in [14C]-acetate labeling of neutral lipids, while AICAR, an AMPK activator, inhibited this DNL response. Biguanides did not activate AMPK in sebocytes, however, they significantly reduced oxygen consumption rate and increased lactate production. Treatment with biguanides, but not isotretinoin, significantly upregulated ACSS2 gene expression in primary sebocytes and showed synergism with lipogenic activators to induce DNL genes. DISCUSSION: These changes are consistent with an acute increase in sebocyte lipogenesis and support the potential of biguanides to cause an initial flare-up in patients suffering from severe acne.

Characterization of a primary brown adipocyte culture system derived from human fetal interscapular fat.

Brown fat has gained widespread attention as a potential therapeutic target to treat obesity and associated metabolic disorders. Indeed, the anti-obesity potential of multiple targets to stimulate both brown adipocyte differentiation and recruitment have been verified in rodent models. However, their therapeutic potential in humans is unknown due to the lack of a human primary brown adipocyte cell culture system. Likewise, the lack of a well-characterized human model has limited the discovery of novel targets for the activation of human brown fat. To address this current need, we aimed to identify and describe the first primary brown adipocyte cell culture system from human fetal interscapular brown adipose tissue. Pre-adipocytes isolated from non-viable human fetal interscapular tissue were expanded and cryopreserved. Cells were then thawed and plated alongside adult human subcutaneous and omental pre-adipocytes for subsequent differentiation and phenotypic characterization. Interscapular pre-adipocytes in cell culture differentiated into mature adipocytes that were morphologically indistinguishable from the adult white depots. Throughout differentiation, cultured human fetal interscapular adipocytes demonstrated increased expression of classical brown fat markers compared to subcutaneous and omental cells. Further, functional analysis revealed an elevation in fatty acid oxidation as well as maximal and uncoupled oxygen consumption in interscapular brown adipocytes compared to white control cells. These data collectively identify the brown phenotype of these cells. Thus, our primary cell culture system derived from non-viable human fetal interscapular brown adipose tissue provides a valuable tool for the study of human brown adipocyte biology and for the development of anti-obesity therapeutics.

Isolation and characterization of human adipose-derived stem cells for use in tissue engineering.

Human adipose-derived adult stem cells (ASCs) represent a unique population of multipotent stem cells. Their utility in a variety of tissue engineering applications, and as a model system for the study of molecular mechanisms of differentiation, is well established. In addition, their relative abundance, ease of isolation from human subcutaneous lipoaspirates, and functional stability make them an excellent physiologically relevant platform. Here, we describe detailed procedures for handling and purification of ASCs from lipoaspirate, as well as their expansion, cryopreservation, quality control, and functional assays.

Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

High-content screening of human primary muscle satellite cells for new therapies for muscular atrophy/dystrophy.

Myoblast proliferation and differentiation are essential for normal skeletal muscle growth and repair. Muscle recovery is dependent on the quiescent population of muscle stem cells - satellite cells. During muscle injury, satellite cells become mitotically active and begin the repair process by fusing with each other and/or with myofibers. Aging, prolonged inactivity, obesity, cachexia and other muscle wasting diseases are associated with a decreased number of quiescent and proliferating satellite cells, which impedes the repair process. A high-content/high-throughput platform was developed and utilized for robust phenotypic evaluation of human primary satellite cells in vitro for the discovery of chemical probes that may improve muscle recovery. A 1600 compound pilot screen was developed using two highly annotated small molecule libraries. This screen yielded 15 dose responsive compounds that increased proliferation rate in satellite cells derived from a single obese human donor. Two of these compounds remained dose responsive when counter-screened in 3-donor obese superlot. The Alk-5 inhibitor LY364947, was used as a positive control for assessing satellite cell proliferation/delayed differentiation. A multivariate approach was utilized for exploratory data analysis to discover proliferation vs. differentiation-dependent changes in cellular phenotype. Initial screening efforts successfully identified a number of phenotypic outcomes that are associated with desired effect of stimulation of proliferation and delayed differentiation.

Feed-forward inhibition of androgen receptor activity by glucocorticoid action in human adipocytes.

We compared transcriptomes of terminally differentiated mouse 3T3-L1 and human adipocytes to identify cell-specific differences. Gene expression and high content analysis (HCA) data identified the androgen receptor (AR) as both expressed and functional, exclusively during early human adipocyte differentiation. The AR agonist dihydrotestosterone (DHT) inhibited human adipocyte maturation by downregulation of adipocyte marker genes, but not in 3T3-L1. It is interesting that AR induction corresponded with dexamethasone activation of the glucocorticoid receptor (GR); however, when exposed to the differentiation cocktail required for adipocyte maturation, AR adopted an antagonist conformation and was transcriptionally repressed. To further explore effectors within the cocktail, we applied an image-based support vector machine (SVM) classification scheme to show that adipocyte differentiation components inhibit AR action. The results demonstrate human adipocyte differentiation, via GR activation, upregulates AR but also inhibits AR transcriptional activity.

EGF receptor (ERBB1) abundance in adipose tissue is reduced in insulin-resistant and type 2 diabetic women.

CONTEXT: Indications of adipose tissue dysfunction correlate with systemic insulin resistance and type 2 diabetes. It has been suggested that a defect in adipose tissue turnover may be involved in the development of these disorders. Whether this dysfunction causes or exacerbates systemic insulin resistance is not fully understood. OBJECTIVES, PARTICIPANTS, AND MEASURES: We tested whether the expression of members of the mitogenic ErbB family was reduced in adipose tissue of insulin-resistant individuals and whether ErbB1 and ErbB2 were involved in adipogenesis. Thirty-two women covering a wide range of body mass index values and insulin sensitivity participated in the cross-sectional portion of this study. We also studied preadipocytes isolated from 12 insulin-sensitive individuals to evaluate the impact of ErbB1 or ErbB2 inhibition on adipogenesis in vitro. For this purpose, we measured phospho-ErbB1 and phospho-ErbB2 levels using ELISA and the expression of peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ-regulated genes by real-time PCR. RESULTS: Among the ErbB family members, only ErbB1 expression was correlated with insulin sensitivity. Additionally, ErbB1 levels correlated positively with PPARγ and several PPARγ-regulated genes including acyl-coenzyme A synthetase long-chain family member 1 (ACSL1), adiponectin, adipose tissue triacylglycerol lipase (ATGL), diacylglycerol acyl transferase 1 (DGAT1), glycerol-3-phosphate dehydrogenase 1 (GPD1), and lipoprotein lipase (LPL), but negatively with CD36 and fatty acid-binding protein 4 (FABP4). In preadipocyte culture, ErbB1, but not ErbB2, inhibition was associated with a reduction in the expression of all the above-mentioned genes. CONCLUSIONS: These findings demonstrate a key role for ErbB1 in adipogenesis and suggest that lower ErbB1 protein abundance may lead to adipose tissue dysfunction.

Tools for the identification of bioactives impacting the metabolic syndrome: screening of a botanical extract library using subcutaneous and visceral human adipose-derived stem cell-based assays.

Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis.

Metabolic remodeling of human skeletal myocytes by cocultured adipocytes depends on the lipolytic state of the system.

OBJECTIVE: Adipocyte infiltration of the musculoskeletal system is well recognized as a hallmark of aging, obesity, and type 2 diabetes. Intermuscular adipocytes might serve as a benign storage site for surplus lipid or play a role in disrupting energy homeostasis as a result of dysregulated lipolysis or secretion of proinflammatory cytokines. This investigation sought to understand the net impact of local adipocytes on skeletal myocyte metabolism. RESEARCH DESIGN AND METHODS: Interactions between these two tissues were modeled using a coculture system composed of primary human adipocytes and human skeletal myotubes derived from lean or obese donors. Metabolic analysis of myocytes was performed after coculture with lipolytically silent or activated adipocytes and included transcript and metabolite profiling along with assessment of substrate selection and insulin action. RESULTS: Cocultured adipocytes increased myotube mRNA expression of genes involved in oxidative metabolism, regardless of the donor and degree of lipolytic activity. Adipocytes in the basal state sequestered free fatty acids, thereby forcing neighboring myotubes to rely more heavily on glucose fuel. Under this condition, insulin action was enhanced in myotubes from lean but not obese donors. In contrast, when exposed to lipolytically active adipocytes, cocultured myotubes shifted substrate use in favor of fatty acids, which was accompanied by intracellular accumulation of triacylglycerol and even-chain acylcarnitines, decreased glucose oxidation, and modest attenuation of insulin signaling. CONCLUSIONS: The effects of cocultured adipocytes on myocyte substrate selection and insulin action depended on the metabolic state of the system. These findings are relevant to understanding the metabolic consequences of intermuscular adipogenesis.

Homeostatic levels of SRC-2 and SRC-3 promote early human adipogenesis.

The related coactivators SRC-2 and SRC-3 interact with peroxisome proliferator activated receptor γ (PPARγ) to coordinate transcriptional circuits to promote adipogenesis. To identify potential coactivator redundancy during human adipogenesis at single cell resolution, we used high content analysis to quantify links between PPARγ, SRC-2, SRC-3, and lipogenesis. Because we detected robust increases and significant cell-cell heterogeneity in PPARγ and lipogenesis, without changes in SRC-2 or SRC-3, we hypothesized that permissive coregulator levels comprise a necessary adipogenic equilibrium. We probed this equilibrium by down-regulating SRC-2 and SRC-3 while simultaneously quantifying PPARγ. Individual or joint knockdown equally inhibits lipid accumulation by preventing lipogenic gene engagement, without affecting PPARγ protein levels. Supporting dominant, pro-adipogenic roles for SRC-2 and SRC-3, SRC-1 knockdown does not affect adipogenesis. SRC-2 and SRC-3 knockdown increases the proportion of cells in a PPARγ(hi)/lipid(lo) state while increasing phospho-PPARγ-S114, an inhibitor of PPARγ transcriptional activity and adipogenesis. Together, we demonstrate that SRC-2 and SRC-3 concomitantly promote human adipocyte differentiation by attenuating phospho-PPARγ-S114 and modulating PPARγ cellular heterogeneity.

Adipogenic differentiation of adipose-derived stem cells.

The primary physiological function of adipose-derived stem cells (ASCs) is to differentiate into adipose tissue. It is now possible to isolate, expand, and cryopreserve ASC from adipose depots of many animal species. These ASC can be induced to undergo adipogenic differentiation in vitro by exposure to a cocktail of chemical agents or inductive growth factors. The current chapter describes methods to induce adipogenesis and to quantify this differentiation process in vitro.

Use of adipose-derived stem cells in high-throughput screening to identify modulators of lipogenesis.

Drug discovery efforts have an increasing focus on functional cell-based screening to identify compounds that modulate targets presented in a relevant format. Historically, immortalized cell lines have been used in primary and secondary screens due to their ease of manipulation, transformation, and propagation. However, more researchers are using primary cells that present their drug targets in their natural context. Human primary cell isolation and propagation procedures have become efficient enough to provide these cells in the necessary scale for early stage drug discovery. Adult human stem cells provide an opportunity for investigating multiple pathways of differentiation, development, regeneration, and toxicity using a single cell source and type. Adipose-derived stem cells (ASCs) are an attractive adult human primary stem cell for drug discovery due their abundance in adipose tissue, ease of isolation, and propagation in culture. They can be expanded in high numbers and retain their unique properties to differentiate into multiple lineages. In this chapter, we describe a protocol to identify modulators of human ASC lipogenesis following partial differentiation to adipocytes.

Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.

Quantification of lipid droplets and associated proteins in cellular models of obesity via high-content/high-throughput microscopy and automated image analysis.

Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease, and infectious diseases (eg, HCV and tuberculosis). To develop high-content analysis (HCA) techniques to analyze lipid droplets and associated proteins, primary human preadipocytes were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPAR-(c) agonist that promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone-sensitive lipase (HSL). The cells were imaged via automated digital microscopy and algorithms were developed to quantify lipid droplet (Lipid Droplet algorithm) and protein expression and colocalization (Colocalization algorithm). The algorithms, which were incorporated into Vala Science Inc's CyteSeer((R)) image cytometry program, quantified the rosi-induced increases in lipid droplet number, size, and intensity, and the expression of perilipin with exceptional consistency (Z' values of 0.54-0.71). Regarding colocalization with lipid droplets, Pearson's correlation coefficients of 0.38 (highly colocalized), 0.16 (moderate), and -0.0010 (random) were found for perilipin, PKC, and HSL, respectively. For hepatocytes (AML12, HuH-7, and primary cells), the algorithms also quantified the stimulatory and inhibitory effect of oleic acid and triacsin C on lipid droplets (Z's > 0.50) and ADFP expression/colocalization. Oleic acid-induced lipid droplets in HeLa cells and macrophages (THP-1) were also well quantified. The results suggest that HCA techniques can be utilized to quantify lipid droplets and associated proteins in many cell models relevant to a variety of diseases.

The molecular mechanisms of coactivator utilization in ligand-dependent transactivation by the androgen receptor.

Androgens drive sex differentiation, bone and muscle development, and promote growth of hormone-dependent cancers by binding the nuclear androgen receptor (AR), which recruits coactivators to responsive genes. Most nuclear receptors recruit steroid receptor coactivators (SRCs) to their ligand binding domain (LBD) using a leucine-rich motif (LXXLL). AR is believed to recruit unique coactivators to its LBD using an aromatic-rich motif (FXXLF) while recruiting SRCs to its N-terminal domain (NTD) through an alternate mechanism. Here, we report that the AR-LBD interacts with both FXXLF motifs and a subset of LXXLL motifs and that contacts with these LXXLL motifs are both necessary and sufficient for SRC-mediated AR regulation of transcription. Crystal structures of the activated AR in complex with both recruitment motifs reveal that side chains unique to the AR-LBD rearrange to bind either the bulky FXXLF motifs or the more compact LXXLL motifs and that AR utilizes subsidiary contacts with LXXLL flanking sequences to discriminate between LXXLL motifs.

Recognition and accommodation at the androgen receptor coactivator binding interface.

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich "NR box" motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.