The authors would like to kindly thank Dr. J. Luther and K. Ubieta for preparing the data and Dr. B. Gr&#246;tsch for proofreading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (BO3811/1-1-Emmy Noether).

<ol>
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The authors have nothing to disclose.

Adipocytes are characterized phenotypically by their size, numbers and area, revealing adipocyte hyperplasia and hypertrophy, induced by excessive energy storage due to over-nutrition 5. These events leading to fatty acid dysregulation and subsequent metabolic syndromes are also states of increased fat mass with preserved metabolisms, which is also referred to as &#34;healthy&#34; fat expansion. For example, Kusminski et al. 21 showed that mice with massive fat expansion remain metabolically healthy, suggesting that fat expansion is not necessarily linked to metabolic syndromes and needs to be carefully determined to evaluate the characteristics of the adipocytes. The adipose tissue (AT) plays a pivotal role in the regulation of body metabolism. AT is the biggest endocrine organ that could influence dyslipidemia, atherosclerosis, hyperinsulinemia and hyperglycemia 3. Evaluating AT homeostasis and the molecular mechanisms regulating it could allow a better understanding of metabolic system disorders. Therefore, unravelling the mechanisms regulating adipocyte differentiation, adipocyte size and fat pad mass would help to develop new therapeutic treatment for obesity disorders. Using in vivo and in vitro methods, it is possible to determine the role of food and gene expression impacts on adipocyte differentiation and activity. To determine the AT homeostasis, determining the balance between adipocyte differentiation, proliferation and apoptosis as suggested by our protocol is as important as analyzing the glucose and insulin metabolic response 22-24.
Expression profiling analyses of genes involved in adipogenesis, lipogenesis, lipolysis, fatty acid uptake, hypoxia, apoptosis and proliferation in primary adipocytes and visceral AT is a high throughput method for obtaining an overview on adipocyte homeostasis and their possible dysfunction. Interesting candidates should be further analyzed at the protein level by western blot or immunohistological staining. To obtain optimal results through real-time PCR system using unsymmetrical cyanine dyes, the concentrations of cDNA ranging from 1 to 10 ng and the optimal primer concentrations ranging from 50 to 900 nM should be tested to minimize nonspecific amplification. The critical components are the primers; for each run, the melting curves need to be strictly controlled to ensure the specificity and to exclude the formation of primer dimers. Therefore, using a negative control with H2O instead of cDNA is recommended. Furthermore, commercial available unsymmetrical cyanine dyes are provided as master mixes that contain a passive reference dye (such as ROX) to provide an internal reference signal. The cDNA signal is normalized during data analysis to the ROX signals to correct well-to-well signal fluctuations. Another point to be considered in order to establish a good qPCR system is the choice of the housekeeping gene. For each condition, several housekeeping genes are used, e.g., HPRT, &#946;-actin, GAPDH, &#946;-2-MG or HSP90.
HIF proteins stabilized by hypoxic conditions are master regulators determining not only adipocytes survival, but also metabolic changes, such as glucose, insulin tolerance and lipid metabolism 11,25,26. To ascertain hypoxic areas in the fat pad, HIF-1&#945; is determined in AT sections by immunohistochemistry. Since HIF proteins are rapidly degraded within 5 to 10 min under normoxic conditions, the procedure and the fixation of the fat pad for the histological analysis should be tightly controlled to avoid latency time. Therefore, to ensure hypoxia not only through HIF staining, pimonidazole is used to determine hypoxic areas in the AT. Pimonidazole is able to distribute into tissues, as it was already shown in bones 27, and effectively mark hypoxic areas by binding to thiol-containing proteins specifically in hypoxic cells, which is further detected in histological sections by specific antibody binding 28. However, other markers and methods can be used to analyze the hypoxia pathway. For example, the involvement of the prolyl hydroxylase (PHD) enzyme, which induce hydroxylation of proline residues under normoxia, as well as the von Hippel-Lindau (VHL) protein, which recognize hydroxylated prolines and induce the poly-ubiquitination to mediate proteasomal HIF degradation, need to be analyzed for a full overview of the pathway 29,30. Moreover, ubiquitous detection of HIFs would also determine the protein stability and degradation that can be alter 31,32.
Furthermore, proliferation by Ki67 and apoptosis by TUNEL staining are determined in vivo through staining of AT histological sections. Quantification of proliferation by Ki67 and apoptosis by TUNEL or Annexin V staining through flow cytometry analysis is also carried out 33. Proliferation could of course be measured by other techniques such as the analyses of the adipocyte cell cycle, which is not addressed by the measurement of Ki67 positive cells. Moreover, apoptosis study by TUNEL can be completed by FACS analyses of Annexin V and TOP-PR-3 which will determine the levels of necrosis versus the apoptosis cell death process. Apoptosis is a fundamental process for the program of cell death, which is important for AT homeostasis. Indeed, dysregulation of adipocyte apoptosis has been implicated previously in processes contributing to obesity and lipodystrophy 34. Moreover, in 2011, Keuper et al. linked adipose tissue inflammation to adipocyte apoptosis. They showed that macrophages induced apoptosis in preadipocytes and adipocytes, which in turn attract macrophages. The recruitment of macrophages accelerates inflammation, which contributes to metabolic syndromes such as glucose and insulin tolerance 35. However, adipocyte apoptosis is still a poorly studied phenomenon, despite the hypothesis that induced adipocyte apoptosis could lead to decreased weight.
The present protocol uses immunohistochemical approaches to study different phenomenon such as proliferation, apoptosis and hypoxia in vivo. Therefore, the tissue was fixed with 4% formaldehyde, which is a critical step. An extended tissue fixation time leads to change of the epitopes, which become non-accessible for the antibody. In contrast, a short fixation time increases the sensitivity of epitopes to reagents. The recommended optimal time of fixation is 24 hr. Moreover, the thickness of the sections also influences the antibodies binding to their epitopes; optimal thickness is between 2 and 5 &#181;m. Sections thicker than 5 &#181;m will give false positive results due to increased binding sites. In contrast, sections thinner than 2 &#181;m contain less binding sites and positive areas are not well defined. Further critical factors are the antibody itself, incubation time, concentration and even temperature, which influence the quality of the specific binding to the epitopes. Therefore, validating antibody concentration and incubation time is necessary for each condition.
To complete the study, we provide an in vitro adipocyte differentiation protocol, which could be extended by different treatments, stimulation or co-cultures. By using in vitro adipocyte cultures, it is feasible to determine defects in adipocyte differentiation and functions. To obtain reliable results, as for all primary cells, the healthy behavior and appearance of the ADSCs and adipocytes is quite important. The granularity, cytoplasmic vacuolations and/or detachment are signs of deterioration, indicating inadequate medium, microbial contamination or senescence of the primary cells. This protocol is using isolated adipocytes from the fat pad tissue, whereas it is as well possible to use mesenchymal stem cell isolated from bone marrow as described by other protocols 36. The latest includes stromal progenitor cells, which might reflect additional differentiation problems occurring at the very early step of adipocyte differentiation, this might be missed in our current protocol. Moreover, ADSCs can be expanded rapidly (more than 10 times within one week), and long-term cultured ADSCs after some passages still retain their mesenchymal pluripotency 37,38. Another advantage using ADSCs is that one can easily switch to human, since ADSCs can be harvested from patients by liposuction which is a simple and minimally invasive method.
As AT influences several other organs in an endocrine manner, the protocol should be extended to adipokines. Adipokines, such as leptin, adiponectin, tumor necrosis factor-&#945; (TNF) and resistin, secreted by adipocytes are known to affect metabolic diseases by controlling fat metabolism, energy homeostasis and insulin sensitivity 39. Therefore, serum and adipocyte secretome analyses should be performed. In the case of AT dysfunction, adipokines and pro-inflammatory cytokines, such as IL-6, can lead to dysregulation of organs such as the liver and pancreas, and of muscle function 4. In order to exclude systemic organ dysfunction, animal models or cell cultures could be tested for their response to glucose stimulation and uptake.
Here we provide a protocol for analyzing the basic state of the AT and adipocytes in vivo and in vitro to reveal molecular mechanisms of adipocyte homeostasis and functionality.

According to the 2014 report from the World Health Organization, 39% of the world&#39;s adult population is overweight, and 13% is obese 1. In the near future, overweight people will comprise a significant proportion of the elderly population. An important feature of obesity and aging is dysregulation of fat in relation to morbidity and mortality 2. Adipokines, proteins secreted by the adipose tissue (AT), can trigger metabolic syndromes such as obesity and type 2 diabetes 3. Metabolic diseases are mostly caused by excessive energy storage in the lipid droplets of adipocytes, which results in AT expansion 4. It is therefore of interest to determine the causes and the molecular mechanisms of AT expansion in order to find opportunities to control it.
Over-nutrition leads to AT expansion, which is regulated by two events: excessive energy storage into the lipid droplets of adipocytes, a process leading to hypertrophy (increase in adipocyte size), and increased adipogenesis, also known as adipocyte hyperplasia 5. Adipogenesis is a process of differentiation of multipotent mesenchymal stem cells (MSC) into adipocytes. Firstly, MSCs develop into preadipocytes during the commitment phase. Secondly, preadipocytes further differentiate to acquire the features of mature and functional adipocytes 6. Several transcription factors have been identified as master regulators for preadipocyte determination, such as zinc finger protein 423 (Zfp423) and early B cell factor 1 (Ebf1). Whereas Zfp423 induces early commitment, Ebf1 is required for the generation of adipocyte progenitors 6. Terminal differentiation is tightly controlled by a transcriptional cascade, whereby peroxisome proliferator-activated receptor &#947; (PPAR&#947;) is the essential transcription factor 7. Further key transcriptional factors are the CCAAT/enhancer-binding protein (C/EBP) family members (i.e., C/EBP&#945;, C/EBP&#946;, and C/EBP&#948;), kruppel-like factors (KLFs), cAMP responsive element binding protein (CREB) and early growth response 20 (Krox20) 6.
Recently, it has been shown that the activator protein-1 (AP-1) family is involved in the adipocyte differentiation process 8,9. The AP-1 family is formed by a dimeric protein complex, composed of Fos, Jun and/or activating transcription factor (ATF) members. Fos-related antigen 1 and 2 (Fra-1 and Fra-2) are able to regulate adipocyte differentiation. Fra-1 impairs adipocyte differentiation by inhibiting C/EBP&#945; 8, whereas Fra-2 controls adipocyte turnover 9. Fra-2 thereby not only decreases the adipocyte number by repressing PPAR&#947;2 expression during adipocyte differentiation, but also decreases adipocyte apoptosis through direct repression of hypoxia-inducible factors (HIFs) expression. The HIF family is a heterodimeric transcription factor complex, composed of HIF-1&#945;, HIF-2&#945; and HIF-1&#946;. The heterodimers consist of an oxygen-sensitive HIF-&#945; protein (HIF-1&#945; or HIF-2&#945;) and the oxygen-insensitive HIF-1&#946; subunit 10. During normoxia, HIF-&#945; proteins are poly-ubiquitinylated and are finally degraded by proteasomes 11. Under hypoxic conditions, occurring in AT during expansion, HIF-&#945; proteins are no longer hydroxylated. They therefore become stabilized and form dimers with the constitutively expressed HIF-1&#946;. Transcriptional activation of genes controlled by the HIF response elements is involved in the regulation of angiogenesis, metabolism, and inflammation 12. Indeed, HIF-1&#945; promotes AT dysfunction by inducing glucose tolerance, inhibiting energy expenditure and peripheral use of lipid, as well as by increasing leptin level and HFD-induced hepatic steatosis 13. Moreover, HIF-1&#945; regulates adipocyte apoptosis in vivo and in vitro9.
The present protocol describes methods for studying AT status to unravel the molecular characteristics of adipocyte homeostasis in adult mice. It shows how apoptosis, proliferation and differentiation of adipocytes in vivo and in vitro can be regulated by hypoxia. To do so, we use mice with adipocyte specific deletion of Fra-2 generated by crossing mice carrying the Fra-2 floxed alleles with Fabp4-CreERT mice 9. By using Fabp4-Cre ERT mice, the deletion is adipocyte specific and inducible by tamoxifen injection 14. For the adult model, intra peritoneal injections of tamoxifen are performed over 5 consecutive days starting at the age of 6 weeks. Thus, the mice are subjected to a normal diet or high-fat diet for 6 weeks before the analysis is done. The mice used in this study were male based on a C57Bl6 background to avoid female hormones, such as estrogens, shown to regulate the body fat distribution 15. Using another genetic background might also alter the metabolic phenotype, due to strain-related differences in lipid management 16.
This protocol demonstrates how to analyze AT under hypoxia using histology and how to quantify adipocyte apoptosis, proliferation and differentiation in vivo using immunohistochemistry and gene profiling analyses. The study is completed by in vitro experiments, showing how to analyze primary adipocyte differentiation and apoptosis altered by exposure to hypoxia.

<table><tbody><tr><td>RNAlater solution</td><td>Ambion</td><td>AM7021</td><td>RNA stabilization solution</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4368813</td><td></td></tr><tr><td>SYBR Select Master Mix</td><td>4472908</td><td></td><td></td></tr><tr><td>Purified Mouse Anti-Human Ki-67</td><td>BD Biosciences</td><td>550609</td><td>Clone B56 (RUO)</td></tr><tr><td>Purified Mouse Anti-Human Ki-67 Clone B56 (RUO)</td><td>550609</td><td>Proliferation marker</td><td></td></tr><tr><td>FITC Annexin V</td><td>BioLegend</td><td>640906</td><td></td></tr><tr><td>Cleaved Caspase-3 Rabbit mAb</td><td>Cell signalling</td><td>9664S</td><td>Clone 5A1E</td></tr><tr><td>Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb</td><td>9664</td><td>Apoptosis marker</td><td></td></tr><tr><td>Lipofectamine2000 Reagent&amp;nbsp;</td><td>Invitrogen</td><td>11668-027</td><td></td></tr><tr><td>TO-PRO-3 Iodide</td><td>T3605</td><td>Nuclear counterstain, Monomeric cyanine nucleic acid stain, Excitation&amp;frasl;Emission: 642&amp;frasl;661 nm</td><td></td></tr><tr><td>Mayer&amp;rsquo;s hemalum</td><td>Merck</td><td>109249</td><td>hematoxylin</td></tr><tr><td>pegGOLD TriFast</td><td>Peqlab</td><td>30-2030</td><td>TRIzol, single-phase solution of guanidine isothiocyanate and phenol</td></tr><tr><td>Percellys Ceramic Kit 1.4&amp;nbsp;mm</td><td>91-PCS-CK14</td><td>tubes containing ceramic beads (1.4 mm)</td><td></td></tr><tr><td>Precellys 24</td><td>91-PCS24</td><td>homogenizer</td><td></td></tr><tr><td>HIF-1 alpha Antibody</td><td>Pierce</td><td>PA1-16601</td><td></td></tr><tr><td>HIF-1 alpha Antibody, 16H4L13</td><td>700505</td><td>Hypoxia marker</td><td></td></tr><tr><td>&lt;em&gt;In Situ&lt;/em&gt; Cell Death Detection Kit, Fluorescein</td><td>Roche</td><td>11 684 795 001</td><td>TdT-mediated dUTP-biotin nick end labeling (TUNEL)&amp;nbsp;</td></tr><tr><td>Eosin</td><td>Sigma</td><td>318906</td><td></td></tr><tr><td>DNase I Solution (1&amp;nbsp;unit/&amp;micro;l)</td><td>Thermo Scientific</td><td>EN0525</td><td></td></tr><tr><td>biotinylated anti mouse IgG (H+L)</td><td>Vector Laboratories</td><td>BA-9200</td><td></td></tr><tr><td>biotinylated anti mouse IgG (H+L)</td><td>BA-1000</td><td></td><td></td></tr><tr><td>Vectastain ABC Kit</td><td>PK-4000</td><td></td><td></td></tr><tr><td>VECTASHIELD Mounting Medium with DAPI</td><td>H-1200</td><td></td><td></td></tr></tbody></table>

mechanism of regulation of adipocyte numbers in adult organisms through differentiation and apoptosis homeostasis

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1&#945; protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

We show how to determine adipocyte homeostasis in vivo and in vitro using the example of Fra-2fl/fl Fabp4-CreERT mice compared to wild-type littermates. Our protocol defines how increased HIF expression by hypoxia is correlated with adipocyte dysfunction as indicated by increased adipocyte apoptosis.
Increased adipocyte size and area in high-fat diet (HFD) treated mice
Over-nutrition, among other factors, results in adipocyte hypertrophy, caused by excessive energy storage in the lipid droplets. The adipocyte size and area is an indicator of hypertrophy. Sections of the fat pad from normal (ND; Figure 6a) and high-fat diet (HFD; Figure 6b) mice as well as quantifications of the adipocyte size and area clearly show adipocyte hypertrophy after 6 weeks of HFD, which is indicated by increased adipocyte size in the HFD treated mice (Figure 6c and d).
Increased hypoxia in the adipose tissue (AT) of adult Fra-2fl/fl Fabp4-CreERT mice leads to increased HIF-1&#945; level and adipocyte apoptosis
To determine the in vivo status of hypoxia in AT, Fra-2fl/fl Fabp4-CreERT mice are analyzed 6 weeks after Fra-2 deletion at the age of 12 weeks and compared with wild-type littermates. Pimonidazole is administered to the mice intraperitoneally as an effective hypoxia marker; it is nontoxic and able to distribute into the AT. Hypoxic adipocytes in the AT in vivo are defined by immunohistochemical antibody staining (Figure 7a). Furthermore, the increased hypoxic status of the AT in mice is accompanied by increased HIF-1&#945; positive adipocytes as indicated by the immunohistochemical staining Figure 7b), which is confirmed by quantification of HIF-1&#945; expression levels and its targets genes 9. Additionally, TUNEL staining of AT sections from Fra-2fl/fl Fabp4-CreERT mice and control littermates (Figure 7c) shows that increased adipocyte apoptosis is correlated with the presence of hypoxia and HIF-1&#945; expression.
Increased HIF-1&#945; expression in primary adipocytes through hypoxia induced adipocyte apoptosis
To analyze adipocyte apoptosis in vitro, we use adipocytes generated from subcutaneous fat pads as described elsewhere 20. As expected, the HIF-1&#945; expression in adipocytes is increased after 24 hr of hypoxia (Figure 8a). To further analyze HIF-1&#945; activities, the RNA levels of HIF target genes such as Inos (inducible nitric oxide synthase) are quantified by qPCR. Figure 8b shows that the increased expression of HIF-1&#945; under hypoxic conditions leads to increased Inos mRNA level. Since we have already shown in vivo (Figure 8) that increased HIF-1&#945; expression in adipocytes correlates with increased adipocyte apoptosis, apoptosis is also quantified in in vitro cultures by Annexin V staining under hypoxic conditions. Consistent with the in vivo data (Figure 7), an increased HIF-1&#945; level is accompanied by increased adipocyte apoptosis induced by hypoxic conditions (Figure 8b). Moreover, to prove that hypoxic-induced apoptosis is HIF-dependent; HIF-1&#945; or HIF-2&#945; is silenced by RNA interference in adipocytes derived from wild-type or Fra-2 deficient mice. The increased adipocyte apoptosis is restored by silencing HIF-1&#945; or HIF-2&#945; as shown by Annexin V staining in Figure 8b, proving that the hypoxia sensor HIF-&#945; regulates the adipocyte apoptosis.
<img alt="Figure 6" src="/files/ftp_upload/53822/53822fig6.jpg" /> 
Figure 6: Increased adipocyte size and area in high-fat diet (HFD) mice. (a, b) H&#38;E staining of sections from the perigonadal fat pad of male wild-type mice fed with normal diet (ND) (a) or high-fat diet (HFD) (b) for 6 weeks. Bars represent 500 &#956;m. (c, d) Quantifications of adipocyte size (c) and area (d) from the perigonadal fat pad of wild-type mice fed with ND (a) or HFD (b) for 6 weeks. n = 10. Data are shown as mean values &#177; SEM. Statistical analysis was performed using Student&#180;s t-test. ***p &#60;0.0001.<a href="https://www.jove.com/files/ftp_upload/53822/53822fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53822/53822fig7.jpg" /> 
Figure 7: Increased HIF-1&#945; level and apoptosis in adipocytes of adult Fra-2fl/fl Fabp4-CreERT mice. Hypoxia (a) and HIF-1&#945; (b) staining in the AT of male Fra-2fl/fl Fabp4-creERT mice and male control littermates 6 weeks after tamoxifen injection. Magnification 20X, insert 40X. Black arrows indicate hypoxic areas and HIF-1&#945; positive cells. (c) TUNEL staining in Fra-2fl/fl Fabp4-CreERT mice and control littermates AT 6 weeks after tamoxifen injection. Magnification 20X, insert 40X. Black arrows indicate TUNEL positive cells. This figure has been modified from Luther et al. 9. <a href="https://www.jove.com/files/ftp_upload/53822/53822fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/53822/53822fig8.jpg" /> 
Figure 8: Increased HIF-1&#945; expression and adipocyte apoptosis in primary adipocytes induced by hypoxia. (a) Real-time PCR analysis of HIF-1&#945; and HIFs target Inos mRNA levels in primary adipocytes placed in hypoxic chambers analyzed at the indicated time points. (b) Quantification of apoptosis by Annexin V FACS staining in primary adipocytes isolated from Fra-2fl/fl Fabp4-CreERT mice or wild-type controls transfected with sh control or sh plasmid against HIF-1&#945; or HIF-2&#945; and placed under hypoxia (1% O2) for 24 hr. This figure has been modified from Luther et al. 9. Data are shown as mean values &#177; S.D. Statistical analysis was performed using Student&#39;s t-test. *P &#60;0.05 and **P &#60;0.01 were accepted as significant. <a href="https://www.jove.com/files/ftp_upload/53822/53822fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of ...

mechanism-regulation-adipocyte-numbers-adult-organisms-through

Research

JoVE Journal

Developmental Biology

15.0K Views.  Universitätsklinikum Erlangen. Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

Video: Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Biology

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Immunology and Infection

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Protein Interaction Analysis in HEK-293 Cells Using Co-Immunoprecipitation

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.

Author Spotlight: Unraveling the Molecular Mechanisms of Brown and Beige Adipocyte Regulation

Protein-protein interactions are important for elucidating the function of target proteins, and co-immunoprecipitation (co-IP) can easily confirm PPIs. We transiently transfected a plasmid encoding an epitope-tagged protein into HEK-293 cells and developed an immunoprecipitation method to easily confirm the binding of two target proteins.

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and ...

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

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