This work was supported by INSERM, the Universit&#233; C&#244;te d&#39;Azur, and the French National Research Agency (ANR) through the program Investments for the future Laboratory of Excellence (Labex SIGNALIFE-ANR-11-LABX-0028-01) and Initiative of Excellence (Idex UCAJEDI ANR-15-IDEX-0001). J.J. is supported by grants from the Soci&#233;t&#233; Francophone du Diab&#232;te (SFD), the Association Fran&#231;aise d&#39;Etude et de Recherche sur l&#39;Ob&#233;sit&#233; (AFERO), the Institut Th&#233;matique Multi-Organismes Technologies pour la Sant&#233; (ITMO), and the Fondation Benjamin-Delessert. J.G. is supported by ANR-18-CE14-0035-01. J-F.T. is supported by ANR grant ADIPOPIEZO-19-CE14-0029-01 and a grant from the Fondation pour la Recherche M&#233;dicale (Equipe FRM, DEQ20180839587). We also thank the Imaging Core Facility of C3M funded by the Conseil D&#233;partemental des Alpes-Maritimes and the R&#233;gion PACA, which is also supported by the GIS IBiSA Microscopy and Imaging Platform C&#244;te d&#39;Azur (MICA).

NOTE: Use sterile techniques to perform all the steps of the protocol in a laminar flow cell culture hood. See Table of Materials for details about all reagents and equipment.
1. Differentiation of murine 3T3-L1 fibroblasts into adipocytes
<ol>
	<li>Grow the 3T3-L1 fibroblasts in 100 mm dishes in culture medium-DMEM without pyruvate, 25 mM glucose, 10% newborn calf serum, and 1% penicillin and streptomycin (Figure 1A). Place the dishes in a tissue culture incubator (7% CO2 and 37 &#176;C).</li>
	<li>Two days after confluence, change the culture medium, replacing with DMEM without pyruvate, 25 mM glucose, 10% fetal calf serum (FCS), and 1% penicillin and streptomycin supplemented with 0.25 mM 3-Isobutyl-1-methylxanthine (IBMX), 0.25 &#181;M dexamethasone, 5 &#181;g/mL insulin, and 10 &#181;M rosiglitazone. 
	NOTE: It takes 5 days to reach confluency when the cells are seeded at 300,000 cells per 100 mm dish.</li>
	<li>Two days later, replace the culture medium with DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin supplemented with 5 &#181;g/mL insulin and 10 &#181;M rosiglitazone and incubate for 2 days. Then, feed the cells every 2 days with DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin (Figure 1B).</li>
	<li>Transfect the 3T3-L1 adipocytes 7-8 days after the beginning of the differentiation protocol. 
	&#8203;NOTE: It is important to reach a high level of differentiation (&#62;80%) before the transfection to avoid the proliferation of the remaining fibroblasts after the transfection, which would lead to a mixed population of cells that might bias the results.</li>
</ol>
2. Preparation of precoated plates
<ol>
	<li>On the day before or a few hours before the transfection, prepare a solution of collagen type I at 100 &#181;g/mL in 30% ethanol from a stock solution at 1 mg/mL. Add 250 &#181;L of collagen per well of a 12-well plate and 125 &#181;L per well of a 24-well plate, and spread the solution over the surface of the well.</li>
	<li>Leave the plate without the lid under the culture hood until the collagen dries. Wash twice with Dulbecco&#39;s phosphate-buffered saline (D-PBS). 
	&#8203;NOTE: Precoated plates are available for purchase.</li>
</ol>
3. Preparation of the transfection mix
NOTE: The final concentration of siRNA is between 1 and 100 nM (1 to 100 pmol of siRNA per well of a 12-well plate). The final concentration of the miR mimic is 10 nM (10 pmol/well). Determine the best concentration of each siRNA, miR mimic, or other oligonucleotide prior to starting the experiment to avoid off-target effects. Perform transfection experiments in triplicate to facilitate statistical analysis of the results. Prepare all reagents in excess to account for normal loss during pipetting.
<ol>
	<li>Mix by pipetting (volume/volume) the siRNA (or other oligonucleotides) with improved Minimal Essential Medium (Table 1). Incubate for 5 min at room temperature.</li>
	<li>Add the transfection reagent and the improved Minimal Essential Medium to the siRNA, and pipet to mix (Table 1). Incubate for 20 min at room temperature (during this time, proceed to section 4). Add the transfection mix to each well of the collagen-coated plate.</li>
</ol>
4. Preparation of the 3T3-L1 adipocytes
<ol>
	<li>Wash the cells in the 100 mm Petri dish twice with D-PBS. Add 5x trypsin to the cells (1 mL per 100 mm dish), making sure to cover all of the surface with the trypsin. Wait for 30 s and carefully remove the trypsin.</li>
	<li>Incubate the Petri dish for 5-10 min at 37 &#176;C in the incubator. Tap the 100 mm dish to detach the cells.</li>
	<li>Add 10 ml of DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin to neutralize the trypsin. Carefully pipet the medium up and down to detach the cells and homogenize the cell suspension.</li>
	<li>Count the cells using a Malassez counting chamber or an automated cell counter, and adjust the concentration of the cells to 6.25 x 105 cells/mL of medium. Seed 800 &#181;L of the cell suspension/well of a 12-well plate (5 x 105 cells) or 400 &#181;l of the cell suspension/well of a 24-well plate (2.5 x 105 cells) containing the transfection mix. 
	NOTE: One 100 mm Petri dish of adipocytes will allow the preparation of one 12-well plate or one 24-well plate. A 100 mm dish usually contains 6-7 x 106 adipocytes, which correspond to 5 x 105 adipocytes per well of a 12-well plate.</li>
	<li>Incubate the plates in a cell culture incubator (7% CO2 and 37 &#176;C), and do not disturb the cells for 24 h. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin. 
	&#8203;NOTE: It is also possible to seed the cells into collagen-precoated 48- and 96-well plates but take more precautions when replacing the media to avoid detachment of the adipocytes.</li>
</ol>
5. Functional analysis of transfected 3T3-L1 adipocytes
<ol>
	<li>Study target knockdown 24-48 h and 48-96 h after siRNA or miR mimic delivery for mRNA and protein, respectively.</li>
	<li>Perform functional analyses of transfected adipocytes to study insulin signaling, glucose uptake, adipokine secretion, lipolysis, and lipogenesis.</li>
</ol>

<ol>
	<li>Kl&#246;ting, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-sensitive+obesity.">Insulin-sensitive obesity.</a> American Journal of Physiology, Endocrinology and Metabolism. 299 (3), 506-515 (2010).</li><li>Weyer, C., Foley, J. E., Bogardus, C., Tataranni, P. A., Pratley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+subcutaneous+abdominal+adipocyte+size,+but+not+obesity+itself,+predicts+type+II+diabetes+independent+of+insulin+resistance.">Enlarged subcutaneous abdominal adipocyte size, but not obesity itself, predicts type II diabetes independent of insulin resistance.</a> Diabetologia. 43 (12), 1498-1506 (2000).</li><li>Bl&#252;her, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+dysfunction+contributes+to+obesity+related+metabolic+diseases.">Adipose tissue dysfunction contributes to obesity related metabolic diseases.</a> Best Practice &amp; Research Clinical Endocrinology &amp; Metabolism. 27 (2), 163-177 (2013).</li><li>Lorente-Cebri&#225;n, S., Gonz&#225;lez-Muniesa, P., Milagro, F. I., Mart&#237;nez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+other+non-coding+RNAs+in+adipose+tissue+and+obesity:+emerging+roles+as+biomarkers+and+therapeutic+targets.">MicroRNAs and other non-coding RNAs in adipose tissue and obesity: emerging roles as biomarkers and therapeutic targets.</a> Clinical Science. 133 (1), 23-40 (2019).</li><li>Jager, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tpl2+kinase+is+upregulated+in+adipose+tissue+in+obesity+and+may+mediate+interleukin-1beta+and+tumor+necrosis+factor-{alpha}+effects+on+extracellular+signal-regulated+kinase+activation+and+lipolysis.">Tpl2 kinase is upregulated in adipose tissue in obesity and may mediate interleukin-1beta and tumor necrosis factor-{alpha} effects on extracellular signal-regulated kinase activation and lipolysis.</a> Diabetes. 59 (1), 61-70 (2010).</li><li>Vergoni, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+and+the+activation+of+the+p53+pathway+mediate+alterations+in+metabolic+and+secretory+functions+of+adipocytes.">DNA damage and the activation of the p53 pathway mediate alterations in metabolic and secretory functions of adipocytes.</a> Diabetes. 65 (10), 3062-3074 (2016).</li><li>Berthou, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tpl2+kinase+regulates+the+COX-2/prostaglandin+E2+axis+in+adipocytes+in+inflammatory+conditions.">The Tpl2 kinase regulates the COX-2/prostaglandin E2 axis in adipocytes in inflammatory conditions.</a> Molecular Endocrinology. 29 (7), 1025-1036 (2015).</li><li>Ceppo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+the+Tpl2+kinase+in+inflammatory+changes+and+insulin+resistance+induced+by+the+interaction+between+adipocytes+and+macrophages.">Implication of the Tpl2 kinase in inflammatory changes and insulin resistance induced by the interaction between adipocytes and macrophages.</a> Endocrinology. 155 (3), 951-964 (2014).</li><li>Jager, J., Gr&#233;meaux, T., Cormont, M., Le Marchand-Brustel, Y., Tanti, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta-induced+insulin+resistance+in+adipocytes+through+down-regulation+of+insulin+receptor+substrate-1+expression.">Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression.</a> Endocrinology. 148 (1), 241-251 (2007).</li><li>Jager, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tpl2+kinase+is+upregulated+in+adipose+tissue+in+obesity+and+may+mediate+interleukin-1beta+and+tumor+necrosis+factor-{alpha}+effects+on+extracellular+signal-regulated+kinase+activation+and+lipolysis.">Tpl2 kinase is upregulated in adipose tissue in obesity and may mediate interleukin-1beta and tumor necrosis factor-{alpha} effects on extracellular signal-regulated kinase activation and lipolysis.</a> Diabetes. 59 (1), 61-70 (2010).</li><li>Hart, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-34a+as+hub+of+T+cell+regulation+networks.">miR-34a as hub of T cell regulation networks.</a> Journal of ImmunoTherapy of Cancer. 7, 187 (2019).</li><li>Brandenburger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-34a+is+differentially+expressed+in+dorsal+root+ganglia+in+a+rat+model+of+chronic+neuropathic+pain.">MiR-34a is differentially expressed in dorsal root ganglia in a rat model of chronic neuropathic pain.</a> Neuroscience Letters. 708, 134365 (2019).</li></ol>

The authors have nothing to disclose.

This paper presents a detailed protocol for the differentiation and transfection of mature adipocytes. This reverse-transfection method is a simple, economical, and highly efficient method to transfect oligonucleotides such as, but not limited to, siRNAs, micro-RNA mimics, and anti-micro-RNAs into 3T3-L1 adipocytes, which is one of the most difficult cell lines to transfect. This method has some limitations that need to be considered. This protocol is not efficient for transfection with plasmid DNA, which limits the utility of this technique for gain-of-function studies. Although murine cell lines, including the 3T3-L1 cell line, have been typically used to study adipocyte function in vitro, micro-RNA expression patterns and activities in murine tissue are often different from those observed in humans; this is also the case with primary cells and cell lines. Moreover, the medium used for the differentiation of 3T3-L1 fibroblast into adipocytes requires an unphysiological hormone cocktail (insulin, dexamethasone, IBMX, and rosiglitazone), and the differentiated cells are morphologically distinct from in vivo mature adipocytes: they present multilocular lipid droplets instead of a unilocular lipid droplet. This could explain some differences in gene expression and cellular responses between in vitro and in vivo studies.
One benefit of using this reverse-transfection protocol compared to electroporation is that this method is cheaper. Indeed, the reagents are less expensive, and the high efficiency of the reverse transfection decreases the quantities of oligonucleotides needed, and there is no need of expensive equipment such as an electroporator. Moreover, using a lower concentration of siRNA is beneficial as it avoids off-target effects. Furthermore, this reverse-transfection is an easy, fast, gentle, and straightforward method of transfection that requires fewer cells and will ensure excellent cell viability and more robust data compared to electroporation. Although the procedure detailed above has been optimized for the differentiation and reverse-transfection of mouse 3T3-L1 cells, human preadipocytes can also be differentiated into adipocytes5 and easily subjected to reverse-transfection using this protocol. This report shows that adipocytes remain viable, healthy, and responsive to insulin after the reverse-transfection using a new generation of a non-liposomal cationic amphiphile transfection reagent. The reverse-transfection could also be performed using other popular transfection reagents. However, the amounts of oligonucleotides and transfection reagent would need to be optimized to ensure good modulation of expression and no adverse effects on cell viability.
This protocol facilitates the study of the role of proteins and micro-RNAs in adipocyte function, but it could also be used to modulate other non-coding RNAs such as lnc-RNAs, Y-RNAs, or eRNAs. There are critical steps in the protocol that can impact the efficiency of the procedure. The differentiation of 3T3-L1 fibroblasts into adipocytes should be carefully monitored. It is important to reach a high level of differentiation to avoid the proliferation of remaining fibroblasts after the transfection, which would bias the results of the experiment. Another important point is to perform the transfection on newly differentiated mature adipocytes; the best timing is 7-8 days after the beginning of the differentiation protocol, which corresponds to 3-4 days after removing the differentiation cocktail. This will ensure robust transfection efficacy and favor good reattachment of the adipocytes to the plate. Finally, the treatment of adipocytes with trypsin should be monitored carefully to ensure detachment of the adipocytes without damaging the cells.

Obesity is considered a major risk factor for numerous metabolic diseases, including insulin resistance (IR), Type 2 Diabetes (T2D), and cardiovascular diseases1. Current therapies have failed to stop the constantly rising prevalence of these diseases, and the management of the IR of obese and diabetic patients remains an important clinical issue. Adipose tissue plays a crucial role in the control of energy homeostasis, and its pathological expansion during obesity contributes to the development of IR and T2D2,3. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Many research studies have investigated the role of protein-coding RNAs in adipocyte physiology and their association with obesity.
More recently, the discovery of non-coding RNAs (ncRNAs), especially micro-RNAs (miRs), has forged novel concepts related to the mechanism of the regulation of gene expression programs. Studies have shown that ncRNAs are important regulators of adipocyte function, and that their dysregulation plays an important role in metabolic diseases4. Thus, the manipulation of proteins and ncRNAs in adipocytes is crucial to decipher their roles in adipocyte function and their impact on pathologies such as T2D. However, manipulating the expression of proteins and ncRNAs in vivo as well as in primary adipocytes remains highly challenging, favoring the use of in vitro adipocyte models.
Murine 3T3-L1 fibroblasts easily differentiate into mature, functional, and insulin-responsive adipocytes, which are a well-characterized cell line used to study adipocyte function (e.g., insulin signaling, glucose uptake, lipolysis and adipokines secretion)5,6,7,8,9,10. These properties make 3T3-L1 adipocytes an attractive model to modulate the expression of protein-coding and nc-RNAs to decipher their role in adipocyte function and their potential role in obesity-related diseases. Unfortunately, whereas 3T3-L1 fibroblasts are easy to transfect using commercially available reagents, differentiated 3T3-L1 adipocytes are one of the most difficult cell lines to transfect. This is why numerous studies manipulating gene expression in 3T3-L1 cells have focused on adipocyte differentiation rather than on adipocyte function.
For a long time, the only efficient technique to transfect adipocytes was electroporation5, which is tedious, expensive, and can cause cell damage. This paper reports a reverse-transfection technique using a common transfection reagent, which reduces hands-on time for transfection, has no effect on cell viability, and is much less expensive than electroporation. This protocol is perfectly suited for the transfection of siRNA and other oligonucleotides such as micro-RNA mimics (miR mimics) and anti-miRs. The principle of the reverse-transfection protocol is to incubate the transfection reagent and the oligonucleotides to form a complex in the cell culture plate and then seed the mature adipocytes into the wells. Then, the adipocytes reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. This simple, efficient, and inexpensive methodology permits the study of the role of protein-coding RNAs and miRs in adipocyte function and their potential role in obesity-related diseases.

<table><tbody><tr><td>12 well Tissue Culture Plate</td><td>Dutscher</td><td>353043</td><td></td></tr><tr><td>2.5% Trypsin (10x)</td><td>Gibco</td><td>15090-046</td><td>diluted to 5x with D-PBS</td></tr><tr><td>2-Propanol</td><td>Sigma</td><td>I9516</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Accell Non-targeting Pool</td><td>Horizon Discovery</td><td>D-001910-10-05</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>Collagen type I from calf skin</td><td>Sigma-Aldrich</td><td>C8919</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D1756</td><td></td></tr><tr><td>D-PBS</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Dulbecco&#039;s&amp;nbsp; Modified Eagles&#039;s Medium (DMEM)</td><td>Gibco</td><td>41965062</td><td>4.5 g/L D-Glucose; L-Glutamine; no Pyruvate</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>51976</td><td></td></tr><tr><td>FAM-labeled Negative Control si-RNA</td><td>Invitrogen</td><td>AM4620</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Free Glycerol Reagent</td><td>Sigma-Aldrich</td><td>F6428</td><td></td></tr><tr><td>Glycerol Standard Solution</td><td>Sigma-Aldrich</td><td>G7793</td><td></td></tr><tr><td>HSP90 antibody</td><td>Santa Cruz</td><td>sc-131119</td><td>Dilution : 0.5 &amp;micro;g/mL</td></tr><tr><td>Improved Minimal Essential Medium (Opti-MEM)</td><td>Gibco</td><td>31985-047</td><td></td></tr><tr><td>Insulin, Human Recombinant</td><td>Gibco</td><td>12585-014</td><td></td></tr><tr><td>miRIDIAN micro-RNA mimics</td><td>Horizon Discovery</td><td></td><td></td></tr><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>miScript II RT Kit</td><td>Qiagen</td><td>218161</td><td></td></tr><tr><td>miScript Primer Assays Hs_RNU6-2_11</td><td>Qiagen</td><td>MS00033740</td><td></td></tr><tr><td>miScript Primer Assays Mm_miR-34a_1</td><td>Qiagen</td><td>MS00001428</td><td></td></tr><tr><td>miScript SYBR Green PCR Kit</td><td>Qiagen</td><td>219073</td><td></td></tr><tr><td>Newborn Calf Serum</td><td>Gibco</td><td>16010-159</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O0625</td><td></td></tr><tr><td>ON-TARGETplus Mouse Plin1 si-RNA SMARTpool</td><td>Horizon Discovery</td><td>L-056623-01-0005</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Perilipin-1 antibody</td><td>Cell Signaling</td><td>3470</td><td>Dilution : 1/1000</td></tr><tr><td>Petri dish 100 mm x 20 mm</td><td>Dutscher</td><td>353003</td><td></td></tr><tr><td>PKB antibody</td><td>Cell Signaling</td><td>9272</td><td>Dilution : 1/1000</td></tr><tr><td>PKB Phospho Thr308 antibody</td><td>Cell Signaling</td><td>9275</td><td>Dilution : 1/1000</td></tr><tr><td>Rosiglitazone</td><td>Sigma-Aldrich</td><td>R2408</td><td></td></tr><tr><td>Transfection reagent (INTERFERin)</td><td>Polyplus</td><td>409-10</td><td></td></tr><tr><td>&amp;alpha;-tubulin antibody</td><td>Sigma aldrich</td><td>T6199</td><td>Dilution : 0.5 &amp;micro;g/mL</td></tr><tr><td>Vamp2 antibody</td><td>R&amp;amp;D Systems</td><td>MAB5136</td><td>Dilution : 0.1 &amp;micro;g/mL</td></tr></tbody></table>

an adipocyte cell culture model to study the impact of protein and micro-rna modulation on adipocyte function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Using the procedure of reverse-transfection described here to modulate the expression of proteins or micro-RNAs in 3T3-L1 adipocytes, the adipocytes have been shown to preserve their morphology after the transfection (Figure 1B,C). Indeed, 2 days after the transfection, the adipocytes were well-spread and attached to the plate and presented multilocular lipid droplets that are a characteristic of mature 3T3-L1 adipocytes. The lipid content was not different between the transfected and non-transfected adipocytes (Figure 1D,E). Moreover, the mRNA expression of differentiation markers such as peroxisome proliferator-activated receptor gamma 2 (Ppar&#947;2), adiponectin (Adipoq), glucose transporter 4 or solute carrier family 2 member 4 (Slc2a4), insulin receptor substrate 1 (Irs1), perilipin-1 (Plin1) was unchanged in transfected cells compared to that in non-transfected adipocytes (Figure 1F). This reverse-transfection protocol is efficient as &#62;70% of the adipocytes were transfected (Figure 1G,H).
Perilipin-1 is an adipocyte-specific protein known to promote lipid droplet formation and inhibit lipolysis. Here, 3T3-L1 adipocytes were transfected with scrambled siRNA (si-SCR) or siRNA against Plin1 (si-PLIN1). Three days after the transfection with si-PLIN1, the mRNA level of Plin1 had decreased by 70% (Figure 2A) and the protein level by 63% (Figure 2B,C). PLIN1 expression was also analyzed by fluorescence microscopy 4 days after the transfection and was found to have decreased by 92% compared to its expression in control adipocytes (Figure 2D-F), thus demonstrating the efficacy of both the transfection protocol and the si-PLIN1.
This protocol was also used to perform reverse-transfection of adipocytes with micro-RNA mimicking (miR mimics) oligonucleotides to upregulate the expression of miR-34a (Figure 3A). The overexpression of miR-34a led to the decrease in VAMP2 protein expression by 50% (Figure 3B,C), a confirmed target of miR-34a11,12. Finally, this study shows that reverse-transfection of 3T3-L1 adipocytes preserves their function and responsiveness to insulin stimulation. Indeed, knockdown of Plin1 in 3T3-L1 adipocytes led to an increase in basal lipolysis (Figure 4A). Moreover, the overexpression of miR-34a in 3T3-L1 adipocytes led to the inhibition of insulin-induced protein kinase B phosphorylation (Figure 4B,C) and glucose uptake (Figure 4D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61925/61925fig01.jpg" /> 
Figure 1: Differentiation of 3T3-L1 fibroblasts into mature adipocytes. (A) 3T3-L1 fibroblasts were seeded at a density of 3 x 105 cells per 100 mm dish. Representative 10x brightfield image of 3T3-L1 fibroblasts 2 days later. (B) Two days after confluency (day 0), the 3T3-L1 fibroblasts were differentiated into adipocytes using a differentiation cocktail mix for 4 days (until day 4). Representative 10x brightfield image of 3T3-L1 fibroblasts differentiated into adipocytes (day 7). Adipocytes with multilocular lipid droplets are easily discernable. (C) The 3T3-L1 adipocytes were transfected with si-SCR on day 7. Representative 10x brightfield images of transfected 3T3-L1 adipocytes (day 9). The morphology of the transfected adipocytes is comparable to that of the non-transfected adipocytes, implying that the transfection method is gentle and not toxic to the adipocytes. Scale bars: 50 &#181;m. (D&#8211;E) The 3T3-L1 adipocytes were transfected with si-SCR on day 7 (upper panels); non-transfected adipocytes (lower panels). Two days later, the cells were incubated with Oil Red O to stain lipids. (D) Representative images of the stained cells in the plate and representative 10x brightfield images are shown. Scale bars: 50 &#181;m. (E) The Oil Red O incorporated into the cells was eluted with 2-propanol and quantified using a spectrophotometer. Data are expressed in arbitrary units, with the absorbance of the non-transfected cells normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed by Student&#8217;s t-test. (F) The 3T3-L1 adipocytes were transfected with si-SCR. Three days after the transfection, the cells were harvested to isolate total RNA. The expression of adipocyte differentiation markers was measured by qRT-PCR and normalized using 36B4 RNA levels. The data represent the mRNA expression in transfected cells relative to that in non-transfected cells (normalized to 1, represented by the dotted line) and are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed by Student&#8217;s t-test. (G&#8211;H) The 3T3-L1 adipocytes were transfected with si-SCR or fluorescent dye (FAM)-labeled si-RNA and plated on coverslips. 3T3-L1 adipocytes were analyzed 8 h later by fluorescence microscopy. (G) Representative single-plane image of transfected 3T3-L1 cells is shown. (H) Quantification of the FAM-positive cells relative to the total number of cells. Data is expressed as percentage of cells containing fluorescent si-RNA. Statistical analysis was performed using Mann-Whitney test, ****p &#60; 0.0001. Abbreviations: si-SCR = scrambled siRNA; SEM = standard error of the mean; qRT-PCR = quantitative reverse-transcription polymerase chain reaction; FAM = fluorescein amidite; DAPI = 4&#8242;,6-diamidino-2-phenylindole; si-FAM = FAM-labeled si-RNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61925/61925fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61925/61925fig02.jpg" /> 
Figure 2: Protein-silencing in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with scrambled si-RNA (si-SCR) or si-RNA against Plin1 (si-PLIN1). (A) Three days after the transfection, mRNA expression of Plin1 was measured by qRT-PCR. The mRNA expression was normalized using 36B4 RNA levels and expressed in arbitrary units, with the si-SCR-treated cells normalized to 1. Results are expressed as the mean + SEM of four independent experiments. Statistical analysis was performed using Student&#8217;s t-test, **p &#60; 0.01. (B&#8211;C) Three days after the transfection, protein lysates were subjected to western blotting with antibodies directed against PLIN1 and HSP90 (loading control). Representative immunoblots are shown. (C) The amount of PLIN1 was quantified by densitometry scanning analysis and normalized using the amount of HSP90. Data are expressed in arbitrary units, with the si-SCR-treated cells normalized to 1. Results are expressed as the mean + SEM of four independent experiments. Statistical analysis was performed using Student&#8217;s t-test, *p &#60; 0.05. (D&#8211;F) The 3T3-L1 adipocytes were transfected with si-SCR or si-PLIN1 and plated on coverslips. The expression of PLIN1 was analyzed 96 h later by fluorescence microscopy. (D) Representative single-plane images of 3T3-L1 adipocytes stained with anti-perilipin antibody and an anti-rabbit-Alexa647-conjugated antibody are shown. Scale bars = 10 &#181;m. (E) Three-dimensional (3D) volume-rendering of 3T3-L1 adipocytes segmented in 3D using commercial software. Scale bars = 10 &#181;m. (F) The quantification of PLIN1 signal intensity relative to the total number of cells. Data are expressed in arbitrary units, with the si-SCR-treated cells normalized to 100%. Statistical analysis was performed using Mann-Whitney test, ****p &#60; 0.0001. Abbreviations: si-SCR = scrambled siRNA; si-PLIN1 = siRNA against Plin1; Plin1 = perilipin-1; HSP90 = heat shock protein 90; SEM = standard error of the mean; qRT-PCR = quantitative reverse-transcription polymerase chain reaction; FAM = fluorescein amidite; DAPI = 4&#8242;,6-diamidino-2-phenylindole; IB = immunoblotting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61925/61925fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61925/61925fig3v2.jpg" /> 
Figure 3: micro-RNA overexpression in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with control micro-RNA mimic (miR-control) or micro-RNA 34a mimic (miR-34a). Three days after the transfection, the cells were harvested for (A) RNA extraction or (B) preparation of protein lysates. (A) The expression of miR-34a was measured by qRT-PCR. The miR expression was normalized using U6 small RNA levels and expressed in arbitrary units, with the miR-control-treated cells normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed using Student&#8217;s t-test, *p &#60; 0.05. (B) Protein lysates were subjected to western blotting with antibodies directed against VAMP2 and TUBULIN (loading control). Representative immunoblots of three independent experiments are shown. (C) The amount of VAMP2 was quantified by densitometry scanning analysis and normalized using the amount of TUBULIN. Data are expressed in arbitrary units, with the miR-control cells normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed by Student&#8217;s t-test, *p &#60; 0.05. Abbreviations: SEM = standard error of the mean; qRT-PCR = quantitative reverse-transcription polymerase chain reaction; VAMP2 = vesicle-associated membrane protein 2; IB = immunoblotting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61925/61925fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61925/61925fig4v2.jpg" /> 
Figure 4: Effects of protein or micro-RNA modulation in 3T3-L1 adipocyte&#160;function. (A) 3T3-L1 adipocytes were transfected with si-SCR or si-PLIN1. The medium was changed 24 h after the transfection and then collected 48 h later to measure basal lipolysis. Results are expressed as glycerol released in the media (&#181;g/mL), and as the mean + SEM of four independent experiments. Statistical analysis was performed using Student&#8217;s t-test, ***p &#60; 0.001. (B) 3T3-L1 adipocytes were transfected with miR-control or miR-34a. The medium was changed 24 h after the transfection, and then, 48 h later, the medium was changed to the depletion medium (DMEM without pyruvate, 25 mM glucose, 1% penicillin and streptomycin, and 0.5% BSA) for 6 h. Then, the cells were treated with 0.5 nM insulin for 5 min. Cells were harvested to prepare protein lysates for western blotting with antibodies directed against phospho-PKB and PKB (loading control). Representative immunoblots of three independent experiments are shown. (C) The amount of phospho-PKB was quantified by densitometry scanning analysis and normalized using the total amount of PKB. Data are expressed in arbitrary units, with the miR-control cells treated with insulin normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed using the two-way ANOVA test, *p &#60; 0.05 compared to miR-control cells treated with insulin. (D) 3T3-L1 adipocytes were transfected with miR-control or miR-34a. The media was changed 24 h after the transfection, and then, 48 h later, the medium was changed to the depletion medium for 6 h. Then, the cells were treated with 0.5 nM insulin for 20 min. Uptake of (2-3H)deoxyglucose was measured over 3 min. Data are expressed in arbitrary units, with the basal glucose uptake in miR-control-treated cells normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed using the two-way ANOVA test, *p &#60; 0.05 compared to miR-control cells treated with insulin. Abbreviations: si-SCR = scrambled siRNA; si-PLIN1 = siRNA against Plin1; SEM = standard error of the mean; PKB = protein kinase B; p-PKB = phosphor-PKB; miR-CTL = miR-control; DMEM = Dulbecco&#8217;s modified Eagle&#8217;s medium; BSA = bovine serum albumin; ANOVA = analysis of variance; IB = immunoblotting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61925/61925fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Per well (12-well plate)</td>
			<td>Per well (24-well plate)</td>
		</tr>
		<tr>
			<td>Oligonucletides</td>
			<td rowspan="2">20 &#181;L</td>
			<td rowspan="2">10 &#181;L</td>
		</tr>
		<tr>
			<td>(si-RNA, miR&#8230;)</td>
		</tr>
		<tr>
			<td>Improved Minimal Essential Medium</td>
			<td>20 &#181;L</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>Transfection reagent</td>
			<td>5.6 &#181;L</td>
			<td>2.8 &#181;L</td>
		</tr>
		<tr>
			<td>Improved Minimal Essential Medium</td>
			<td>154.4 &#181;L</td>
			<td>77.2 &#181;L</td>
		</tr>
		<tr>
			<td>Total volume of transfection mix</td>
			<td>200 &#181;L</td>
			<td>100 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Transfection reagents required for 12-well and 24-well plate formats.

Watch this Scientific Journal Video about An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function at JoVE.com

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

an-adipocyte-cell-culture-model-to-study-impact-protein-micro-rna

Nanyang Technological University

Research

JoVE Journal

Biology

3.5K Views.  Université Côte d’Azur, Inserm, C3M, Team Cellular and Molecular Pathophysiology of Obesity and Diabetes, Nice, France. Dit protocol maakt het mogelijk om eiwitten, of microRNA-expressie, in gedifferentieerde adipocyten te manipuleren om hun rol in de adipocytenfunctie te bestuderen. Deze omgekeerde transfectiemethode is een eenvoudige, economische en zeer efficiënte methode om oligonucleotiden te transfecteren in muizen 3T3-L1-adipocyten. Deze methode kan ook worden toegepast op menselijke preadipocyten die zijn gedifferentieerd in adipocyten.Melanie Gaudfrin, een technicus van ons laboratorium, demon...

Video: An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

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.

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 ...

Developmental Biology

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 ...

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

Behavior

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Bioengineering

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 ...

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Medicine

Three-Dimensional Adipocyte Culture as a Model to Study Cachexia-Induced White Adipose Tissue Remodeling

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic wasting has been proposed to be stimulated by several inflammatory mediators, which may disrupt the integrative physiology of adipose tissues and other tissues such as the brain and muscle. In this scenario, the tumor can survive at the host&#39;s expense. In recent clinical research, the intensity of depletion of the different fat deposits has been negatively correlated with the patient&#39;s survival outcome. Studies have also shown that various metabolic disorders can alter white adipose tissue (WAT) remodeling, especially in the early stages of cachexia development. WAT dysfunction resulting from tissue remodeling is a contributor to overall cachexia, with the main modifications in WAT consisting of morpho-functional changes, increased adipocyte lipolysis, accumulation of immune cells, reduction of adipogenesis, changes in progenitor cell population, and the increase of &#34;niches&#34; containing beige/brite cells.
To study the various facets of cachexia-induced WAT remodeling, particularly the changes&#160;progenitor cells and beige remodeling, two-dimensional (2D) culture has been the first option for in vitro studies. However, this approach does not adequately summarize WAT complexity. Improved assays for the reconstruction of functional AT ex vivo&#160;help the comprehension of physiological interactions between the distinct cell populations. This protocol describes an efficient three-dimensional (3D) printing tissue culture system based on magnetic nanoparticles. The protocol is optimized for investigating WAT remodeling induced by cachexia induced factors (CIFs). The results show that a 3D culture is an appropriate tool for studying WAT modeling ex vivo and may be useful for functional screens to identify bioactive molecules for individual adipose cell populations applications and aid the discovery of WAT-based cell anticachectic therapy.

This protocol describes a three-dimensional (3D) magnetic printing culture system that permits dissection of white adipose tissue (WAT) remodeling induced by a conditioned medium from cancer cells. Using a 3D culture system of UCP1+ adipocytes that express a green fluorescent protein (GFP) allows the study of beige adipocytes contributing to adipose tissue remodeling.

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic ...

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 ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

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