We thank Daniel Mondrag&#243;n, Antonio Prado, Fernando L&#243;pez-Barrera, Mart&#237;n Garc&#237;a-Serv&#237;n, Alejandra Castilla, and Mar&#237;a Antonieta Carbajo for their technical assistance, and Jessica Gonzalez Norris for critically editing the manuscript. This protocol was supported by Consejo Nacional de Ciencia y Tecnolog&#237;a de M&#233;xico (CONACYT), Fondo Sectorial de Investigacio&#769;n para la Educacio&#769;n&#160;Grant 284771 (to Y.M.).

All rodent experiments were approved by the Bioethics Committee of the Institute of Neurobiology of the UNAM, protocol number 075.
1. Isolation of mouse adipocyte precursor cells
<ol>
	<li>Euthanize 8-10-week-old male C57BL/6 mice (e.g., by CO2&#160;inhalation and subsequent cervical dislocation) (four animals per isolation). Disinfect the mice by rubbing with 70% ethanol and obtain the adipose tissue immediately after sacrifice. 
	NOTE: After the euthanasia of rodents, isolate the adipose tissue immediately and perform all steps inside a sterile laminar flow hood. Mice are kept under 12 h light/dark cycles with free access to food and water.</li>
	<li>Dissect the inguinal subcutaneous adipose tissue from each mouse. Remove only the adipose tissue, avoiding the removal of muscle, skin, or any other tissue, and collect it in a 50 mL conical tube containing 15 mL of type 1 collagenase solution (1.5 mg/mL) on ice. Dissolve type 1 collagenase in Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose-1% bovine serum albumin (BSA) and filter through 0.2 &#181;m syringe filters.</li>
	<li>Cut the adipose tissue into small pieces with sterile surgical scissors and digest the samples by incubation with type 1 collagenase solution at 37 &#176;C in an orbital shaker (150 rpm) for 30 min (place the tube containing fat and collagenase in a horizontal position for a greater shaking surface). Check the digestion every 10 min to make sure it works (tissue degradation is visible) and to prevent overdigestion (see Supplementary Figure S1).</li>
	<li>Filter using a 200 &#181;m mesh-syringe (previously autoclaved) to eliminate tissue not digested with collagenase. Pass the filter over the edge of the tube to drain as many cells as possible into the solution. Add 15 mL of cold DMEM-1% BSA to stop digestion and centrifuge at 400 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the top layer containing mature adipocytes and most of the liquid layer; avoid touching or disturbing the pellet. Add 20 mL of cold phosphate buffered saline (PBS)-2% fetal bovine serum (FBS) and resuspend the pellet. Centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant, aspirating first the upper layer to eliminate the remaining adipocytes and fat. Resuspend the pellet in 1 mL of ammonium chloride potassium (ACK) lysing buffer (to lyse red blood cells) and incubate on ice for 5 min. Add 10 mL of PBS-2% FBS, mix, and centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 200 &#181;L of anti-Fc solution (purified rat anti-mouse CD16/CD32 diluted in PBS-2% FBS [1:150]) to reduce Fc receptor-mediated binding by antibodies of interest. Incubate on ice for 5 min. Transfer the cell suspension to a 5 mL tube that fits into the prechilled racks of a magnetic cell separator and centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Eliminate the supernatant, add 200 &#181;L of the mixture of CD31(PECAM-1) monoclonal antibody (390)-biotin (1:100) and CD45 monoclonal antibody (30-F11)-biotin (1:100), mix well, and incubate on ice for 15 min (prepare antibody dilutions in anti-Fc solution; see Table 1). Add 400 &#181;L of PBS-2% FBS and centrifuge at 400 &#215; g for 5 min at 4 &#176;C. 
	NOTE: CD31 and CD45 are markers of endothelial cells and hematopoietic cells, respectively.</li>
	<li>Aspirate the supernatant and incubate in 100 &#181;L of anti-biotin microbeads (1:5) for 15 min on ice (prepare antibody dilutions in anti-Fc solution, see Table 1). Add 400 &#181;L of PBS-2% FBS and centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in 350 &#181;L of PBS-2% FBS. Remove cell aggregates or large particles from the single-cell suspensions with a preseparation filter (70 &#181;m). First, activate the filter with 100 &#181;L of PBS-2% FBS, pass the cell suspension through the filter (collect in a clean tube), and finally wash the filter with 100 &#181;L of PBS-2% FBS.</li>
	<li>Perform magnetic separation of cells using the negative separation strategy with a magnetic cell separator.
	<ol>
		<li>Place the sample in position A of the chill rack (ensure that the rack is precooled) and two empty tubes in position B and C to recover the non-labeled and labeled cells, respectively.</li>
		<li>Load the washing buffer and running buffer into the corresponding bottles before turning on the equipment. Program the equipment to perform the magnetic separation of cells with the DEPLETE protocol. 
		NOTE: This protocol is for depletion in standard mode for the removal of cells with normal antigen expression (in this case, cells labeled with anti-CD31 and anti-CD45), if recovery is the highest priority.</li>
		<li>In the separation section, select the number of samples to be separated and select the DEPLETE protocol. Press Run and start the separation. At the end of the program, recover the unlabeled cells and centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Remove the supernatant and resuspend the pellet in 500 &#181;L of growth medium (Table 1).</li>
	<li>Seed the APCs in one well of a 12-well plate previously coated with 2.5% basement membrane matrix (approximately 50,000-75,000 cells are obtained). Add 400 &#181;L of 2.5% basement membrane matrix to cover the entire surface of each well. Immediately after removing the excess solution and leaving only a small layer, let the plate dry (without a lid) inside the laminar flow hood for at least 1 h. Incubate at 37 &#176;C in a 5% CO2 atmosphere. Change the medium every 48 h until the cells reach 80% confluence.</li>
	<li>At 80% confluence, remove the medium completely and wash with 350 &#181;L of PBS-2% FBS. Harvest the cells with 350 &#181;L of 0.05% trypsin-EDTA for 2 min at 37 &#176;C. Add 2 mL of growth medium and collect the cells in a new 50 mL conical tube, then centrifuge at 400 &#215; g for 5 min.</li>
	<li>Passage the APCs to 12-well plates (10,000-20,000 cells per well) previously coated with 2.5% basement membrane matrix. Incubate at 37 &#176;C with 5% CO2. Change the medium every 48 h until cells reach 80% confluence. 
	NOTE: APCs can be passaged once. Subsequently, they lose their ability to proliferate and differentiate. See the workflow for the APCs isolation process in Supplementary Figure S2.</li>
</ol>
2.&#160;Adipocyte differentiation and induction of insulin resistance
NOTE: Maintain cells at 37 &#176;C in 5% CO2&#160;and perform steps involving change of medium and treatments with TNF-&#945; and insulin inside a sterile hood.
<ol>
	<li>At 80% confluence, begin the differentiation process; aspirate off any growth medium and replace it with 500 &#181;L of differentiation medium (Table 1) containing 3.3 nM bone morphogenetic protein (BMP4) in each well.</li>
	<li>After 48 h, replace the medium with the differentiation medium (500 &#181;L per well) and the differentiation cocktail (Table 1).</li>
	<li>Remove the medium 72 h later and add 100 nM of insulin in 500 &#181;L of fresh differentiation medium. 
	NOTE: Cells begin to show a round appearance with small lipid droplets inside.</li>
	<li>Aspirate off any differentiation medium and replace it with 500 &#181;L of simple medium-2% FBS (Table 1) 48 h later. Induce insulin resistance with 4 ng/mL of TNF-&#945;. Include control wells without TNF-&#945; treatment.</li>
	<li>After 24 h of incubation, add 4 ng/mL TNF-&#945; in simple medium-0% FBS (Table 1). Maintain control wells without TNF-&#945; treatment.</li>
	<li>Activate the insulin signaling pathway 24 h later by adding 100 nM insulin and incubate for 15 min at 37 &#176;C in a 5% CO2 atmosphere. Remove the medium and wash with 500 &#181;L of PBS. Include control wells without insulin treatment.</li>
</ol>
3. Evaluation of insulin signaling pathway by western blot
<ol>
	<li>Protein extraction and quantification
	<ol>
		<li>Wash each well of cells with 500 &#181;L of PBS and keep on ice to lyse the cells in 50 &#181;L of RIPA buffer (Table 1) containing 1% protease inhibitor cocktail added just prior to sample isolation. Scrape the entire surface of the well with a tip and transfer the lysate to a 0.6 mL microcentrifuge tube (keep on ice).</li>
		<li>Incubate for 1 h at 4 &#176;C in a rotating shaker,&#160;mix by vortexing, and centrifuge at 8,000 &#215; g for 15 min at 4 &#176;C and recover the supernatant (keep on ice).</li>
		<li>Determine the protein concentration using the Bradford assay (do not dilute the sample to quantify).</li>
		<li>Take the necessary volume corresponding to 40-60 &#181;g and denature with 6x Laemmli buffer (Table 1) by&#160;incubating at 97 &#176;C for 15 min while shaking at 550 rpm. Freeze until use.</li>
	</ol>
	</li>
	<li>SDS-polyacrylamide gel electrophoresis
	<ol>
		<li>Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with a 7.5% polyacrylamide gel of 1.5 mm thickness. Insert the gel into the tank and fill with running buffer (Table 1).</li>
		<li>Add 3 &#181;L of prestained protein standard to the first well of the gel and load each sample into subsequent wells.</li>
		<li>Run the gel at 80 V for approximately 15 min to ensure the sample enters the polyacrylamide concentrator gel matrix. Thereafter, increase the voltage to 90 V for 2.5 h or until the 37 kDa molecular weight marker can be seen at the bottom edge of the gel.</li>
		<li>Transfer the proteins from the gel to a nitrocellulose membrane in a semi-dry chamber for 35 min at 25 V. Beforehand, submerge the gel, membrane, and filters in transfer buffer (Table 1) for a few minutes.</li>
	</ol>
	</li>
	<li>Western blot
	<ol>
		<li>After transfer, wash the membrane with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T 0.1%) (Table 1) for 3 min with shaking at 30 rpm.</li>
		<li>Block the membrane with blocking solution (Table 1) at 4 &#176;C for 1 h in constant rotation.</li>
		<li>Cut the membrane into three parts according to the molecular weight marker to incubate overnight with different primary antibodies (diluted in blocking solution) at 4 &#176;C in constant rotation: Phospho-IRS1 (Tyr608) antibody (1:1,000), expected band at 180 kDa; Phospho-Insulin receptor &#946; (Tyr1150/1151) antibody (1:1,000), expected band at 95 kDa; Phospho-Akt (Ser473) antibody (1:1,000), expected band at 60 kDa.</li>
		<li>Wash the membranes 5x for 5 min each with TBS-T 0.1%.</li>
		<li>Incubate the membranes with the corresponding peroxidase-coupled secondary antibody (diluted in blocking solution) for 2 h at room temperature in constant rotation: peroxidase affiniPure donkey anti-rabbit IgG (H+L) (1:5,000).</li>
		<li>Wash the membranes 5x for 5 min each with TBS-T 0.1%.</li>
		<li>Detect the proteins using the chemiluminescence detection system. Prepare the substrate according to the manufacturer&#39;s instructions.</li>
		<li>Place the blot in a plastic bag and add 200 &#181;L of chemiluminescent substrate to completely cover the membrane. Drain excess substrate and remove any air bubbles.</li>
		<li>Expose each blot for 30-60 s in a high-performance imaging system compatible with chemiluminescence.</li>
		<li>Strip the membranes (steps 10-13 of the western blot section) to break the antibody-antigen interactions and thus allow nitrocellulose membranes to be re-blotted. Recover the membranes and wash 3x for 5 min each with TBS-T 1% at 50 rpm.</li>
		<li>Incubate for 7 min with 10 mL of 0.2 M NaOH at 50 rpm.</li>
		<li>Wash 3x for 5 min each with tap water at 50 rpm.</li>
		<li>Wash for 10 min with TBS-T 1% at 50 rpm.</li>
		<li>Repeat steps 2-9 of the western blot section to incubate the membrane with anti-beta tubulin (55 kDa) as a loading control using 1:1,000 dilution.</li>
		<li>Perform densitometric analysis25&#160;for each protein using ImageJ software (https://imagej.nih.gov/).</li>
	</ol>
	</li>
</ol>

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R., Van Parijs, L., Lodish, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F.Tumor+necrosis+factor-a+suppresses+adipocyte-specific+genes+and+activatesexpression+of+preadipocyte+genes+in+3T3-L1+adipocytes:+nuclear+factor-kappaB+activation+by+TNF-a+is+obligatory.">F.Tumor necrosis factor-a suppresses adipocyte-specific genes and activatesexpression of preadipocyte genes in 3T3-L1 adipocytes: nuclear factor-kappaB activation by TNF-a is obligatory.</a> Diabetes. 51 (5), 1319-1336 (2002).</li><li>Jager, J., Gre &#769;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-1b-induced+insulin+resistance+in+adipocytes+throughdown-regulation+of+insulin+receptor+substrate-1+expression.">Interleukin-1b-induced insulin resistance in adipocytes throughdown-regulation of insulin receptor substrate-1 expression.</a> Endocrinology. 148 (1), 241-251 (2007).</li><li>Rotter, V., Nagaev, I., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-6+(IL-6)+induces+insulinresistance+in+3T3-L1+adipocytes+and+is,+like+IL-8+and+tumor+necrosis+factor-a,overexpressed+in+human+fat+cells+from+insulin-resistant+subjects.">Interleukin-6 (IL-6) induces insulinresistance in 3T3-L1 adipocytes and is, like IL-8 and tumor necrosis factor-a,overexpressed in human fat cells from insulin-resistant subjects.</a> The Journal of Biological Chemistry. 278 (46), 45777-45784 (2003).</li><li>Isidor, M. S., 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+resistance+rewires+the+metabolic+gene+program+and+glucose+utilization+in+human+white+adipocytes.">Insulin resistance rewires the metabolic gene program and glucose utilization in human white adipocytes.</a> International Journal of Obesity. 46 (3), 535-543 (2021).</li><li>Houstis, N., Rosen, E. D., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+have+a+causal+role+in+multiple+forms+of+insulin+resistance.">Reactive oxygen species have a causal role in multiple forms of insulin resistance.</a> Nature. 440 (7086), 944-948 (2006).</li></ol>

The authors declare no conflicts of interest.

This paper provides a method for studying insulin resistance that uses primary adipocytes in culture treated with TNF-&#945;. This model has the advantage that primary adipocytes can be cultured under defined conditions for long periods of time with a tight control of cellular environmental factors26. The assay duration is 15-20 days, although variations in the percentage of differentiated adipocytes can occur between experiments. Primary adipocytes have advantages over cell lines since they have ...

Insulin is an anabolic hormone produced by pancreatic islet &#946;-cells and the key regulator of glucose and lipid metabolism. Among its many functions, insulin regulates glucose uptake, glycogen synthesis, gluconeogenesis, protein synthesis, lipogenesis, and lipolysis1. The initial molecular signal after insulin interaction with its receptor (IR) is the activation of the intrinsic tyrosine protein kinase activity of IR2, resulting in its autophosphorylation3&#160;and the subsequent activation of a family of proteins known as insulin receptor substrates (IRS), which binds to adaptor proteins leading to activation of a cascade of protein kinases4. Insulin activates two main signaling pathways: phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)&#160;and Ras-mitogen-activated protein kinase (MAPK). The former constitutes a major branch point or node4,5&#160;for the activation of numerous downstream effectors involved in diverse physiological functions, including the regulation of fuel homeostasis, whereas the latter regulates cell growth and differentiation4,6. Insulin actions ultimately depend on the cell type and physiological context7.
One of the main insulin-responsive metabolic tissues is the adipose tissue. White adipose tissue is the most abundant type of fat in humans and rodents, distributed within subcutaneous fat (between the skin and muscles) and visceral fat (around the organs in the abdominal cavity). Given their large volume, adipocytes or fat cells are the most abundant cell type in adipose tissue. These fat cells can be brown/beige (thermogenic), pink (in the mammary gland), and white8,9. White adipocytes keep the main energy reserves in the body in the form of triglycerides, an insulin-dependent process. Insulin promotes glucose transport and lipogenesis, while it inhibits lipolysis or lipid breakdown7,10. It also facilitates the differentiation of preadipocytes into adipocytes&#160;&#8212; the mature fat-storing cells11.
Insulin resistance occurs when a normal insulin level produces an attenuated biological response, resulting in compensatory hyperinsulinemia12. Insulin resistance is a condition associated with overweight and obesity5, that when combined leads to type 2 diabetes (T2D) and other metabolic diseases13. Hyperinsulinemia compensates insulin resistance in peripheral tissues to maintain normal blood glucose levels14. However, eventual &#946;-cell loss or exhaustion, together with exacerbated insulin resistance, leads to elevated blood glucose levels consistent with T2D5. Therefore, insulin resistance and hyperinsulinemia can contribute to the development of obesity-derived metabolic diseases15. Furthermore, obesity may cause chronic low-grade local inflammation promoting insulin resistance in adipose tissue15,16,17. In addition, obesity-derived alterations in adipose tissue, such as fibrosis, inflammation, and reduced angiogenesis and adipogenesis, lead to lower adiponectin serum levels (an insulin sensitizer) and increased secretion of factors such as plasminogen activator inhibitor 1 (PAI-1), free fatty acids, and exosomes into the bloodstream, exacerbating insulin resistance17.
Many aspects underlying insulin resistance remain unknown. In vitro and in vivo models have been developed to study the mechanisms mediating insulin resistance in major target tissues, including adipose tissue. The advantage of in vitro models is that researchers have more control of the environmental conditions and can evaluate insulin resistance in specific cell types. Particularly, adipocyte precursor cells (APCs) have the individual phenotype of the donor tissue, which might reflect physiology better than adipocyte cell lines. A main factor inducing insulin resistance in vitro is tumor necrosis factor-&#945; (TNF-&#945;). TNF-&#945; is a proinflammatory cytokine secreted by adipocytes and macrophages in adipose tissue18. While it is required for proper adipose tissue remodeling and expansion19, long-term exposure to TNF-&#945; induces insulin resistance in adipose tissue in vivo and in adipocytes in vitro20. Chronic TNF-&#945; treatment of several cell types leads to increased serine phosphorylation of both IR and IRS-1, thereby promoting decreased tyrosine phosphorylation21. Increased phosphorylation of IRS-1 on serine residues inhibits the IR tyrosine kinase activity and may be one of the key mechanisms by which chronic TNF-&#945; treatment impairs insulin action22,23. TNF-&#945; activates pathways involving the serine/threonine kinase inhibitor of nuclear factor &#312;B kinase &#946; (IKK&#946;) and c-Jun N terminal kinase (JNK)24. JNK induces a complex proinflammatory transcriptional program but also directly phosphorylates IRS-16.
Understanding the pathogenesis of insulin resistance has become increasingly important to guide the development of future therapies against T2D. APCs have proven to be an excellent model for the study of fat cell biology, including the sensitivity and resistance to insulin, and for identifying the intrinsic properties of adipocytes independent of the systemic environment. APCs can be easily obtained from different adipose depots, and under the appropriate conditions, differentiated into mature adipocytes. With this method, direct effects on insulin resistance/sensitivity can be evaluated in adipocytes.

<table><tbody><tr><td>&lt;strong&gt;1. Isolation mouse adipocyte precursor cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ACK lysing buffer&amp;nbsp;</td><td>LONZA</td><td>10-548E</td><td></td></tr><tr><td>Anti-Biotin Microbeads&amp;nbsp;</td><td>Miltenyi</td><td>130-090-485</td><td></td></tr><tr><td>Anti-CD31</td><td>eBioscience</td><td>13-0311-85</td><td></td></tr><tr><td>AutoMACS Pro Separator</td><td>Miltenyi</td><td></td><td></td></tr><tr><td>Basement membrane matrix (matrigel)</td><td>Corning</td><td>354234</td><td></td></tr><tr><td>bFGF</td><td>Sigma</td><td>F0291</td><td>Growth factor</td></tr><tr><td>BSA</td><td>Equitech-Bio, Inc.</td><td>BAC63-1000</td><td></td></tr><tr><td>CD45 Monoclonal Antibody (30-F11) - Biotin&amp;nbsp;</td><td>eBioscience</td><td>13-0451-85</td><td></td></tr><tr><td>Collagenase, Type 1</td><td>Worthington Biochem</td><td>LS004197</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D1756</td><td></td></tr><tr><td>DMEM</td><td>GIBCO</td><td>12800017</td><td></td></tr><tr><td>DMEM low glucose</td><td>GIBCO</td><td>31600-034</td><td></td></tr><tr><td>EGF</td><td>Peprotech</td><td>315-09</td><td>Growth factor</td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140-079</td><td></td></tr><tr><td>ITS mix</td><td>Sigma</td><td>I3146</td><td></td></tr><tr><td>L-ascorbic acid 2-phosphate</td><td>Sigma</td><td>A8960</td><td></td></tr><tr><td>LIF</td><td>Millipore</td><td>ESG1107</td><td>Growth factor</td></tr><tr><td>Linoleic acid-albumin</td><td>Sigma</td><td>L9530</td><td></td></tr><tr><td>MCDB 201 medium</td><td>Sigma</td><td>M6770</td><td></td></tr><tr><td>Normocin</td><td>InvivoGen</td><td>ant-nr-2</td><td></td></tr><tr><td>PDGF-BB&amp;nbsp;</td><td>Peprotech</td><td>315-18</td><td>Growth factor</td></tr><tr><td>Peniciline-Streptomycine</td><td>BioWest</td><td>L0022-100</td><td></td></tr><tr><td>Pre-Separation Filters (70 &amp;micro;m)</td><td>Miltenyi</td><td>130-095-823</td><td></td></tr><tr><td>Purified Rat Anti-Mouse CD16 / CD32&amp;nbsp;</td><td>BD Pharmingen</td><td>553142</td><td></td></tr><tr><td>Trypsin-EDTA&amp;nbsp;</td><td>GIBCO</td><td>25300062</td><td></td></tr><tr><td>&lt;strong&gt;2. Adipocyte differentiation and insulin resistance induction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine [IBMX]</td><td>Sigma</td><td>I5879</td><td>Differentiation cocktail</td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>5020-BP-010</td><td>Differentiation cocktail</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D1756</td><td>Differentiation cocktail</td></tr><tr><td>Insulin</td><td>Sigma</td><td>I9278</td><td></td></tr><tr><td>Rosiglitazone</td><td>Cayman</td><td>71742</td><td>Differentiation cocktail</td></tr><tr><td>TNF&amp;alpha;</td><td>R&amp;amp;D Systems</td><td>210-TA-005&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;3. Evaluation of insulin signaling pathway by western blot&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-beta tubulin antibody</td><td>Abcam</td><td>ab6046</td><td></td></tr><tr><td>Bromophenol blue</td><td>BioRad</td><td>161-0404</td><td>Laemmli buffer</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td>RIPA buffer</td></tr><tr><td>EGTA</td><td>Sigma</td><td>E4378</td><td>RIPA buffer</td></tr><tr><td>FluorChem E system</td><td>ProteinSimple</td><td></td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G6279</td><td>Laemmli buffer</td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7126</td><td>Running and Transfer buffer</td></tr><tr><td>Igepal</td><td>Sigma</td><td>I3021</td><td>RIPA buffer</td></tr><tr><td>2- mercaptoethanol</td><td>Sigma</td><td>M3148</td><td>Laemmli buffer</td></tr><tr><td>Methanol</td><td>JT Baker</td><td>907007</td><td>Transfer buffer</td></tr><tr><td>NaCl</td><td>JT Baker</td><td>3624-05</td><td>TBS-T</td></tr><tr><td>NaF</td><td>Sigma</td><td>77F-0379</td><td>RIPA buffer</td></tr><tr><td>NaOH&amp;nbsp;</td><td>JT Baker</td><td>3722-19</td><td></td></tr><tr><td>Na4P2O7</td><td>Sigma</td><td>114F-0762</td><td>RIPA buffer</td></tr><tr><td>Na3VO4</td><td>Sigma</td><td>S6508</td><td>RIPA buffer</td></tr><tr><td>Nitrocellulose membrane&amp;nbsp;</td><td>BioRad</td><td>1620112</td><td></td></tr><tr><td>Nonfat dry milk</td><td>BioRad</td><td>1706404</td><td>Blocking solution</td></tr><tr><td>Prestained protein standard&amp;nbsp;</td><td>BioRad</td><td>1610395</td><td></td></tr><tr><td>Protease inhibitor cocktail&amp;nbsp;</td><td>Sigma</td><td>P8340-5ML</td><td></td></tr><tr><td>Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L)&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>711-035-132</td><td></td></tr><tr><td>Phospho- Insulin Receptor &amp;beta;&amp;nbsp;</td><td>Cell signaling</td><td>3024</td><td></td></tr><tr><td>Phospho-Akt (Ser473) Antibody</td><td>Cell signaling</td><td>9271</td><td></td></tr><tr><td>Phospho-IRS1 (Tyr608) antibody</td><td>Millipore</td><td>9432</td><td></td></tr><tr><td>Saccharose&amp;nbsp;</td><td>JT Baker</td><td>407205</td><td>RIPA buffer</td></tr><tr><td>SDS</td><td>BioRad</td><td>1610302</td><td>Running and laemmli buffer</td></tr><tr><td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td><td>Thermo Scientific</td><td>34577</td><td></td></tr><tr><td>Tris-base</td><td>Promega</td><td>H5135</td><td>Running, transfer and laemmli buffer</td></tr><tr><td>Tris-HCl</td><td>JT Baker</td><td>4103-02</td><td>RIPA buffer - TBS</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379</td><td>TBS-T</td></tr></tbody></table>

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.

Over the last few years, the increased prevalence of obesity and T2D has prompted an intense search for the mechanisms mediating insulin resistance in adipose tissue. With the protocol described here, APCs can be differentiated into mature adipocytes to evaluate insulin resistance and sensitivity. Once the APCs reach confluence, it takes 10 days to complete their differentiation into mature adipocytes and their TNF-&#945;-mediated induction of insulin resistance (Figure 1).
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Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

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 ...

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2.5K Views.  Universidad Nacional Autónoma de México (UNAM). 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. 

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

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

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Immunology and Infection

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 ...

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 ...

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.

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 ...

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 ...

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 ...

Culturing Murine Adipocytes and Explants for Functional and Imaging Studies

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte&#160;progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and ...