Name: Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance (Video) | JoVE Journal
Uploaded: 2023-02-17T14:00:00.000+00:00
Duration: 9 min 48 s
Description: Learn to isolate and differentiate mouse preadipocytes from subcutaneous fat for studying insulin resistance using western blot analysis. This primary cell model enables precise investigation of metabolic disease mechanisms.

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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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<li>Kirkland, I. M., Tchkonia, T., Pirtskhalava, T., Han, J., Karagiannides, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipogenesis+and+aging%3A+does+aging+make+fat+go+MAD%5BTitle%5D)+AND+%22Experimental+Geronotology%22%5BJournal%5D)">Adipogenesis and aging: does aging make fat go MAD.</a> Experimental Geronotology. 37 (6), 757-767 (2002).</li>
<li>Guo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aging+results+in+paradoxical+susceptibility+of+fat+cell+progenitors+to+lipotoxicity%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Endocrinology+and+Metabolism%22%5BJournal%5D)">Aging results in paradoxical susceptibility of fat cell progenitors to lipotoxicity.</a> American Journal of Physiology. Endocrinology and Metabolism. 292 (4), 1041-1051 (2007).</li>
<li>Ruan, H., Hacohen, N., Golub, T. R., Van Parijs, L., Lodish, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((F.Tumor+necrosis+factor-a+suppresses+adipocyte-specific+genes+and+activatesexpression+of+preadipocyte+genes+in+3T3-L1+adipocytes%3A+nuclear+factor-kappaB+activation+by+TNF-a+is+obligatory%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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 ́meaux, T., Cormont, M., Le Marchand-Brustel, Y., Tanti, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin-1b-induced+insulin+resistance+in+adipocytes+throughdown-regulation+of+insulin+receptor+substrate-1+expression%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">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/pubmed?term=((Interleukin-6+%28IL-6%29+induces+insulinresistance+in+3T3-L1+adipocytes+and+is%2C+like+IL-8+and+tumor+necrosis+factor-a%2Coverexpressed+in+human+fat+cells+from+insulin-resistant+subjects%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Insulin+resistance+rewires+the+metabolic+gene+program+and+glucose+utilization+in+human+white+adipocytes%5BTitle%5D)+AND+%22International+Journal+of+Obesity%22%5BJournal%5D)">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/pubmed?term=((Reactive+oxygen+species+have+a+causal+role+in+multiple+forms+of+insulin+resistance%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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

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-α-mediated induction of insulin resistance (Figure 1).
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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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JoVE Journal

Biology

This video demonstrates the differentiation of bone marrow stromal cells (BMSCs) into adipocytes. In the two-step process, in the presence of specific growth factors and hormones, undifferentiated BMSCs first differentiate into preadipocytes. The second step involving treatment with glucose and insulin leads to the formation of mature adipocytes with multiple lipid droplets.

In Vitro Mouse Bone Marrow Stromal Cell Differentiation into Adipocytes

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons.  We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy.  A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells.  Treatment is administered via oral gavage.  Tumor growth is monitored by in vivo imaging system.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Learn to isolate and differentiate mouse preadipocytes from subcutaneous fat for studying insulin resistance using western blot analysis. This primary cell model enables precise investigation of metabolic disease mechanisms.

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

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