Name: an adipocyte cell culture model to study the impact of protein and micro-rna modulation on adipocyte function
Uploaded: 2021-05-04T14:00:00
Description: 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

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 tiss...

<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>

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 util...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;s&#160; Modified Eagles&#39;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 &#181;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>&#945;-tubulin antibody</td>
			<td>Sigma aldrich</td>
			<td>T6199</td>
			<td>Dilution : 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Vamp2 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB5136</td>
			<td>Dilution : 0.1 &#181;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 wa...

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 ...

This protocol makes it possible to manipulate proteins, or microRNA expression, in differentiated adipocytes to study their role in adipocytes function. This reverse-transfection method is a simple, economical, and highly efficient method to transfect oligonucleotides into mouse 3T3-L1 adipocytes. This method can also be applied to human preadipocytes differentiated into adipocytes.Demonstrating the procedure will be Melanie Gaudfrin, a technician from our laboratory. Grow the 3T3-L1 fibroblasts in 100 millimeter dishes in DMEM without pyruvate, 25 millimolar glucose, 10%newborn calf serum, and one percent penicillin and streptomycin. Place the dishes in a tissue culture incubator at 37 degrees Celsius and seven percent carbon dioxide.Two days after confluence, change the culture medium, replacing it with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin supplemented with 0.25 millimolar IBMX, 0.25 micromolar dexamethasone, five micrograms per milliliter insulin, and 10 micromolar rosiglitazone. Incubate the dishes for two days. Two days later, replace the culture medium with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin supplemented with five micrograms per milliliter insulin and 10 micromolar rosiglitazone and incubate for another two days.Feed the cells every two days with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin and keep the cells in the incubator. One day or a few hours before the transfection, prepare a solution of collagen type I at 100 micrograms per milliliter in 30%ethanol from a stock solution at one milligram per milliliter. Add 250 microliters of collagen per well of a 12-well plate or 125 microliters per well of a 24-well plate and spread the solution over the surface of the well.Leave the plate without the lid under the culture hood until the collagen dries. Then wash it twice with D-PBS. Pipette the siRNA with improved minimum essential medium to mix and incubate for five minutes at room temperature.Add the transfection reagent and the improved minimal essential medium to the siRNA and pipette to mix. Incubate it for 20 minutes at room temperature. Then add the transfection mix to each well of the collagen-coated plate.Wash the cells in the 100 millimeter Petri dish twice with D-PBS. Add 5x trypsin to the cells, making sure to cover the entire surface with the trypsin. Wait for 30 seconds and carefully remove the trypsin.Incubate the Petri dish for five to 10 minutes at 37 degrees Celsius in the incubator. Then tap the dish to detach the cells, avoiding cell damage. Add 10 milliliters of DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin to neutralize the trypsin.Carefully pipette the medium up and down to detach the cells and homogenize the cell suspension. Count the cells using a Malassez counting chamber or an automated cell counter and adjust the concentration of the cells to 0.625 million cells per milliliter of medium. Seed 800 microliters of the cell suspension per well of a 12-well plate or 400 microliters of the cell suspension per well of a 24-well plate containing the transfection mix.Incubate the plates in a cell culture incubator and do not disturb them for 24 hours. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin and return the cells to the incubator. Typical time points for detecting target knockdown after siRNA or miR delivery are 24 to 48 hours for mRNA and 48 to 96 hours for protein.Functional experiments can be performed on transfected adipocytes including insulin signaling, glucose uptake, adipokine secretion, lipolysis, and lipogenesis. The adipocytes have been shown to preserve their morphology after the transfection. Two days after transfection, the adipocytes presented multilocular lipid droplets with lipid content not different between the transfected and non-transfected adipocytes.The mRNA expression of various differentiation markers was unchanged in transfected cells compared to that in non-transfected adipocytes. The reverse-transfection protocol is efficient as more than 70%of the adipocytes were transfected. Three days after the transfection with siRNA against Perilipin-1, the mRNA level of Perilipin-1 had decreased by 70%and the protein level by 63%Perilipin-1 expression was also analyzed by fluorescence microscopy four days after the transfection and was found to have decreased by 92%compared to its expression in control adipocytes.The reverse-transfection of adipocytes with microRNA mimicking oligonucleotides to up-regulate the expression of miR-34a is shown here. The over-expression of miR-34a led to the decrease in VAMP2 protein expression by 50%The knockdown of Perilipin-1 in 3T3-L1 adipocytes led to an increase in basal lipolysis while the over-expression of miR-34a led to the inhibition of insulin-induced protein kinase B phosphorylation and glucose uptake. It's important to reach a high level of adipocytes differentiation to perform the transfection on newly differentiated adipocytes and to carefully monitor the treatment of adipocytes with trypsin.Following the transfection, it is possible to perform functional experiments on the adipocytes to study the impact of proteins or microRNA manipulation on insulin signaling, glucose uptake, lipogenesis, and lipolysis.

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