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

Summary

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.

Abstract

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.

Introduction

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 T2D^2,3. 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.

Protocol

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

1. Grow the 3T3-L1 fibroblasts in 100 mm dishes in culture medium-DMEM without pyruvate, 25 mM glucose, 10% newborn calf serum, and 1% penicillin and streptomycin (Figure 1A). Place the dishes in a tissue culture incubator (7% CO₂ and 37 °C).
2. Two days after confluence, change the culture medium, replacing with DMEM without pyruvate, 25 mM glucose, 10% fetal calf serum (FCS), and 1% penicillin and streptomycin supplemented with 0.25 mM 3-Isobutyl-1-methylxanthine (IBMX), 0.25 µM dexamethasone, 5 µg/mL insulin, and 10 µM rosiglitazone.
   NOTE: It takes 5 days to reach confluency when the cells are seeded at 300,000 cells per 100 mm dish.
3. Two days later, replace the culture medium with DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin supplemented with 5 µg/mL insulin and 10 µM rosiglitazone and incubate for 2 days. Then, feed the cells every 2 days with DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin (Figure 1B).
4. Transfect the 3T3-L1 adipocytes 7-8 days after the beginning of the differentiation protocol.
   ​NOTE: It is important to reach a high level of differentiation (>80%) before the transfection to avoid the proliferation of the remaining fibroblasts after the transfection, which would lead to a mixed population of cells that might bias the results.

2. Preparation of precoated plates

1. On the day before or a few hours before the transfection, prepare a solution of collagen type I at 100 µg/mL in 30% ethanol from a stock solution at 1 mg/mL. Add 250 µL of collagen per well of a 12-well plate and 125 µL per well of a 24-well plate, and spread the solution over the surface of the well.
2. Leave the plate without the lid under the culture hood until the collagen dries. Wash twice with Dulbecco's phosphate-buffered saline (D-PBS).
   ​NOTE: Precoated plates are available for purchase.

3. Preparation of the transfection mix

NOTE: The final concentration of siRNA is between 1 and 100 nM (1 to 100 pmol of siRNA per well of a 12-well plate). The final concentration of the miR mimic is 10 nM (10 pmol/well). Determine the best concentration of each siRNA, miR mimic, or other oligonucleotide prior to starting the experiment to avoid off-target effects. Perform transfection experiments in triplicate to facilitate statistical analysis of the results. Prepare all reagents in excess to account for normal loss during pipetting.

1. Mix by pipetting (volume/volume) the siRNA (or other oligonucleotides) with improved Minimal Essential Medium (Table 1). Incubate for 5 min at room temperature.
2. Add the transfection reagent and the improved Minimal Essential Medium to the siRNA, and pipet to mix (Table 1). Incubate for 20 min at room temperature (during this time, proceed to section 4). Add the transfection mix to each well of the collagen-coated plate.

4. Preparation of the 3T3-L1 adipocytes

1. Wash the cells in the 100 mm Petri dish twice with D-PBS. Add 5x trypsin to the cells (1 mL per 100 mm dish), making sure to cover all of the surface with the trypsin. Wait for 30 s and carefully remove the trypsin.
2. Incubate the Petri dish for 5-10 min at 37 °C in the incubator. Tap the 100 mm dish to detach the cells.
3. Add 10 ml of DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin to neutralize the trypsin. Carefully pipet the medium up and down to detach the cells and homogenize the cell suspension.
4. Count the cells using a Malassez counting chamber or an automated cell counter, and adjust the concentration of the cells to 6.25 x 10⁵ cells/mL of medium. Seed 800 µL of the cell suspension/well of a 12-well plate (5 x 10⁵ cells) or 400 µl of the cell suspension/well of a 24-well plate (2.5 x 10⁵ cells) containing the transfection mix.
   NOTE: One 100 mm Petri dish of adipocytes will allow the preparation of one 12-well plate or one 24-well plate. A 100 mm dish usually contains 6-7 x 10⁶ adipocytes, which correspond to 5 x 10⁵ adipocytes per well of a 12-well plate.
5. Incubate the plates in a cell culture incubator (7% CO₂ and 37 °C), and do not disturb the cells for 24 h. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin.
   ​NOTE: It is also possible to seed the cells into collagen-precoated 48- and 96-well plates but take more precautions when replacing the media to avoid detachment of the adipocytes.

5. Functional analysis of transfected 3T3-L1 adipocytes

1. Study target knockdown 24-48 h and 48-96 h after siRNA or miR mimic delivery for mRNA and protein, respectively.
2. Perform functional analyses of transfected adipocytes to study insulin signaling, glucose uptake, adipokine secretion, lipolysis, and lipogenesis.

Results

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...

Discussion

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...

Materials

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