Live-Cell Förster Resonance Energy Transfer Imaging of Metabolically Regulated Akt Activation Dynamics in HepG2 Cells

Summary

Here, we present a protocol to quantify the spatiotemporal dynamics of Akt activation and phosphorylation in live HepG2 cells. Förster resonance energy transfer (FRET) imaging is a powerful tool that provides valuable insights into insulin signaling pathways and metabolic regulation in cancer cells.

Abstract

Metabolically regulated Akt activation is a critical node in the insulin signaling cascade and provides valuable insights into the relationship between diabetes and cancer. To precisely quantify Akt activity in HepG2 cells, we developed a robust, reproducible protocol utilizing Förster Resonance Energy Transfer (FRET) with genetically encoded Akt-specific biosensors. This protocol outlines detailed steps for cell culture, imaging dish preparation, and transfection of HepG2 cells to express FRET-based biosensors, alongside specific guidelines for laser scanning confocal microscope hardware and software configuration. The results demonstrated unique patterns of insulin signaling in HepG2 cells, which exhibit an irreversible switch characterized by constitutive Akt activation with a defined switch-on threshold but no switch-off threshold. In contrast, myotubes display a reversible switch. The persistent Akt activation in HepG2 cells suggests mechanisms underlying insulin resistance and metabolic dysregulation in hepatic cells, with broader implications for understanding the progression of metabolic disorders and cancer. This protocol offers a valuable framework for exploring Akt-related signaling pathways and cellular behaviors across various disease contexts.

Introduction

Diabetes mellitus poses a major global health challenge, characterized by insulin resistance and impaired glucose homeostasis¹. A comprehensive understanding of insulin signaling pathways is crucial for elucidating the pathophysiology of this disease, as insulin plays a pivotal role in glucose metabolism, cell growth, and survival². Numerous studies have demonstrated that insulin signaling significantly impacts various cancers, linking insulin resistance to tumor progression and poor patient outcomes^3,4,5,

Protocol

An overview of the experimental steps involved in FRET live-cell imaging to monitor Akt phosphorylation in single HepG2 cells is shown in Figure 1.

1. Plasmid acquisition, propagation, and purification

NOTE: This section outlines the essential steps for acquiring, amplifying, and purifying the plasmid required for single-cell FRET analysis.

1. Utilize the pEevee-iAkt-NES-YPet plasmid (Figure 2A).
   NOTE: The plasmid was generously provided by Prof. Kazuhiro Aoki at the National Institute for Basic Biology (NIBB), Japan.Plasmi....

Results

To investigate Akt activation in HepG2 cells, the cells were seeded onto pre-coated imaging dishes and transfected with the FRET-based biosensor pEevee-iAkt-NES (Figure 2A), designed to enable real-time monitoring of Akt phosphorylation. Following transfection, the cells underwent serum starvation for 4 h in a serum-free medium to synchronize their metabolic state and minimize basal insulin signaling.

The cells were subsequently exposed to varying insulin concentr.......

Discussion

The protocol for live-cell FRET imaging to monitor Akt phosphorylation in HepG2 cells involves several key steps to ensure reliable and reproducible results. The first critical step is cell culture, which includes routine cell maintenance, coating of imaging dishes, and cell seeding. Proper coating is essential for cell attachment during time-lapse imaging experiments, as it ensures stable cell adherence, prevents detachment, and minimizes drift, which can lead to inconsistent data^9,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA	Gibco	Cat#25200-056	Use ice-cold PBS for cell wash
15 mm glass bottom cell culture dish	NEST	Cat#801001
2 mL Nalgene cryogenic vials	Thermo Scientific	Cat#5012-0020
5 mL Stripette Serological Pipets	Corning	Cat#4487
95% Ethanol	Kermel	Cat#C028005
A1 HD25/A1R HD25 confocal microscope	Nikon	https://www.nikon.com/	Magnification: 40×, Numerical Aperture (NA): 1.30, Pixel Dwell Time: 2.4 ms, Pixel Size: 1024
Ampicillin	Sigma-Aldrich	Cat#A9393
Bovine serum albumin (BSA)	VWR Life Science	Cat#N208-10g
Corning 25 cm2 rectangular culture flasks	Corning	Cat#430639
Countess 3 automated cell counter	Thermo Scientific	http://www.thermofisher.com/#AMQAX2000
Countess cell counting chamber slides	Thermo Scientific	http://www.thermofisher.com/#C10228
Digital vortex mixers	Thermo Scientific	https://www.thermofisher.com/
Dimethyl sulfoxide	Sigma-Aldrich	Cat#D2650
Eppendorf Safe-Lock Tubes 1.5 mL	Eppendorf	Cat#022363204
EZ-PCR mycoplasma detection kit	Biological Industries	Cat# 20-700-20
Fetal Bovine Serum, qualified, Australia	Gibco	Cat#10099141
GlutaMAX Supplement	Gibco	Cat#35050061
GraphPad Prism 9	GraphPad Software	https://www.graphpad.com/
HepG2	National Collection of Authenticated Cell Cultures	#CSTR:19375.09.3101HUMSCSP510  http://www.cellbank.org.cn/
Immersion Oil Type 37	Cargille Laboratories	Cat #16237
Insulin	Sigma-Aldrich	Cat#I5500-50MG	Warm to 37 °C before use
LB Broth (1x)	Invitrogen	Cat#10855001
Minimum Essential Medium (MEM)	Gibco	Cat#11095080	Warm to 37 °C before use
mySPIN 12 mini centrifuge	Thermo Scientific	https://www.thermofisher.com/
NanoDrop One	Thermo Scientific	https://www.thermofisher.com
Nikon Plan Fluor 40×/1.30 Oil Lens	Nikon	https://www.nikon.com/
NIS-Elements-AR	Nikon	https://www.nikon.com/
Non-Essential Amino Acids (NEAA) (100x)	Gibco	Cat#11140050
One Shot LB Agar Plates	Invitrogen	Cat#A55802
One Shot Stbl3 chemically competent E. coli	Invitrogen	Cat#C737303
Parafilm	PARAFILM	Cat#B8R05606
PBS (phosphate buffered saline)	Gibco	Cat#10010023
pEevee-iAkt-NES (7,033 bp)	Miura et al31	https://benchling.com/s/seq-q46zFYCfl0swLAun0t28/edit
Penicillin-streptomycin	Gibco	Cat#15070063
Plasmocin prophylactic	InvivoGen	Cat#ant-mpp
Poly-L-Lysine Hydrobromide	Sigma-Aldrich	Cat#P4832
Precision general purpose baths	Thermo Scientific	https://www.thermofisher.com/
QIAprep spin miniprep kit	QIAGEN	Cat#27106
SnapGene	SnapGene by Dotmatics	https://www.snapgene.com
Sodium Pyruvate (100 mM)	Gibco	Cat#11360070
Syringe filter unit, 0.22 μm	Millipore	Cat#SLGP033RS
Tokai Hit stage top incubator	TOKAI HIT	https://www.tokaihit-livecell.com/stagetopincubator
UltraPure DNase/RNase-free distilled water	Invitrogen	Cat#10977015
Xfect Transfection Reagent	Takara Bio	Cat#631317