Name: Live-Cell Förster Resonance Energy Transfer Imaging of Metabolically Regulated Akt Activation Dynamics in HepG2 Cells (Video) | JoVE Journal
Uploaded: 2025-05-23T13:50:00.000+00:00
Duration: 8 min 3 s
Description: Learn FRET imaging to visualize Akt activation dynamics in live HepG2 cells for studying insulin signaling and metabolic regulation in cancer. This protocol enables real-time analysis of phosphorylation events.

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5 minutes

This work was partly supported by the Natural Science Foundation of Shenzhen (JCYJ20240813113606009), the Shenzhen-Hong Kong Cooperation Zone for Technology and Innovation (HZQB-KCZYB-2020056), National Natural Science Foundation of China (32070681), National Key R&#38;D Program of China (2019YFA0906002), and Shenzhen Peacock Plan (KQTD2016053117035204).

The authors declare no competing interests.

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 data9,<su...

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 outcomes3,4,5,6. HepG2 cells, a commonly used hepatocellular carcinoma cell line, serve as a valuable model for studying insulin resistance and the interplay between metabolic dysregulation and cancer development7. Traditionally, researchers have viewed insulin responses as graded; however, recent studies have revealed that individual cells can exhibit bistable responses, displaying salient transitions between unresponsiveness and full response occurring at specific insulin concentration thresholds8,9.
F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for studying the spatio-temporal distribution of biomolecules in living cells10. By extracting information from molecular dynamics, FRET provides insights into processes such as Akt activation in real time, making it an invaluable technique for studying living cells11,12. This imaging method has proven essential in studying cellular dynamics, particularly in metabolic diseases and cancer, where precise molecular interactions are crucial13. FRET also enables real-time monitoring of molecular interactions, shedding light on mechanisms such as insulin resistance and tumor progression14,15. FRET biosensors are crucial in cancer research for studying tumor microenvironments, drug resistance, and metabolic disorders16. FRET detection methods, such as sensitized emission (SE), acceptor bleaching (AB), fluorescence lifetime imaging microscopy (FLIM), and spectroscopy, each offer distinct advantages to quantify molecular interactions17. SE measures energy transfer between donor and acceptor fluorophores, resulting in a measurable shift in emission spectra that correlates with the proximity of interacting biomolecules18. AB uses selective photobleaching of the acceptor fluorophore and tracks changes in donor fluorescence, which allows researchers to assess interaction kinetics and distances19. FLIM evaluates fluorescence decay rates of the donor fluorophore, directly influenced by FRET efficiency, to provide precise nanoscale measurements of molecular interactions20.
Using FRET techniques, we recently demonstrated bistable insulin responses in C2C12-derived myotubes8,9,21,22,23,24. The distinct switch-on and switch-off thresholds for Akt activation, as we discovered, suggest that the graded whole-body insulin dose-response belies the complexity of the subcellular signaling cascade starting from insulin stimulus, which culminates in an all-or-none response at the single-cell level21,22,23,24. To test the presence of bistability in other cell types, we stimulated HepG2 cells with insulin and recorded their response using single-cell FRET imaging. We stimulated HepG2 cells with varying insulin concentrations and monitored Akt activity at the single-cell level using an Akt biosensor. The Akt biosensor comprises enhanced cyan fluorescent protein (ECFP)25 as the donor fluorophore and the brightest variant of yellow fluorescent protein (YPet)26 as the acceptor fluorophore, linked by an Eevee linker containing the peptide sequence SGRPRTTTFADSCKP. This peptide acts as a substrate for phosphorylated Akt (pAkt), optimized from human glycogen synthase kinase 3&#946; (GSK3&#946;). In its unphosphorylated state, the spatial separation between the donor and acceptor fluorophores exceeds the F&#246;rster radius, which inhibits energy transfer. Upon insulin stimulation, Akt phosphorylation occurs and leads to the phosphorylation of SGRPRTTTFADSCKP. This process induces a conformational change that brings the donor and acceptor within the F&#246;rster radius, enabling FRET27. As a result, the FRET signal intensity correlates with the amount of phosphorylated Akt molecules and allows real-time quantification of insulin-mediated cellular responses.
This protocol, initially developed to study insulin signaling in C2C12-derived myotubes, has been successfully applied to HepG2 cells and utilized across different hardware and software platforms, thus demonstrating its applicability, adaptability, and versatility. HepG2 cells exhibit constitutive Akt activity, which makes them an ideal in vitro model to study liver-specific insulin signaling and metabolic processes. The key features of the protocol are described step-by-step in the protocol section.

<table><tbody><tr><td>0.25% trypsin-EDTA</td><td>Gibco</td><td>Cat#25200-056</td><td>Use ice-cold PBS for cell wash</td></tr><tr><td>15 mm glass bottom cell culture dish</td><td>NEST</td><td>Cat#801001</td><td></td></tr><tr><td>2&amp;nbsp;mL Nalgene cryogenic vials</td><td>Thermo Scientific</td><td>Cat#5012-0020</td><td></td></tr><tr><td>5 mL Stripette Serological Pipets</td><td>Corning</td><td>Cat#4487</td><td></td></tr><tr><td>95% Ethanol</td><td>Kermel</td><td>Cat#C028005</td><td></td></tr><tr><td>A1 HD25/A1R HD25 confocal microscope</td><td>Nikon</td><td>https://www.nikon.com/</td><td>Magnification: 40&amp;times;, Numerical Aperture (NA): 1.30, Pixel Dwell Time: 2.4 ms, Pixel Size: 1024</td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>Cat#A9393</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>VWR Life Science</td><td>Cat#N208-10g</td><td></td></tr><tr><td>Corning 25&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt; rectangular culture flasks</td><td>Corning</td><td>Cat#430639</td><td></td></tr><tr><td>Countess 3 automated cell counter</td><td>Thermo Scientific</td><td>http://www.thermofisher.com/#AMQAX2000</td><td></td></tr><tr><td>Countess cell counting chamber slides</td><td>Thermo Scientific</td><td>http://www.thermofisher.com/#C10228</td><td></td></tr><tr><td>Digital vortex mixers</td><td>Thermo Scientific</td><td>https://www.thermofisher.com/</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>Cat#D2650</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tubes 1.5&amp;nbsp;mL</td><td>Eppendorf</td><td>Cat#022363204</td><td></td></tr><tr><td>EZ-PCR mycoplasma detection kit</td><td>Biological Industries</td><td>Cat# 20-700-20</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, Australia</td><td>Gibco</td><td>Cat#10099141</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>Cat#35050061</td><td></td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad Software</td><td>https://www.graphpad.com/</td><td></td></tr><tr><td>HepG2</td><td>National Collection of Authenticated Cell Cultures</td><td>#CSTR:19375.09.3101HUMSCSP510&amp;nbsp; http://www.cellbank.org.cn/</td><td></td></tr><tr><td>Immersion Oil Type 37</td><td>Cargille Laboratories</td><td>Cat #16237</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>Cat#I5500-50MG</td><td>Warm to 37 &amp;deg;C before use</td></tr><tr><td>LB Broth (1x)</td><td>Invitrogen</td><td>Cat#10855001</td><td></td></tr><tr><td>Minimum Essential Medium (MEM)</td><td>Gibco</td><td>Cat#11095080</td><td>Warm to 37 &amp;deg;C before use</td></tr><tr><td>mySPIN 12&amp;nbsp;mini centrifuge</td><td>Thermo Scientific</td><td>https://www.thermofisher.com/</td><td></td></tr><tr><td>NanoDrop One</td><td>Thermo Scientific</td><td>https://www.thermofisher.com</td><td></td></tr><tr><td>Nikon Plan Fluor 40&amp;times;/1.30 Oil Lens</td><td>Nikon</td><td>https://www.nikon.com/</td><td></td></tr><tr><td>NIS-Elements-AR</td><td>Nikon</td><td>https://www.nikon.com/</td><td></td></tr><tr><td>Non-Essential Amino Acids (NEAA) (100x)</td><td>Gibco</td><td>Cat#11140050</td><td></td></tr><tr><td>One Shot LB Agar Plates&amp;nbsp;</td><td>Invitrogen</td><td>Cat#A55802</td><td></td></tr><tr><td>One Shot Stbl3 chemically competent E.&amp;nbsp;coli</td><td>Invitrogen</td><td>Cat#C737303</td><td></td></tr><tr><td>Parafilm</td><td>PARAFILM</td><td>Cat#B8R05606</td><td></td></tr><tr><td>PBS (phosphate buffered saline)</td><td>Gibco</td><td>Cat#10010023</td><td></td></tr><tr><td>pEevee-iAkt-NES (7,033&amp;nbsp;bp)</td><td>Miura et&amp;nbsp;al&lt;sup&gt;31&lt;/sup&gt;</td><td>https://benchling.com/s/seq-q46zFYCfl0swLAun0t28/edit</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>Cat#15070063</td><td></td></tr><tr><td>Plasmocin prophylactic</td><td>InvivoGen</td><td>Cat#ant-mpp</td><td></td></tr><tr><td>Poly-L-Lysine Hydrobromide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Cat#P4832</td><td></td></tr><tr><td>Precision general purpose baths</td><td>Thermo Scientific</td><td>https://www.thermofisher.com/</td><td></td></tr><tr><td>QIAprep spin miniprep kit</td><td>QIAGEN</td><td>Cat#27106</td><td></td></tr><tr><td>SnapGene</td><td>SnapGene by Dotmatics</td><td>https://www.snapgene.com</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Gibco</td><td>Cat#11360070</td><td></td></tr><tr><td>Syringe filter unit, 0.22&amp;nbsp;&amp;mu;m</td><td>Millipore</td><td>Cat#SLGP033RS</td><td></td></tr><tr><td>Tokai Hit stage top incubator</td><td>TOKAI HIT</td><td>https://www.tokaihit-livecell.com/stagetopincubator</td><td></td></tr><tr><td>UltraPure DNase/RNase-free distilled water</td><td>Invitrogen</td><td>Cat#10977015</td><td></td></tr><tr><td>Xfect Transfection Reagent</td><td>Takara Bio</td><td>Cat#631317</td><td></td></tr></tbody></table>

live-cell f&#246;rster resonance energy transfer imaging of metabolically regulated akt activation dynamics in hepg2 cells

Live-Cell F&#246;rster Resonance Energy Transfer Imaging of Metabolically Regulated Akt Activation Dynamics in HepG2 Cells

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 F&#246;rster 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.

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...

Here, we present a protocol to quantify the spatiotemporal dynamics of Akt activation and phosphorylation in live HepG2 cells. F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool that provides valuable insights into insulin signaling pathways and metabolic regulation in cancer cells.

Metabolically regulated Akt activation is a critical node in the insulin signaling cascade and provides valuable insights into the relationship between ...

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Learn FRET imaging to visualize Akt activation dynamics in live HepG2 cells for studying insulin signaling and metabolic regulation in cancer. This protocol enables real-time analysis of phosphorylation events.

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

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