Hello all,
I am trying to study growth-regulating effects of Insulin in mammalian cells, by dual luciferase assays. I worked on HeLa cells but the results are not reproducible, even when I do the assay in the exact same way (experimental conditions, reagents, timings etc are all maintained constant between 2 experiments). This makes me wonder, do HeLa cells have insulin receptor to show a response to insulin?
I am looking for other mammalian cell lines that are best study insulin signaling. Many research papers suggested HepG2 cells, but I've heard they are very difficult to transfect (I have to transfect my luciferase reporter vector into the mammalian cells).
Although, I originally intended to work with HEK293 cells (which may be ideal for insulin studies involving transfection), when I aspirate media out of the 96 well plates to put in serum-free media (for serum starvation, to clear out the background signal and to control the amount of insulin in the system, which I add after 2h of serum starvation), the cells detach and the results are very haphazard. Kindly give your suggestions.
yes all mammalian cells do most likely to have GLUT4 receptors, though will have expression of others too
Amended on 25 February 2018
I have searched among my protein database for insulin receptor based upon the Edman degradation method in vain (PDMD method; please see file, HepG2 fucoidan).
However, Insulin-related proteins have been detected.
Hepatoma HepG2 (cultured without fucoidan) has Insulin receptor substrate 2/IRS-2 at 15.0 μg/mg cell protein. Interestingly, IRS-2 is present in the membrane sub-fraction as determined by the ultracentrifugation at 100,000 x g, 90 min, at 4 C.
Healed HepG2 (cultured with fucoidan) has IGF2R/CI Man-6-P receptor at 11.3 μg/mg cell protein. IGF2R of this cell comes to the soluble fraction.
Normal liver tissue (Pseudo liver-cancer) has Insulinoma-associated protein 1/Zinc finger protein IA-1 at 0.44 μg/mg tissue protein.
HCC tissue with PBC has Insulin-like growth factor-binding protein 5/IGFBP-5/IBP-5 at 3.7 μg/mg tissue protein.
LC tissue with leprosy has IGF2R at 21.8 μg/mg tissue protein. This protein comes to soluble fraction.
LC tissue (named as No.6) has Insulin-degrading enzyme/Insulinase/IDE at 4.0 μg/mg tissue protein.
HCC tissue (No.6) has Insulin-degrading enzyme/Insulinase/IDE at 13.3 μg/mg tissue protein and IGF2R at 27.0 μg/mg tissue protein. IGF2R of this tissue also comes to the soluble fraction.
Serum of 1y11mo girl (with excess biotin and anorexia) has Insulin-responsive sequence DNA-binding protein 1/SNED1/IRE-BP1/Sushi, nidogen and EGF-like domain-containing protein 1 at 2.4 μg/mg serum protein. The link between SNED1 and anorexia is not yet clear.
Serum of 12mo girl (with common cold) has Insulin-like growth factor 1A/IGF-I at 6.7 μg/mg serum protein. Her body temperature may be high by the common cold induced by co-infection of Helicobacter pylori J99 and Mycoplasma pulmonis.
Glucose transporting proteins are found as follows.
Normal liver tissue (Pseudo liver-cancer) has Sodium-glucose cotransporter/SLC5A1 at 11.2 μg/mg tissue protein.
LC tissue with leprosy has Solute carrier family 2, facilitated glucose transporter member 1/GLUT1/SLC2A1/Glucose transporter type 1, erythrocyte/brain at 4.9 μg/mg tissue protein.
LC tissue (No.6) has GLUT1/SLC2A1/Glucose transporter type 1, erythrocyte/brain at 2.1 μg/mg tissue protein.
HCC tissue (No.6) has Sodium/glucose cotransporter-like protein at 1.2 μg/mg tissue protein.
Serum of 1y11mo girl (with excess biotin and anorexia) has Sodium/myo-inositol cotransporter 2 at 11.1 μg/mg serum protein.
It is interesting that the another growth-factor of biotin (vitamin H) than insulin seems to be strongly linked to the expression of insulin-related proteins as has been shown in the serum of 1y11mo girl (with excess biotin and anorexia).
By the way, I have recently added biotin (5.0 μg/mL) to the culture medium of Caco-2 cells, since biotin is a low price drug than insulin (please see file; JCB Fucoidan transport). Biotin has growth promoting effect onto other cultured cell lines such as Rat TRL1215, Human Hc, HuH-6, HeLa, HuH-7, HepG2, JMSU1, 293T cells (our unpublished observation at The Gunma University, Maebashi, Gunma, Japan), however fungal contamination is usually occurred in this biotin-added medium. To prevent fungal contamination, the addition of Amphotericin B becomes to be necessary to the biotin-added medium.
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