Materials and Reagents

Primary mouse hepatocytes
Medium199 (Life Technologies, Invitrogen™, catalog number: 11150-059 )
Fetal bovine serum (FBS)
bicinchoninic acid (BCA)
Penicillin-Streptomycin, Liquid (Life Technologies, Invitrogen™, catalog number: 15140-122 )
PBS^+/+ (Sigma-Aldrich, catalog number: D8662 )
Dulbecco’s modified eagle’s medium (DMEM), without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate, powder (Sigma-Aldrich, catalog number: D5030 )
Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
Sodium L-lactate (Sigma-Aldrich, catalog number: L7022 )
Sodium pyruvate (Life Technologies, Invitrogen™, catalog number: 11360-070 )
100 mM MEM sodium pyruvate solution (100x), liquid (Life Technologies, Invitrogen™, catalog number: 11360-070 )
200 mM L-Glutamine (100x), liquid (Life Technologies, Invitrogen™, catalog number: 25030-081 )
1 M HEPES buffer solution (Life Technologies, Invitrogen™, catalog number: 15630-080 )
pCPT-cAMP (Sigma-Aldrich, catalog number: C3912 )
Insulin (Sigma-Aldrich, catalog number: I9278 )
Autokit glucose (Wako, catalog number: 439-90901 )
BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23227 )
HEPES
Glucose production buffer (see Recipes)

Equipment

iMark Microplate Absorbance Reader (Bio-Rad, catalog number: 168-1135 )
BD BioCoat^™Collagen I 6-well Plates (BD Biosciences, catalog number: 356400 )

Procedure

Primary mouse hepatocytes were cultured in BD BioCoat^™Collagen I 6-well plates (1 x 10⁶ cells per well) in Medium199 supplemented with 5% FBS, penicillin (100 units/ml) and streptomycin (100 μg/ml).
6-48 h after plating, serum-starved overnight in 2 ml/well of Medium199 supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml) without FBS.
Wash the cells by 2 ml/well of warm (37 °C) PBS^+/+ twice.
Replace PBS^+/+with 1 ml of glucose production buffer consisting of glucose-free DMEM (pH 7.4) without phenol red supplemented with 20 mM sodium lactate, 2 mM sodium pyruvate, 2 mM L-glutaminie and 15 mM HEPES.
Incubate cells at 37 °C for 6 h with or without 0.1 mM pCPT-cAMP and/or 100 mM insulin, 0.2 ml of medium was collected and the glucose concentration was measured with a colorimetric glucose assay kit.
Collect 0.2 ml of medium from each well.
Measure the glucose concentration with a colorimetric glucose assay kit following manufacturer’s instruction.
Normalized the readings to the total protein content determined from the whole-cell lysates by bicinchoninic acid (BCA) protein assay kit following manufacturer’s instruction.

Recipes

Glucose production buffer (Please note some component come with the DMEM as noted in paresis)
Components (g/L)
L-Arginine^.HCl 0.084 (contained in DMEM)
L-Cystine^.2HCl 0.0626 (contained in DMEM)
Glycine 0.030 (contained in DMEM)
L-Histidine^.HCl^.H₂O 0.042 (contained in DMEM)
L-Isoleucine 0.105 (contained in DMEM)
L-Leucine 0.105 (contained in DMEM)
L-Lysine^.HCl 0.146 (contained in DMEM)
L-Methionine 0.03 (contained in DMEM)
L-Phenylalanine 0.066 (contained in DMEM)
L-Serine 0.042 (contained in DMEM)
L-Threonine 0.095 (contained in DMEM)
L-Tryptophan 0.016 (contained in DMEM)
L-Tyrosine^.2Na^.2H₂O 0.10379 (contained in DMEM)
L-Valine 0.094 (contained in DMEM)
Choline chloride 0.004 (contained in DMEM)
Folic acid 0.004 (contained in DMEM)
Myo-Inositol 0.0072 (contained in DMEM)
Niacinamide 0.004 (contained in DMEM)
D-Pantothenic acid (Hemicalcium) 0.004 (contained in DMEM)
Pyridoxal^.HCl 0.004 (contained in DMEM)
Riboflavin 0.0004 (contained in DMEM)
Thiamine^.HCl 0.004 (contained in DMEM)
Calcium chloride (anhydrous) 0.2 (contained in DMEM)
Ferric nitrate^.9H₂O 0.0001 (contained in DMEM)
Magnesium sulfate (Anhydrous) 0.09767 (contained in DMEM)
Potassium chloride 0.4 (contained in DMEM)
Sodium chloride 6.4 (contained in DMEM)
Sodium phosphate monobasic (Anhydrous) 0.109 (contained in DMEM)
L-Glutamine 0.584
Sodium bicarbonate 3.7
Sodium lactate 2.24
Sodium pyruvate 0.22
HEPES 3.575
Adjust pH to 7.3, filter using a 0.45 μm filter and store at 4 °C.

Acknowledgments

This protocol was adapted from Sakai et al. (2012). The development of this protocol was supported by a Grant-in-Aid for Creative Scientific Research (to M.K.) and a Grant-in-Aid for Scientific Research (C) (21591155 to M.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a grant from the National Center for Global Health and Medicine (21S116 to M.M.), a grant from Takeda Science Foundation (to M.M.) and a Novo Nordisk Pharma Insulin Award (to M.M.).

References

Matsumoto, M., Pocai, A., Rossetti, L., Depinho, R. A. and Accili, D. (2007). Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver. Cell Metab 6(3): 208-216.
Sakai, M., Matsumoto, M., Tujimura, T., Yongheng, C., Noguchi, T., Inagaki, K., Inoue, H., Hosooka, T., Takazawa, K., Kido, Y., Yasuda, K., Hiramatsu, R., Matsuki, Y. and Kasuga, M. (2012). CITED2 links hormonal signaling to PGC-1alpha acetylation in the regulation of gluconeogenesis. Nat Med 18(4): 612-617.
Yoon, J. C., Puigserver, P., Chen, G., Donovan, J., Wu, Z., Rhee, J., Adelmant, G., Stafford, J., Kahn, C. R., Granner, D. K., Newgard, C. B. and Spiegelman, B. M. (2001). Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413(6852): 131-138.