L alanyl l glutamine

B16F10 (mouse melanoma; from the Cell Resource Center for Biomedical Research Institute of Development, Japan), MC38 (mouse colon carcinoma; kindly provided by Dr. H. Tahara, The Institute of Medical Science, University of Tokyo), LLC (mouse lung carcinoma; kindly provided by Dr. M. Sakata-Yanagimoto, Department of Hematology, University of Tsukuba) and MB49 (mouse bladder carcinoma; kindly provided by Dr. W.T. Godbey, Department of Chemical & Biomolecular Engineering, Tulane University) were cultivated in DMEM (Thermo Fisher) with 100 mM sodium pyruvate (Wako), 1% MEM nonessential amino acids solution (Wako), and 100 mM L-alanyl-L-glutamine (Wako), 4T1 (American Type Culture Collection) was cultivated in RPMI 1640 (Wako) at 37°C under 5% CO2. These media were supplemented with 10% FBS and 1% penicillin-streptomycin (Wako),

The INS1E cell line, a gift from Prof. Pierre Maechler,¹⁴ (link) was maintained in RPMI-1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Gibco), 0.5 mM monothioglycerol (Antioxidant, Wako, Osaka, Japan), 2 mM L-alanyl-L-glutamine (Wako), and 50 μg/ml gentamicin sulfate (Wako). Rat pancreatic fibroblasts were cultured under the same conditions as INS1E cells. Rat neonatal pancreases were collected and enzymatically digested in a collagenase/proteinase cocktail (0.1% collagenase L, Nitta Gelatin, Osaka, Japan, 0.2% dispase II, Gibco in HBSS, Gibco) for 60 mins in a reciprocating water bath shaker at 37°C. Undigested debris was removed using a nylon mesh and the cells were cultured as described below. Attached fibroblasts proliferated continuously and were sequentially passaged (sub-cultured in another dish), effectively diluting any contaminating islets and blood cells. Pancreatic fibroblasts exhibited cellular senescence after 3 to 6 passages, and were used before cell division had slowed down.

The rat insulinoma cell line INS-1E was grown in advanced RPMI-1640 medium (Thermo Fisher
Scientific Inc., Waltham, MA, USA) containing 5% FBS, 0.5 mM monothioglycerol (FUJIFILM
Wako Pure Chemical Corp.) and 2 mM L-alanyl-L-glutamine (FUJIFILM Wako Pure Chemical
Corp.) [18 (link)]. Cells were seeded in 24-well plates (1
× 10⁵ cells per well) and cultured for 2 days at 37°C in 5% CO₂. The
medium was removed and INS-1E cells were preincubated for 1 h (5% CO₂, 37°C) in
RPMI-1640 (glucose free; FUJIFILM Wako Pure Chemical Corp.) with 5% FBS and 100 mg/dl
glucose. Next, the cells were incubated for 1 h in a fresh batch of the same composition
medium with added 1,5-AG or TI. To determine the effects of 1,5-AG and TI on insulin
secretion, INS-1E cells were incubated in RPMI-1640 containing various concentrations of
1,5-AG (0, 10, 100 and 1,000 µM) and TI (0, 1, 10 and 100
µg/ml). Samples were collected in Protein LoBind^® tubes and
insulin in the medium was determined with a Rat Insulin ELISA Kit (Morinaga Institute of
Biological Science, Inc.) according to the manufacturer’s protocol. To each well,
200-µl of M-PER^TM Mammalian Protein Extraction Reagent
(Thermo Fisher Scientific Inc.) was added to dissolve cells for the determination of
cellular protein content with a Pierce^TM BCA Protein Assay Kit (Thermo Fisher
Scientific Inc.).