Ad293 cells

Cell surface expression of MTLR (WT) and mutants was performed using AD-293 cells (Agilent) cultured in Dulbecco’s Modified Eagle’s Medium containing 10% (v/v) fetal bovine serum at 37°C in 5% CO₂. Cells grown to a density of 200,000 to 250,000 cells/ml were transiently transfected with WT or mutant plasmids of MTLR for 24 hours. The transfected cells were collected and blocked with 5% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 15 min at room temperature before incubating with primary anti-Flag antibody (diluted with PBS containing 5% BSA at a ratio of 1:150; ABclonal) for 1 hour at room temperature. Cells were washed three times with PBS containing 1% (w/v) BSA and then incubated with anti-mouse Alexa Fluor 488–conjugated secondary antibody diluting at a ratio of 1:1000 (Invitrogen) for 2 hours at 4°C in the dark. After another three washes, cells were suspended in 200 μl of PBS containing 1% BSA. The surface expression of the MTLR was monitored by detecting the fluorescent intensity of Alexa Fluor 488 using a BD Accuri C6 (excitation, 488 nm and emission, 519 nm). Data were analyzed by BD Accuri C6 software 1.0.264.

MIN6-K8 cells were established as described previously [24 (link)]. Got1 KO-1 and -2 cells were established by sub-cloning of MIN6-K8 cells transfected with Cas9 nickase and guide RNA pairs targeting mouse Got1 as described previously [25 (link)]. Got1 KO-1 and -2 cells refer to clones A60 and A64, in Murao et al. [25 (link)], respectively. MIN6-K8 and Got1 KO cells were cultured in Dulbecco's Modified Eagle Medium (high glucose) (DMEM-HG, Sigma) containing 4500 mg/L glucose supplemented with 10% fetal bovine serum (FBS) (BioWest) and 5 ppm 2-mercaptoethanol. For treatment with HG, LD, or 2DG (Figure 6A, Supplementary Table 5), the respective culture media were prepared by adding glucose, l-glutamine, and 2-deoxy-d-glucose (2DG) (Wako) to DMEM without glucose (Sigma) according to the manufacturer's instructions. AD293 cells were purchased from Agilent and cultured in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 5 ppm 2-mercaptoethanol. All cells were maintained at 37 °C with 5% CO₂.

Adenoviral shRNA expression constructs targeting mouse Nmnat2 mRNA and scramble shRNA were purchased from VectorBuilder. Adenoviruses were generated by transfection of the plasmid constructs into AD293 cells (Agilent) using Lipofectamine 2000 (Thermo). The viruses were collected according to the manufacturer's protocol and purified by using Vivapure AdenoPACK 20 (Sartorius).
For the measurement of insulin secretion and content, the cells were plated in 24-well plates at 2 × 10⁵ cells/well. For the other experiments, the cells were plated in 6-well plates at 4 × 10⁵ cells/well. Following a 4-day culture, the cells were infected with adenoviruses at multiplicity of infection (MOI) of 5. Forty-eight hours after the first infection, the culture medium was discarded and the cells were again infected with the same amount of viruses. Forty-eight hours after the second infection, the cells were subjected to analysis.