D glucose

Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed using HFD‐fed Wwp1 WT and KO mice at 13–15 weeks old. Prior to GTT and ITT, mice were fasted for 24 h. d‐glucose (1.0 g/kg body weight, Wako) or insulin (1.0 U/mL body weight, Wako) were injected intraperitoneally for GTT or ITT, respectively. Next, serial blood sampling from the tail vein was performed at 0, 30, 60, and 120 min after injection. Blood glucose levels were measured using an Accu‐chek^® aviva blood glucose meter (Roche).

Cerebral cortices (i.e., the dorsal pallium) were dissected manually under a microscope from E14.5 and E16.5 Bcl11b-IRES-EGFP knock-in mice (Figure 1C). Harvested cortices were gently dissociated into single cell suspensions by Neuron Dissociation Solution S (Wako, Japan) and resuspended in fluorescence activated cell sorting (FACS) Buffer [phenol-free, Ca²⁺Mg²⁺-free Hank’s balanced salt solution (HBSS; Invitrogen, Waltham, MA, United States)] containing 2% FBS (HyClone, United States), 20 mM D-glucose (Wako), and 50 mg/ml penicillin/streptomycin (Invitrogen). Samples were filtered through cell-strainer caps (35 μm mesh; BD Biosciences, Franklin Lakes, NJ, United States) into FACS Buffer. The cells were analyzed and sorted by a FACS AriaIII cell sorter and FACSDiva software (BD Biosciences). A 100 μm ceramic nozzle (BD Biosciences) with a sheath pressure of 20–25 psi and an acquisition rate of 1500–3000 events/s was used for the sorting. Gates were set as follows. A positive gate was set so that less than 0.1% of events exceeded the threshold in GFP-negative population samples, and a negative gate was set so that less than 0.1% of GFP-positive cells were included in the analysis of the GFP-negative sorted cells (Figure 1D). Sorted cells were collected in Nerve Cell Culture Medium (DS Pharma Biomedical, Japan) and centrifuged for RNA extraction.

All blood samples were collected following an overnight fast. Blood glucose levels and insulin concentrations were measured using the commercial kits, Glucose CII test Wako kit (Mutarotase-GOD method, Wako Pure Chemical Industries, Osaka, Japan) and Morinaga Ultra-Sensitive Mouse/Rat Insulin ELISA kit (MIoBS, Yokohama, Japan), respectively. For the intraperitoneal glucose tolerance test (ipGTT), mice were subjected to fasting for 16 h, and intraperitoneally injected with 1.5 g/kg of D-glucose (Wako). Blood samples were collected before and then sequentially after injection. The ipGTT tests were done on the same day for mice from the same age group of both diet groups.

Twenty μL of bacterial culture was added to 2 mL of BHI containing 1% D-glucose (Wako, Osaka, Japan) in 6-well plates (Thermo Fisher Scientific, NY, USA). After incubation, media were discarded and the wells were washed four times using physiological saline. White patches left on the bottom surfaces of the wells after washing were considered “biofilm.”

d-Allulose was provided by Kagawa University Rare Sugar Research Center and Matsutani Chemical Industry Co. Ltd. The purity of d-allulose was higher than 98%. d-glucose, d-mannitol, and lithium chloride were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). GLP-1(7-36 amide) and oxytocin were from Peptide Institute (Osaka. Japan). Porcine insulin was from Sigma (MO), and exendin(9-39) (Ex(9-39)) was from Abgent (CA).