Insulin

As a microscopic assay for the insulin-stimulated cell surface expression of GLUT4, FlagGLUT4-mCherry/IR-T7 transgenic CHO-K1 cells were washed twice with phosphate-buffered saline (PBS), and the cells were further incubated with Ham's F-12 medium (without serum) for 3 h. Subsequently, the cells were exposed to 1 μg/ml insulin (Wako) for 30 min as indicated. After insulin stimulation, the cells were chilled immediately on ice. The cells were fixed with 4% paraformaldehyde for 30 min on ice and treated with 50 mM glycine at 4°C for 10 min as a quenching procedure. Cells were then reacted with anti-Flag M2 monoclonal IgG as a primary antibody, following incubation with Alexa Fluor 488 goat anti-mouse IgG as a secondary antibody. Note that the plasma membrane was intentionally left non-permeabilized in this assay in order to detect cell surface GLUT4 exclusively. Cell surface immunofluorescent images were obtained using a BZ-X700 fluorescence microscope (Keyence). Cell number was measured using the Hybrid Cell Count software of BZ-Analyzer. ImageJ 1.45s (National Institutes of Health) was used for image processing. Statistical significance was calculated by the chi-square test. To observe the nucleus, cells were treated with 2.5 μg/ml Hoechst 33342.

MCF10A cells (ATCC) were cultured in DMEM/F12 (Nacalai Tesque) supplemented with 5% horse serum (Gibco), 20 ng/ml EGF (PeproTech), 0.5 µg/ml hydrocortisone (Sigma), 10 µg/ml insulin (Wako), 100 ng/ml Cholera toxin (Bio Academia), and a 1% penicillin-streptomycin solution (Nacalai Tesque). MCF7, T47D, MDA-MB-231 cells (ATCC) and Hs578T cells (JCRB) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). BT-549 cells (JCRB) were cultured in RPMI1640 (Nacalai Tesque) containing 10% FBS, 0.023 U/ml insulin (Wako), and a 1% penicillin-streptomycin solution (Nacalai Tesque). Normal human epidermal keratinocyte (NHEK) cells were purchased from PromoCell and cultured in keratinocyte growth medium 2 (PromoCell). The cells were maintained in a CO₂ incubator at 37°C and 5% CO₂. Recombinant human TGF-β1 (CHO derived) was purchased from PeproTech.

White adipocyte differentiation of hASCs was performed using the DMEM/F12-based medium as previously described according to the regularly used protocol with slight modifications to ensure successful adipocyte induction [62 ]. Adipogenic differentiation was initiated on the second day after cell confluence, followed by a 7-day induction period and a 14-day maintenance period. The induction cocktail contained 100 nM insulin (093-06351, Wako, Japan), 1 μM dexamethasone (10008980, Cayman Chemical, Ann Arbor, MI, USA), 0.5 mM IBMX (10008978, Cayman Chemical, USA), and 1 μM rosiglitazone. For the maintenance medium, 100 nM insulin and 1 μM dexamethasone were prepared with a DMEM-F12-based medium. To generate white adipocytes, hASCs were seeded at a density of 5 × 10⁴ cells per cm² and grown to confluence. Both the induction and maintenance medium was subsequently changed every 4 days for a total of 21 days.
For induced adipocytes from d-hASCs, the cells were seeded in the preadipocyte growth medium (PGM-2 ^TM Bulletkit, Lonza, Walkersville, MD, USA). When the cells reach 80% confluency, change the growth medium to adipogenic differentiation medium to induce adipogenicity. Rhinsulin, dexamethasone, IBMX, and indomethacin were added to the adipogenic differentiation medium. Refresh the medium every 10 to 12 days [14 (link)].

Reporter cell lines that respond to the expression and transcriptional activity of c-Myc (E-H1) or HNF1B (D-D1) derived from NMuMG cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), supplemented with 1% insulin (FUJI FILM Wako Pure Chemical, Osaka, Japan) and 0.5% penicillin-streptomycin (P/S; Invitrogen, Carlsbad, CA, USA) [29 (link)]. The E-H1 and D-D1 cells express c-Myc and HNF1B under the Tet-ON system and the fluorescent protein monomeric Keima (mKeima). Since the ORF of mKeima was inserted after the c-Myc ORF or HNF1B following the internal ribosomal entry site, c-Myc or HNF1B expression could be monitored through the mKeima expression. These proteins were induced by the addition of Doxycycline (DOX, 100 ng/mL) [29 (link)]. The cancer cell lines HeLa, PANC-1, MIA PaCa-2, DU145, and A549 were cultured in DMEM supplemented with 10% FBS and 0.5% P/S, and HCT116 and HL-60 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% FBS and 0.5% P/S. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

MECs were co-cultured with mitomycin C-treated C3H10T1/2 in DMEM/F12 supplemented with 10% FBS, 10 ng/mL human epidermal growth factor (hEGF; BD Biosciences, Tokyo, Japan), 5 μg/mL insulin (Wako), 0.5 μg/mL hydrocortisone (Sigma), 5 μM forskolin (Wako), 1.8 × 10⁻⁴ M adenine (Sigma), 100 μg/mL streptomycin (Meiji Seika Pharma), 100 U/mL penicillin G (Meiji Seika Pharma), 50 μg/mL gentamycin (Nakalai Tesque, Tokyo, Japan), 10 μM Rho-associated coiled-coil-forming kinase inhibitor (ROCKi; Y-27632; LC Laboratories, New Boston, MA, USA) [20 (link), 21 (link)], and 10% Matrigel (growth factor reduced; BD Biosciences) at 5% CO₂ and 37 °C for 7 days. The numbers of MECs and C3H10T1/2 were 5000 and 6.25 × 10⁴, respectively, in 250 μL of culture medium in a 48-well plate. When wells of different sizes were used, these numbers were changed proportionately relative to the area of the well.

All mice were weighted 2 times per week, and food intake was monitored. After 12 weeks of HFD, GTT and ITT were carried out. For GTT, overnight fasted mice received glucose at 1 g/kg bodyweight (BW) intraperitoneally (i.p.), and glucose levels were measured at 0, 15, 30, 60, and 90 min time points (Terumo). For ITT, mice were fasted for 5 hrs before receiving insulin (Wako) at 0.75 U/kg BW i.p., and glucose levels were measured at 0 and 15 min time points. Adipose tissue was collected, weighed and fixed in formalin prior to paraffin mounting, sectioning and staining with hematoxylin and eosin (H&E). Livers were collected after perfusion with PBS. Parts of liver tissue were frozen in Tissue-Tek OCT compound (Sakura Finetek) in liquid nitrogen and sections were stained with Oil-Red-O (Sigma) for measurement of fat deposition. The degree of fat deposition was measured by Oil-Red-O staining intensity around three portal-tract areas per slide.