Recombinant insulin

MCF-7, BT474, T47D, BT20 and MDA-MB-231 breast cancer cells were authenticated by SNP analysis (LGC standards, Wesel, Germany or Genolytic, Leipzig, Germany). SKBR3 cells were purchased from ATCC (American Type Culture Collection). Cells were maintained in RPMI medium supplemented with 10% fetal calf serum (FCS, Pan Biotech) in the absence of antibiotics. Fulvestrant-resistant T47D cells, T47D/182^R-1 and T47D/182^R-2, were generated as previously described [45 (link)]. A fulvestrant-resistant MCF-7 subline (MCF-7/FulvR) was established in our laboratory (Halle) by growing MCF-7 cells long-term in 100 nM fulvestrant (LKT, Laboratories). All cell lines were kept in the same batch of FCS. For treatment with insulin, recombinant insulin (Sigma-Aldrich) was added to cells at a final concentration of 8 μg/ml (~90 μIU/ml).

Mice were fasted for 16 h (GTT) or for 6 h (ITT) with fresh water in new cages. After measurement of fasting BW and an initial blood-Glucose level, either 2 g/kg d-Glucose (G8280, Sigma Aldrich) in PBS or 0.5 U/kg recombinant insulin (91077C, Sigma Aldrich) was intraperitoneally injected, before measurements of blood-Glucose levels taken at 0, 30, 60, and 120 min after the injection.

Rapamycin (cat. no. 553210‐10MG), doxycycline (cat. no. D9891‐1G), streptozotocin (cat. no. S0130‐500MG), TWEEN 80 (cat. no. P1754‐500ML), and recombinant insulin (catalog no. I0516), were all purchased from Sigma Aldrich (Buchs, Switzerland).

Adipogenic differentiation is almost identical to osteogenic induction with slight modification; the cell density was adjusted and fixed at 3 × 10⁴/cm² at day 0, cultured with standard adipogenic induction medium containing 1 μM dexamethasone, 200 μM Indomethacin (Cat. I7378, Sigma-Aldrich, St. Louis, MO, USA), 10 μM recombinant insulin (Cat. 91077C, Sigma-Aldrich, St. Louis, MO, USA), and 0.5 mM 3-Isobutyl-1-methylxanthine (Cat. I7018, Sigma-Aldrich, St. Louis, MO, USA). The medium was changed every 3–4 days, continued to 21 days, fixed, and stained with 0.6% Oil Red O (Cat. O0625, Sigma-Aldrich, Saint Louis, MO, USA) in compliance with the manufacturer’s instruction. The Oil Red O-stained oil droplets were further extracted by 100% isopropanol (Cat. 190764, Sigma-Aldrich, St. Louis, MO, USA), followed by absorbance measurement at O.D. 492 nm through a spectrophotometer (BioTek Synergy H1, Agilent, Santa Clara, CA, USA).

In order to promote the establishment of cell–cell interactions and the formation of cell clumps which will then be embedded into the collagen matrix, fresh or cryopreserved hepatocytes were first incubated overnight (i.e. 12–15 h) in ultra low-attachment plate (LAP) (Corning Costar) at the concentration of 2 × 10⁶ cells/well of a MW6 plate in the medium described previously before being embedded in the collagen matrix. Collagen type I (Sigma-Aldrich) was diluted into the culture medium and the pH was adjusted at 7.4 before the cells were added at a concentration of 3.5 × 10⁵ cells/ml. This mix of cells and collagen was poured into 96-well plates (100 μl) or 48-well plates (250 μl) and incubated at 37 °C, 5% CO₂. After polymerization, an equal volume of medium was added. Cells were maintained in the medium described previously supplemented with hydrocortisone hemisuccinate 1.08 μM, ITS (Recombinant insulin 10 μg/ml, transferrin 5.5 μg/ml, sodium selenite 5 ng/ml) (Sigma-Aldrich), rhHGF (2.5 ng/ml) (BioLegend) and rhEGF (50 ng/ml) (PeproTech). The medium was renewed every 48–72 h. This method is protected under an international patent (EP2018030560320180516/WO2019219828)⁶² .