Bovine insulin

If the SVFs were evenly distributed in the culture dish and reached approximately 85% confluence, the growth medium was replaced with an induction medium, which contains 10 mM rosiglitazone (Cayman Chemical, Ann Arbor, MI, USA), 1.4 mg/mL 3-isobutyl-1-methylxanthine (Solarbio, Beijing, China), 1 mg/mL dexamethasone (Solarbio, Beijing, China)), and 3 mg/mL bovine insulin (Solarbio, Beijing, China). This induction was maintained for 10 days of differentiation. The SVFs’ growth state and the generation of lipid droplets were continuously monitored.
When a large number of lipid droplets appeared in the cells, the induction of differentiation of ovine SVFs was stopped. ORO staining was used to identify the lipid droplet distribution. To stain the lipids, cells were washed with cold PBS three times and fixed in 4% paraformaldehyde overnight at 4 °C. The cells were then incubated with the ORO working solution for 30 min. After repeated cleaning with double-distilled water, images were obtained with a microscope (Leica, Wetzlar, Germany).

The IST was determined as previously described with a slight modification^{58 (link)}. Briefly, on experimental days 40, 80 and 120, six layers in each group were selected for insulin treatment. Insulin-treated chickens received 100 µg/kg BW bovine insulin (Solarbio, China; diluted in 1X PBS) via i.p. injection, and then blood glucose concentration was determined at 0 min, 15 min, 30 min, 60 min, and 120 min. AUCs were calculated by GraphPad Prism7.

Cancer tissues obtained from mPTC^GFP were minced in lysis buffer (Collagenase I (Sigma) 1 mg/ml; Dispase II (Invitrogen) 0.5 mg/ml in PBS) and shaked on an orbital shaker at 37 °C for 60 min. Dissociated tumor cells were filtered with 40 μm cell strainer (#352340, BD Falcon) and centrifugated at 500 g for 5 min, the pellet was resuspended in cold Ham’s F-12 Nutrient Mix (#11765062, Gibco). The isolation of tumor cells was next performed on FACSAria Cell Sorter (Becton Dickinson) according to GFP signal, and positive cells were washed with PBS and then cultured in F-12 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 5 mg/L transferrin (Sigma-Aldrich), 10 mg/L bovine insulin (Solarbio), 3.5 mg/L hydrocortisone (Sigma-Aldrich), 10 mg/L somatostatin (Sigma-Aldrich), 0.02 mg/L Gly-His-Lysacetate (Sigma-Aldrich) and 1 IU/L bovine thyroid stimulating hormone (TSH) (Sigma-Aldrich) related to the 6H medium at 37 °C⁵⁹ . Next, Primary cells treated with 10 μM PLX4032 or 2 μM SCH772984 were subjected to western blot and RT-qPCR analysis. Primary cells from mPTC mice were directly cultured in complete F-12 medium. Thyrocytes were confirmed by immunofluorescence staining of thyroglobulin and representative photos were taken by a fluorescence microscopy.

If the SVFs were evenly distributed in the culture dish and reached approximately 85% confluence, the growth medium was replaced with an induction medium, which contains 10 mM rosiglitazone (Cayman Chemical, Ann Arbor, MI, USA), 1.4 mg/mL 3-isobutyl-1-methylxanthine (Solarbio, Beijing, China), 1 mg/mL dexamethasone (Solarbio, Beijing, China)), and 3 mg/mL bovine insulin (Solarbio, Beijing, China). This induction was maintained for 10 days of differentiation. The SVFs’ growth state and the generation of lipid droplets were continuously monitored.
When a large number of lipid droplets appeared in the cells, the induction of differentiation of ovine SVFs was stopped. ORO staining was used to identify the lipid droplet distribution. To stain the lipids, cells were washed with cold PBS three times and fixed in 4% paraformaldehyde overnight at 4 °C. The cells were then incubated with the ORO working solution for 30 min. After repeated cleaning with double-distilled water, images were obtained with a microscope (Leica, Wetzlar, Germany).

3T3-L1 cells were purchased from the China Infrastructure of Cell Line Resource (Beijing, China) and maintained in DMEM (Hyclone, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; Gibco, Langley, OK, United States) in an incubator with 5% CO₂ at 37°C. The culture medium was changed every second day. When confluence reached 70–80%, cells were detached by trypsin with 0.05% EDTA (Gibco, Carlsbad, CA, United States) and reseeded in new culture plates at a density of 5 × 10⁶ cells/plate. To induce differentiation to adipocytes, the preadipocytes were cultured in complete medium to a confluence of 70%, and then for two more days to reach contact inhibition. The culture medium was then replaced with MDI (methylisobutylxanthine, dexamethasone, insulin) induction medium (it is recorded as 0 d), a complete medium supplemented with 5 μg/mL bovine insulin (Solarbio, Beijing, China), 0.5 μmol/L 3-isobutyl-1-methylxanthine (Sigma, Shanghai, China), and 1 μmol/L dexamethasone (Sigma). The cells were cultured for 48 h in this medium, which was then replaced with insulin medium (DMEM + 10% FBS + 10 μg/mL insulin) to maintain differentiation for 48 h. Finally, the medium was replaced with a complete medium, and the cells were incubated until day 8. The adipocytes were harvested and stained by Oil Red O following published protocols (Ramírez-Zacarías et al., 1992 (link)).

To determine whether exogenous insulin had a regulatory role on hemolymph glucose and affected the expressions of glucose metabolism genes in shrimp, 90 shrimp were fasted for 12 h and then refed with commercial food. After 1 h, they were divided into two groups. The experimental group shrimp were injected with bovine insulin (Solarbio Science & Technology Co. Ltd., Beijing, China) at 1.5 IU/kg body weight, and the control group was injected with an equal volume of H₂O. Samples were collected at 0, 10, 30, 60, and 120 min after injection. The experimental steps for sampling and detection were the same as those in Section 4.4.