I2643

Mito-QC animals were used to determine mitophagy in retinal ex vivo explant cultures under different treatment conditions as described. After sacrificing the animals through cervical dislocation, retinae were isolated from the eyes as described previously [58 (link)]. Retinae were placed in 24 well plates and incubated with DMEM (Dulbecco’s Modified Eagle Medium; 41966-029, Gibco) with 1% glutamine (2 mM, 25,030, Gibco), 1% penicillin-streptomycin (0.5 mg/mL, 11,568,876, Gibco) and 1 µM of insulin (I2643, Sigma) with the indicated treatments and time. After incubation flatmounts were washed with PBS and fixed in 3.7% PFA containing 200 mM HEPES for 1 h at RT and subsequently washed 3× 5 min with PBS. Flatmounts were mounted in Vectashield (H-1000-10, Vector Laboratories INC, Burlingame, CA, USA). Pictures were taken with an SP5 microscope (Leica TSC SP5 confocal microscope) with a ×63 objective.

In unpublished pilot studies conducted in our laboratory, we established that a single intravenous injection of 5 U/kg of insulin in anesthetized, overnight fasted rats produced a consistent and highly reproducible level of hypoglycemia in both Chow- and KD-fed groups. Such a consistent degree of hypoglycemia allowed tracking the changes in ASNA with changes in blood glucose levels over time without the need for a hypoglycemic-hyperinsulinemic clamp. In the current study, hypoglycemia was induced in the Chow-fed (n = 9) and KD-fed (n = 7) rats by a single intravenous injection of insulin (human recombinant, 5 U/kg, I2643, Sigma-Aldrich, North Ryde, NSW, Australia). The control Chow (n = 3) and KD-fed (n = 3) rats were injected the same volume of PBS. Peripheral blood glucose and BHB were measured from a drop of blood, obtained via a tail nick, at 0 (pre insulin administration), 15, 30, 45, 60, 90, and 120 min. Baseline (0 min, pre insulin administration) blood glucose and BHB were consistently measured between 11:00 a.m. and 12:00 p.m. An Accu-check Performa glucose meter (Roche Diabetes Care, North Ryde, NSW, Australia) and corresponding glucose strips were used for glucose determination, and an Abbott Optium Neo glucose and ketone meter with Freestyle Optium ketone strips (Abbott Diabetes Care, Doncaster, VIC, Australia) were used for BHB measurements.

A published collagenase perfusion technique was employed for PHH isolation from liver wedges [13 (link)]. Briefly liver was digested and centrifuged to isolate PHH. These cells were resuspended and then plated on 24-well plates previously coated with rat tail collagen type 1 at a density of 3x10⁵ cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM) (Catalogue number [CN]:41965–039; Gibco laboratories, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS) (CN:10270106; Gibco) and 5% glutamine/penicillin/ streptomycin (GPS) (CN:10378016; Gibco). After 2 hours the cells were washed with Phosphate-buffered saline (PBS) (CN:10010023; Gibco) and the media changed to our standard medium for PHH culture, constituted of Arginine-/Glutamine-free Williams E (CN:12551032; Gibco) with 1% GPS, hydrocortisone (2μg/ml) (H4001; Sigma-Aldrich, St. Louis, MO., USA), insulin (0.124 U/ml) (I2643; Sigma-Aldrich) and L-ornithine (400μM) (O6503; Sigma-Aldrich), subsequently the cells were kept at 37°C in 95% air/ 5% CO₂.

In the third and fourth week of the feeding trial with the different diets (WD, WD+PF, WD+FF, WD+BF), assays for glucose homeostasis and insulin sensitivity were carried out, respectively. For the oral glucose tolerance test (OGTT), animals were fasted for 6 hours and a fixed dose of D-glucose (Sigma-Aldrich, 2 g/kg, meaning 300 μl of glucose solution 20% per mouse) was administered per os and blood (~3–5 μL) was collected from tail at different time points. Blood glucose levels were measured by a glucometer (Accu-Check ®, Roche Diagnostics) at baseline and 15, 30, 60 and 120 minutes after glucose administration. For the insulin tolerance test, mice fasted for 4 hours before human insulin (I2643, Sigma-Aldrich) was injected i.p. (0.25 U/kg). Blood glucose was measured at the baseline and 30, 60, 120 minutes after insulin injection.

iHMuSCs were seeded at 12,000 cells per cm² in 12-well plates containing glass coverslips and were grown in growth medium for 4 days. Growth medium was replaced by differentiation medium (skeletal muscle basal medium containing 100 µg/mL transferrin (Sigma-Aldrich #T3309), 10 µg/mL human insulin (Sigma-Aldrich #I2643), 100 U/mL penicillin and 100 µg/mL streptomycin) and cells were further incubated for 5 days. Cells were washed with PBS, fixed in formaldehyde 4% for 10 min at RT and were permeabilized in triton X-100 0.1% for 10 min at RT. Immunolabeling was carried out using rabbit anti-desmin (1:200, at 4 °C overnight, Abcam #ab32362, Cambridge, UK) and/or rabbit anti-dystrophin (1:50, at 4 °C overnight, Abcam #15277) antibodies revealed with donkey anti-rabbit IgG and/or anti-mouse IgG antibodies. Nuclei were labeled with Hoechst for 10 sec at RT, and mounting was performed in Fluoromount.

Human ovarian granulosa-like tumor cell line KGN, which originated from a stage III invasive ovarian granulosa cell carcinoma in a 63-year-old woman, was considered a model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells (26 (link)). In this study, KGN cells were kindly provided by Clinical Anatomy & Reproductive Medicine Application Institute. KGN cells were cultured with Dulbecco's Modified Eagle's Medium-high glucose (DMEM, Sigma, USA) supplemented with 12% fetal bovine serum (FBS, Invitrogen Gibco, USA) and maintained in an atmosphere of 5% CO₂ at 37°C.
KGN cells were plated in 6-well plates at 10⁵ per well. After starving for 24 h, the cells were treated without or with insulin (I2643; Sigma, USA) at concentrations of 1, 2, and 4 mg/ml. Then, KGN cells were treated with insulin (4 μg/ml) and RES at concentrations of 10, 25, 50, and 100 μM for 24 h. In addition, the cells were also treated with AGK2 (10 μM, A8231; Sigma, USA) and RES (25 μM) for 24 h.