Proinsulin

Plasma fetuin-A (R&D system, Minnesota, USA), proinsulin (Mercodia, Uppsala, Sweden), and IGF-II (R&D system, MI, USA) were measured by enzyme-linked immunosorbent assay (ELISA) kits, and the absorbance was determined using a microplate spectrophotometer (Beckman CX7, USA). Serum insulin and insulin-like growth factor 1 (IGF-I) concentrations were detected by automated chemiluminescent assays (ADVIA Centaur and Immulite 2000, SIEMENS, Germany). The minimal detectable concentrations were 0.62 ng/ml for fetuin-A, 3.5 pmol/L for insulin, 1.7 pmol/L for proinsulin, 25 ng/ml for IGF-I and 1.88 pg/ml for IGF-II, respectively. The intra-assay and inter-assay coefficients of variation were in the ranges of 0.5–8.3% for fetuin-A, 2.0–6.5% for insulin and IGF-I, 0.34–5.0% for proinsulin, and 2.4–9.3% for IGF-II, respectively. We measured not only insulin but also proinsulin because both are positively associated with birth weight (23 (link)).

Human c-peptide and proinsulin levels were measured from plasma samples and cell supernatants with Ultrasensitive C-PEPTIDE ELISA (Mercodia, Sweden) and PRO-INSULIN (Mercodia, Sweden) according to manufacturer's instructions. Human MANF levels were measured using in-lab ELISA (Galli et al. 2016).

Serum insulin level obtained was determined using blood collected from the saphenous vein in 2 mL heparin-coated Eppendorf tubes and measured using an ultrasensitive mouse insulin ELISA Kit in a semi-fasted state (i.e., removal of food from the cage) (#80-INSMSU-E01/E10, ALPCO, Salem, New Hampshire, USA). Glycogen was measured using frozen liver samples according to the manufacturer’s instructions (Cayman Chemicals, Ann Arbor, MI, USA). Glucagon (Crystal Chemicals, Elk Grove Village, IL, USA) concentrations were determined using commercially available kits (Pro-insulin: #10–1,232-01, Mercodia, Winston Salem, NC, insulin-degrading enzyme (IDE): LS-F5702, LSBio, Seattle, WA, USA, and c-peptide:#80-CPTMS-E01, ALPCO, Salem, New Hampshire, USA). Serum samples were diluted per kit recommendation, if applicable. All samples were read at OD 450 nm using BioTek microplate reader (Santa Clara, CA, USA). Total serum concentration of Insulin-like growth factor-1 (IGF-1) was measured using Mouse IGF-1 ELISA Kit (#RAB (1)0229, MilliporeSigma, Oakville, ON, Canada). Samples were prepared following manufacturers’ standard procedures and read at OD 450 nm on the BioTek microplate reader (Santa Clara, CA, USA).

Stage 7 cells were rinsed twice with Krebs buffer (129 mM NaCl, 5 mM NaHCO₃, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1 mM Na₂HPO₄, 1.2 mM KH₂PO₄, 10 mM HEPES, 0.1% BSA) in deionized water and then sterile filtered. The cells were pre-incubated in Krebs buffer for 30 min. Followed by incubation in Krebs buffer spiked with 1.67 mM glucose (Krebs buffer low glucose) for 40 min. Media was collected after 20 min and 40 min, centrifuged to remove unwanted cells and supernatant was transferred to a new vial. After each incubation, we added the same amount of fresh media to the cells. After low glucose incubation, the cells were incubated in Krebs buffer spiked with 20 mM glucose (Krebs buffer high glucose) for 60 min, and media was collected every 10 min. Followed by incubation in Krebs buffer spiked with 20 mM glucose and 10 nM Exendin-4 for 20 min, with media collection after 10 min and 20 min. As a last stage of stimulation of insulin secretion, cells were incubated in Krebs buffer spiked with 20 mM glucose and 30 mM KCl for 20 min, we collected media after 10 min and 20 min. All supernatants were frozen at −80 °C, and Ultrasensitive C-peptide levels (#10-1141-01;Mercodia), Insulin (#10-113-01; Mercodia) were measured with ELISA. Proinsulin (#10-1118-01; Mercodia) was measured as a negative control, to check that cells were removed from the media.