Hi 14k

EndoC-βH1 and βH2 cells were seeded onto coated 24 well plates at a density of 300,000 cells/well. The night before experiment, the cells were incubated in 2.8 mmol/L glucose culture medium. Prior to the experiment, the cells were incubated in a modified Krebs-Ringer buffer (KRB) medium consisting of (mmol/L) 138 NaCl, 3.6 KCl, 0.5 MgSO₄, 0.5 NaH₂PO₄, 5 NaHC0₃, 1.5 CaCl₂ and 5 HEPES (adjusted to pH 7.4 with NaOH) and supplemented with 0.2% w/v BSA. The cells were washed with the glucose-free medium, preincubated for 15 min at 1 mmol/L glucose before a 40 min test incubation at either 1, 6 or 20 mmol/L glucose and with added tolbutamide (0.2 mmol/L) or diazoxide (0.5 mmol/L) as indicated. Supernatants (0.3 ml) were taken for determination of insulin release. Cellular insulin content was extracted by acid ethanol treatment. The samples were frozen pending later analysis which was carried out using commercial ELISA (Alpha Laboratories) or radioimmunosassay (HI-14 K, Merck). Insulin secretion is expressed as a percentage of cellular insulin content and fold-increase in secretion as a ratio with basal secretion (1 mmol/L). The somatostatin receptor 2 (SSTR2) antagonist CYN154806 was obtained from TOCRIS (Abingdon, UK).

To define the dynamic effects of VCS and TAC on insulin secretion from human islets, we used human islet perifusion studies (7 (link)). We assessed the effects of TAC (10 ng/mL, 30 ng/mL) and VCS (20 ng/mL, 60 ng/mL) on the dynamics of insulin secretion in response to 2 stimuli that are diagnostic of changes in metabolism/signaling or exocytosis. Our standard approach (7 (link), 15-20 (link)) compared the response to 15 mM glucose stimulation and direct depolarization with 30 mM KCl. More specifically, 65 islets per column were perifused (0.4 mL/min) with 3 mM glucose KRB solution as described previously (7 (link)) for 60 minutes to equilibrate the islets to the KRB and flow rate, and then with the indicated condition (as described in corresponding figure legends). First-phase insulin release was defined as the amount of insulin secreted during the first 20 minutes of 15 mM glucose stimulation, while the remaining 40 of stimulation were defined as second-phase. Dimethyl sulfoxide (DMSO), VCS, and TAC were present at the indicated concentrations during the entirety of the perifusion experiment. We repeated this experiment on islets from 17 donors to take into account the significant variability between human islet preparations (11 (link)). Samples were stored at −20 °C and insulin secretion was quantified using human insulin radioimmunoassay kits (Millipore Cat# HI-14K) (21 ).

Plasma glucose was measured in duplicate by the glucose oxidase method using an automated glucose analyzer (YSI 2300; Yellow Springs Instruments, Yellow Springs, OH, USA). Additional blood samples were collected into tubes on ice containing ethylenediamine tetra-acetate and protease inhibitor cocktail and, for the MMTT test, dipeptidyl peptidase-4 inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Samples were centrifuged at 4°C, separated, and frozen at −80°C for subsequent analysis. Plasma insulin (Millipore Cat# HI-14 K, RRID: AB_2801577) and C-peptide (Millipore Cat# HCP-20 K, RRID: AB_2891151) were assayed in duplicate by double-antibody radioimmunoassay (Millipore, Billerica, MA, USA).

Plasma glucose and insulin concentrations were analyzed using commercially available kits (ref. no. A11A01667, Glucose HK CP, ABX Diagnostics; and ref. no. HI-14 K, Millipore, respectively). Plasma amino acid concentrations were determined by UPLC-MS, as previously described (26 (link)). Plasma l-[ring-¹³C₆]-phenylalanine enrichments were determined by GC-MS (Agilent 7890A GC/5975C MSD; Agilent Technologies), as previously described (26 (link)).
Basal muscle protein synthesis rates were assessed to confirm that protein ingestion increases muscle protein synthesis rates. The single biopsy approach was applied to assess postabsorptive muscle protein synthesis rates without the need to collect an additional muscle biopsy, as previously described (26 (link), 30 (link)).