Dextrose

Daidzein was purchased from Agilent Technologies; p-coumaric acid and naringenin were purchased from Sigma. E. coli DH5α (Zymo Research) or TOP10 (Thermo Fisher) were used for DNA manipulation and amplification. E. coli was cultured at 37 °C in Luria–Bertani media (Fisher Scientific) with 100 mg/L carbenicillin (GoldBio) or 50 mg/L kanamycin (Sigma) for plasmid maintenance. All engineered yeast strains described in this work, as listed in Supplementary Table S1, were constructed within the CEN.PK2–1D strain (MATα; his3Δ1; leu2–3,112; ura3–52; trp1–289; MAL2–8c; SUC2). All yeast strains were grown at 30 °C in yeast extract peptone medium (YP) supplemented with 2% (w/v) dextrose (all components from Fisher Scientific), synthetic drop-out media (SD) [containing yeast nitrogen base (YNB) without amino acids (Sunrise Science Products), ammonium sulfate (Thermo Fisher), and the appropriate dropout mixture (Takara Bio) for plasmid maintenance] supplemented with 2% (w/v) dextrose. 200 mg/L Hygromycin B (Santa Cruz Biotechnology) was additionally used with SD media to select for the correct integrants.

Methods for preparing brain slices for electrophysiological recordings were as previously described (Dorris et al., 2014 (link)). Rats were deeply anesthetized with isoflurane gas and killed by decapitation. Trunk blood was collected at sacrifice for serum hormone levels assessment. The brain was dissected rapidly into ice-cold, oxygenated sucrose artificial CSF (s-ACSF) containing (in mM): 75 sucrose, 1.25 NaH₂PO₄, 3 MgCl₂, 0.5 CaCl₂, 2.4 Na pyruvate, 1.3 ascorbic acid from Sigma-Aldrich, St. Louis, MO, and 75 NaCl, 25 NaHCO₃, 15 dextrose, 2 KCl from Fisher, Pittsburg, PA; osmolarity 295–305 mOsm, pH 7.2–7.4. Serial 300 μm coronal brain slices containing the caudate-putamen were prepared using a vibratome and incubated in regular ACSF containing (in mM): 126 NaCl, 26 NaHCO₃, 10 dextrose, 3 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 295–305 mOsm, pH 7.2–7.4) for 30 minutes at 35°C, and at least 30 minutes at room temperature (21–23 °C). Slices were stored submerged in room temperature, oxygenated ACSF for up to 5 hours after sectioning in a large volume bath holder.

Sodium tetraborate, dextrose, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) were from Fisher Scientific (Pittsburg, PA). Cosmic Calf Serum was from HyClone Laboratories (South Logan, UT). Sodium hydroxide (NaOH) and boric acid were purchased from EMD chemicals (San Diego, CA). RPMI 1640 was from Mediatech (Manassas, Va). Acetonitrile (ACN) was from Avantor Performance Materials (Center Valley, PA). Gentamicin was from Lonza (Walkersville, MD). Collagenase P was purchased from Roche Diagnostics (Indianapolis, IN). All other chemicals were from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. All solutions were made with ultrapure DI water (NANOpure® Diamond system, Barnstead International, Dubuque, IA) and filtered with 0.2 μm nylon syringe filters from Pall Corporation (Port Washington, NY) unless otherwise noted. The pH for all solutions were adjusted using 1 or 5 M NaOH as needed.

Women who met study criteria were mailed instructions to follow an unrestricted diet for 3 days preceding the OGTT and to fast at least 10 hours the night before. At each in-person examination, fasting blood specimens were collected between 7:00 and 10:30 a.m. before consumption of 75 g of dextrose (Fisherbrand). Plasma aliquots obtained from fasting and 2-hour postload blood samples were stored at −70 °C until being transported to the University of Washington Northwest Lipid Metabolism and Diabetes Research Laboratories (Seattle, Washington), which performed enzymatic assays for glucose and double-antibody radioimmunoassays for total insulin developed by the Diabetes Endocrinology Research Center Immunoassay Core Laboratory (Seattle, Washington) (5 (link)).

Cam3.II K13^WT, Cam3.II K13^R539T (RF967), and Cam3.II K13^C580Y are laboratory-adapted Cambodian isolates that were genetically engineered at the K13 locus as described in the literature ^{14 (link)}. Cam3.II K13^C580Y parasites expressing β2 C31Y, β2 C31F, or β5 A20S were obtained from in vitro selection studies with WLL and WLW as described in the literature ^{36 (link)}. Parasite cultures were propagated in O+ red blood cells (RBCs) (anonymous donors, purchased from Interstate Blood Bank, Memphis, Tennessee). RBCs were stored at 50% hematocrit in ADSOL (2 mM adenine (Alfa Aesar, Haverhill, Massachusetts), 111 mM dextrose (Fisher BioReagents, Pittsburgh, Pennsylvania), 41.2 mM mannitol (Acros Organics, Fair Lawn, New Jersey), and 154 mM sodium chloride (Fisher BioReagents) for prolonged RBC vitality as described in ref ^{47 (link)} at 4^oC. Parasites were cultured in complete media (RPMI 1640 media supplemented with 0.01 mg/mL gentamicin (Gibco, Dun Laoghaire, Co Dublin, Ireland), 50 mg/mL hypoxanthine (Acros Organics), and 0.5% Albumax II (Invitrogen, Carlsbad, California)) at 5% hematocrit, and were maintained at 37^oC in a Heracell™ VIOS 160i CO₂ Incubator (Thermo Fisher Scientific, Waltham, Massachusetts) under hypoxic conditions (5% O₂, 5% CO₂, 90% N₂). Gas was purchased from Matheson Gas (Irving, Texas).