GoKawiilAnimals and housing conditions
Experiments were performed in accordance with the Animal Protection Law of the European Union after permission by the Governments of Upper Bavaria, Germany, or Copenhagen, Denmark, or by the Institutional Animal Care and Use Committees of the Universities of Texas Southwestern, Michigan, Duke or Yale, USA. Mice were double- or single-housed and unless otherwise indicated fed ad libitum with either a regular chow (1314, Altromin or 5L0D, LabDiet) or HFD (58% fat, D12331, Research Diets) under constant ambient conditions of 22 ± 2 °C with constant humidity (45–65%) and a 12 h:12 h light:dark cycle. Leptin receptor-deficient db/db mice were purchased from the Jacksons Laboratory (Strain 000697). Doxycyclin-inducible GIPR-overexpressing mice (TRE-GIPR mice) were generated in-house at The University of Texas Southwestern Medical Center as described previously41. C57BL/6J DIR-knockout mice were generated in-house at Helmholtz Munich (Supplementary Information 1).
Pharmacological studies
Indirect calorimetry and assessment of body composition was performed as described in Supplementary information 1. Drug effects were assessed in age-matched male single-, or double-housed C57BL6/J mice that were randomly assigned in groups matched for genotype, body weight and body composition (fat and lean tissue mass). Mice were treated subcutaneously at the indicated doses with 5 μl per g body weight of either Vehicle, Lani (CAS: 927961-18-0, MedChemExpress), semaglutide, GLP-1–Lani, GLP-1–Tesa, GLP-1–GIP, GLP-1–GIP–Tesa, GLP-1–GIP–Lani, or co-administration of either GLP-1–GIP plus Lani or GLP-1–Lani plus acyl-GIP (Extended Data Fig. 1a–f). All peptides were provided by the Novo Nordisk Research Center Indianapolis, IN, USA or the Indiana Biosciences Research Institute, IN, USA (for drug development see Supplementary Information 1). Assessment of drug effects on the cardiovascular system and on POMC neuronal activity was performed as described in Supplementary Information 1.
Glucose and lipid metabolism
Glucose and insulin tolerance was assessed in 6 h-fasted mice injected intraperitoneally with either 1.5–2 g kg−1 of glucose or 0.6–0.75 U kg−1 of insulin (Humalog; Eli Lilly). Glucose-induced insulin secretion was assessed in 6 h-fasted mice orally gavaged with 4 g kg−1 glucose. Pyruvate tolerance was assessed in 12 h-fasted mice injected intraperitoneally with 1.25 g kg−1 sodium pyruvate (11360070, Thermo Fisher). Commercially available ELISAs were used according to the manufacturer’s instruction to measure insulin (90080, Crystal Chem), triglycerides (94501, Fujifilm), cholesterol (293-93601, Fujifilm) and free fatty acids (434-91795, 436-91995, 270-77000, Fujifilm). Hyperinsulinaemic-euglycaemic clamps and assessment of tissue-selective glucose uptake were performed as described in Supplementary Information 1.
Gene expression analysis
Total RNA was isolated using the RNeasy Kit (QIAGEN) according to the manufacturer’s instructions. cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit (QIAGEN) or High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Gene expression was profiled using SYBR green (Thermo Fisher Scientific) and the Quantstudio 7 flex cycler (Applied Biosystems). The relative expression levels of each gene were normalized to the housekeeping gene HPRT. Primer sequences are listed in Supplementary Information 1.
Cell culture studies
HEK293T cells (CRL-3216, ATCC) were cultured in DMEM (11995073, Life Technologies) supplemented with 10% heat-inactivated FBS (10500064, Life Technologies), 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin solution (Pen/Strep, P4333, Sigma Aldrich). Cells (700,000 per well) were seeded in 6-well plates and incubated to 70% confluency in DMEM (10% FBS, 1% Pen/Strep). Twenty-four hours following seeding, transient transfections were performed using Lipofectamine 2000 (11668019, Invitrogen) according to the manufacturer’s instructions without including additional transformation carrier DNA. BRET assays and in vitro quantification of PPAR-responsive genes were performed as described in Supplementary Information 1.
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