X0277_X1_79_S79
Rattus norvegicus
Protocols: The termination was performed by decapitation for blood and tissue collection. Microdissections of the hypothalamus and dorsal vagal complex (DVC) was performed for RNA isolation and subsequent sequencing. Tissues were immediately frozen on dry ice upon collection. Male Sprague Dawley rats were maintained on ad libitum high-fat, high-sucrose diet (HFHS, 59 kcal% fat, #E15772-347, ssniff spezialdiäten GmbH) from 10 weeks of age and for a minimum of 12 weeks. Rats had an average body weight of >700g before initiating pharmacological studies. The rats were double-housed (Department of Experimental Medicine Rigshospitalet Unit) in a humidity- (45-65%) and temperature-controlled environment (21-23 oC) with a 12-hour light/dark cycle (6:00 am – 6:00 pm). At an age of 23 weeks, DIO rats weighing 720g on average were divided into four experimental groups (n = 10): vehicle, 5 nmol kg-1 cagrilintide or retatrutide, and the loose equimolar combination of cagrilintide and retatrutide administered in a single dose. HFHS diet intake was measured and replenished daily for ad libitum feeding followed by measurements of body weight and s.c. o.d. compound administration for 16 days. For RNA extraction, all tissues were homogenized in TRIzol reagent (QIAzol Lysis Reagent, Qiagen, 79306) with a stainless-steel bead (Qiagen, 69989) using a TissueLyser II (Qiagen, 85300) set at 30 Hz for 3 min. Total RNA was isolated according to the manufacturer's protocol using QIAwave RNA Mini Kit (Qiagen, 74534) and RNA was quantified and RNA quality accessed using NanoDrop 2000 (Thermo Fischer, ND-2000). Total RNA was then converted to cDNA by mixing with FS buffer (Thermo Fisher Scientific, 18080-044), dithiothreitol (Thermo Fisher Scientific, 18080-044) and random primers (Sigma-Aldrich, 11034731001) followed by incubation at 70 oC for 3 min in a thermal cycler (Eppendorf Mastercycler Pro) for first strand synthesis. dNTPs (Thermo Fisher Scientific, R0192), RNase out (Thermo Fisher Scientific, 10777019) and Superscript III (Thermo Fisher Scientific, 18080-044) were subsequently added for cDNA synthesis using a thermal cycler (Eppendorf Mastercycler Pro) with the following steps: 5 min at 25 oC, 60 min at 50 oC and 15 min at 70 oC. cDNA was diluted 1:20 for all tissues and stored at−20 oC. Libraries were prepared using the Universal Plus mRNA-seq with NuQuant protocol (Tecan) as recommended by the manufacturer. Libraries were quantified with NuQuant using the CLARIOstar Plate Reader (BMG Labtech) and quality checked using a TapeStation instrument (Agilent Technologies)
strain
Sprague-Dawley
individual
1820
organism
Rattus norvegicus
organism
Rattus norvegicus
collection date
not collected
sex
male
scientific_name
Rattus norvegicus
common name
Norway rat
organism part
DVC
developmental stage
adult
genotype
wild type genotype
isolate
not applicable
rin
9
age
23
geographic location (country and/or sea)
not collected