Step Description Equipment Data collected 1 Administer HFHS diet at 4 wks of age - - 2 Mice weighed weekly, food consumption measured Balance Body weight and food consumption 3 Blood chemistry at 6, 10, 14 wks and at sac (4h fast) after HFHS diet - Plasma glucose, insulin, triglyceride 4 Oral glucose tolerance test (4h fast) after ~14 wks on HFHS diet Kits Plasma glucose and insulin at 0, 5, 15, 30, 60, 120 min 5 Blood chemistry after ~14 wks on HFHS diet - Plasma adiponectin and leptin 6 Mice euthanized and islets harvested Stereomicroscope Number of islets per mouse 7 Ex vivo insulin and glucagon secretion in response to various amounts of glucose and other compounds Kits Insulin and glucagon secretion 8 BMD, organ weights DXA, balance Tibia BMD, heart weight, liver weight, gonadal fat pad weight 9 Bile acids profiled from feces - Quantity of specific bile acids per mg feces
Procedure 1: Assessment of diabetes-associated traits
Definitions & Abbreviations: HFHS: high-fat high-sucrose diet; HOMA: Homeostatic Model Assessment; AUC: area under curve
Fasting (duration): 4h
Equipment, software, and supplies
- Glucose oxidase kit (Thermo Scientific, TR15221)
- Insulin RIA kit (Millipore, SRI-13K, Bedford, MA, USA)
Reagents and solutions
- Amino acids: Ala, Glu, Leu
- Palmitic acid
Holding Area and/or Sample Collection Area
Collection Method: Bleed, Necropsy, Fecal samples
- Mice are weighed weekly and food consumption determined after administration of HFHS diet.
- Four-hour fasting plasma samples are collected for insulin, glucose, and triglyceride measurements (kits are used to quantify) at 6, 10, 14 wks and at sac after HFHS diet.
- Mice are administered an oral glucose tolerance test after ~14 wks on diet (18 wks of age) and glucose and insulin measured at 0, 5, 15, 30, 60, 120 min after glucose administration. Glucose is administered via oral gavage (2g/kg). Blood is collected from a retro-orbital bleed. Area under curve (AUC) is determined for both glucose and insulin.
- HOMA-IR and HOMA-B are determined using fasting plasma values of glucose and insulin at 0 min. HOMA-IR = (glucose x insulin) / 405; HOMA-B = (360 x insulin) / (glucose - 63).
- Plasma adiponectin and leptin levels are determined at the time of the glucose tolerance test.
- Mice are euthanized and organs harvested (pancreas, heart, liver, gonadal fat pads).
- Islets are harvested from pancreas.
- Ex vivo insulin secretion is determined from isolated islets in response to the following: 3.3 mM glucose, 8.3 mM glucose, 16.7 mM glucose, 3.3 mM glucose + 40 mM KCl, 8.3 mM glucose + 100 nM GLP-1, 8.3 mM glucose + amino acids (1.25 mM Ala + 2 mM Glu + 0.5 mM Leu), 16.7 mM glucose + 0.5 mM palmitic acid.
- Ex vivo glucagon secretion is determined from isolated islets in response to 3.3 mM glucose + 40 mM KCl.
- Bone mineral density is determined from isolated tibia.
- Heart, liver, and gonadal fat pads are weighed (pancreas was not weighed as it was inflated in situ with collagenase as the first step to islet isolation).
- Bile acids are measured from feces.
Keller MP, Gatti DM, Schueler KL, Rabaglia ME, Stapleton DS, Simecek P, Vincent M, Allen S, Broman AT, Bacher R, Kendziorski C, Broman KW, Yandell BS, Churchill GA, Attie AD. Genetic Drivers of Pancreatic Islet Function. Genetics. 2018 May;209(1):335-356. doi: 10.1534/genetics.118.300864. Epub 2018 Mar 22.