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Insulin Tolerance Test Protocol Mice

Glucose Tolerance Test In Mice

Glucose Tolerance Test In Mice

Glucose tolerance test is a standard procedure that addresses how quickly exogenous glucose can be cleared from blood. Specifically, uptake of glucose from the blood by cells is regulated by insulin. Impairment of glucose tolerance (i.e, longer time to clear given amount of glucose) indicates problems with maintenance of glucose homeostasis (insulin resistance, carbohydrate metabolism, diabetes, etc). According to the WHO, in a standard oral glucose tolerance test (OGTT), glucose level should be below 7.8 mmol/L (140 mg/dl) at 2 h. Levels between this and 11.1 mmol/L (200 mg/dl) indicate “impaired glucose tolerance”, and any level above 11.1 mmol/L (200 mg/dl) confirms a diagnosis of diabetes. Materials and reagents Mice (~20 C57BL/6J (B6) males of 2-3 months old) 70% ethanol Beta-D(+)-glucose (Sigma-Aldrich, catalog number: G8270 ) NaCl KCl Sodium phosphate Phosphate buffered saline (PBS) (see Recipes) Equipment ACCU-CHEK comfort curve glucometer (Roche Diagnostics, catalog number: 03537536001 ) (this product has been discontinued. Any new product of ACCU-CHEK should work fine as well) Such device quantifies glucose amperometrically by measuring the current produced upon oxidation of glucose to gluconic acid by glucose oxidase, or to gluconolactone by dehydrogenase. 27 gauge needle (Single-Use Needles, supplied by VWR, BD Medical, catalog number: BD305109 ) Microvette CB300 Z serum separator (SARSTEDT, catalog number: 16.440.100 ) Acrodisc 25 mm syringe filters w/ 0.2 μM HT Tuffryn membrane (Pall Corporation, catalog number: 4192 ) Procedure Note: All the following experimental procedures that involve animals (rodents) should receive approval from IACUC or equivalent committee. Humane treatment of animals should be practiced all the time. In the Cavener lab, we us Continue reading >>

Mouse Metabolic Phenotyping Centers Mmpc Protocols

Mouse Metabolic Phenotyping Centers Mmpc Protocols

Intraperitoneal Insulin Tolerance Test Version: 1 Edited by: Fawaz G. Haj Summary: An intraperitoneal insulin tolerance test or ipITT is designed to determine determine the sensitivity of insulin-responsive tissues in the rodent. This is determined by measurement of glucose remaining in the circulation over time after a bolus ip insulin injection. Reagents and Materials: Reagent/Material Vendor Stock Number Humalin® R Eli Lilly R-100 Insulin Syringes Fisher Scientific 14-826-79 Saline Solution Fisher Scientific L97753 Easy Check Glucose test strips JRS Medical 00-101 Easy Check Glucose monitor JRS Medical Y4209 Reagent Preparation: Dilute the stock solution (100 U/ml) with saline to 0.5 U/mL (1/200 dilution) by adding 5μl stock (100 U/mL) to 995 μl 0.9% (w/v) sterile saline Protocol: 1. Fast mice for 4 h only by taking away food early in the morning (7:00am). 2. Calibrate the glucose meter according to the manufacturer’s instructions. 3. Deprive mice from water then remove approximately 5μl of blood (one drop) from the tail via a tail tip cut and transfer directly onto a glucose indicator strip. 4. Measure blood glucose immediately in a glucometer. Mouse Metabolic Phenotyping Centers MMPC Protocols 09/17/12 2 of 2 page(s) 5. 4. Give the mouse an intraperitoneal injection of insulin (0.5 U/kg) with a 27 G needle. 6. 5. Continue to take blood samples from the initial tail cut before the insulin injection and at 15, 30, 45, 60 and 120 min. 7. 6. Between each of these time points, return the mouse to its cage and monitor it every minute. NOTE: 1-The mouse is given an intraperitoneal injection with a 27G needle of insulin. Before performing the experiment, we will have to determine if the mouse strain is insulin resistant or glucose tolerant, so as to avoi Continue reading >>

Methods And Models For Metabolic Assessment In Mice

Methods And Models For Metabolic Assessment In Mice

Journal of Diabetes Research Volume 2013 (2013), Article ID 986906, 8 pages 1Metabolic Unit, ISIB CNR, 35127 Padova, Italy 2Department of Medicine, Lund University, 221 84 Lund, Sweden Academic Editor: Daisuke Koya Copyright © 2013 G. Pacini et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The development of new therapies for the treatment of type 2 diabetes requires robust, reproducible and well validated in vivo experimental systems. Mice provide the most ideal animal model for studies of potential therapies. Unlike larger animals, mice have a short gestational period, are genetically similar, often give birth to many offspring at once and can be housed as multiple groups in a single cage. The mouse model has been extensively metabolically characterized using different tests. This report summarizes how these tests can be executed and how arising data are analyzed to confidently determine changes in insulin resistance and insulin secretion with high reproducibility. The main tests for metabolic assessment in the mouse reviewed here are the glucose clamp, the intravenous and the oral glucose tolerance tests. For all these experiments, including some commonly adopted variants, we describe: (i) their performance; (ii) their advantages and limitations; (iii) the empirical formulas and mathematical models implemented for the analysis of the data arising from the experimental procedures to obtain reliable measurements of peripheral insulin sensitivity and beta cell function. Finally, a list of previous applications of these methods and analytical techniques is provided to better comprehend their use a Continue reading >>

Insulin Tolerance Test

Insulin Tolerance Test

Glucose Test Strips - AccuChek Comfort Curve or equivalent echoMRI if the mice differ in body fat levels (see below) 0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of insulin, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass. This may need to be adjusted depending on the insulin sensitivity of the mice, and this is based on a normal C57BL/6J mouse on chow. In general for insulin resistant mice, such as those >40g on a high fat diet or such, increase the dose to 2 or 2.5U/kg. In general you want the insulin to decrease blood glucose by about 60-70% in the most responsive of your too group so if your response is <20% of >70% change in blood glucose you will probably have to change your dose and retry. The insulin is diluted from Humulin R-100 and is purchased through the veterinary staff. Remove food from mice for about 6h by putting them in a fresh cage. Add do not feed tag to cages, or ideally move cage to procedure room. Try to make sure that the mice are in a quiet, undisturbed temperature controlled room with the lights on. Typically starve the mice at 8AM and aim to start injections at 2PM Prepare a 1 g/10mL solution of glucose in case some animals become hypoglyemic. Weigh mice, mark tails if necessary with different colors for rapid identification and take fasting glucose measurement via a tail clip. Prepare insulin syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL). At approximately 1 min intervals, inject appropriate amount of insulin into interperitoneal cavity of the mouse. Immobilize mouse and restrain tail with one hand Aim needle between the midline and the hip bone Insert syringe (do not inject) in Continue reading >>

Insulin Tolerance Test

Insulin Tolerance Test

An insulin tolerance test (ITT) is a medical diagnostic procedure during which insulin is injected into a patient's vein, after which blood glucose is measured at regular intervals. This procedure is performed to assess pituitary function, adrenal function, and sometimes for other purposes. An ITT is usually ordered and interpreted by endocrinologists. Insulin injections are intended to induce extreme hypoglycemia below 2.2 mmol/l (40 mg/dl). Patient must have symptomatic neuroglycopenia to trigger counter-regulatory cascade. Glucose levels below 2.2 nmol/L are insufficient absent symptoms. The brain must register low glucose levels. In response, adrenocorticotropic hormone (ACTH) and growth hormone (GH) are released as a part of the stress mechanism. ACTH elevation causes the adrenal cortex to release cortisol. Normally, both cortisol and GH serve as counterregulatory hormones, opposing the action of insulin, i.e. acting against the hypoglycemia.[1] Thus ITT is considered to be the gold standard for assessing the integrity of the hypothalamic–pituitary–adrenal axis. Sometimes ITT is performed to assess the peak adrenal capacity, e.g. before surgery. It is assumed that the ability to respond to insulin induced hypoglycemia translates into appropriate cortisol rise in the stressful event of acute illness or major surgery.[2] This test is potentially very dangerous and must be undertaken with great care, because it can iatrogenically induce the equivalent of a diabetic coma. A health professional must attend it at all times. Other provocation tests which cause much less release of growth hormone include the use of glucagon, arginine and clonidine. Side effects[edit] Side effects include sweating, palpitations, loss of consciousness and rarely convulsions due to severe Continue reading >>

Insulin Tolerance Test And Hyperinsulinemic-euglycemic Clamp

Insulin Tolerance Test And Hyperinsulinemic-euglycemic Clamp

Human insulin (Eli Lilly, Indianapolis, IN) [3-3H] glucose (Perkin Elmer, catalog number: NET331A250UC ) 2-deoxy-D-[1-14C] glucose (2-[14C]DG) (PerkinElmer, catalog number: NET328250UC ) C57BL/6J mice were fasted for 6 h and then injected intraperitoneally with insulin (1 U per kg of body weight), and blood glucose concentrations were monitored over time using a Contour blood glucometer on a drop of blood from the tip of the tail. Mice were cannulated in the lateral cerebral ventricle and catheterized in the right internal jugular vein for the hyperinsulinemic-euglycemic clamp (Figure 1)(Thrivikraman et al., 2002). Intravenous infusion of [3-3H] glucose (5 Ci bolus, 0.05 Ci/min) was used. Human insulin (16 mU/kg) was injected intravenously as a bolus, followed by continuous infusion at 2.5 mU/kg/min. Tail blood glucose was measured by glucometer at 10 min intervals, and 20% glucose was infused to maintain blood glucose at euglycemic levels (120 to 140 mg/dl of plasma glucose levels). After steady state had been maintained for 1 h, the glucose uptake in various tissues was determined by injecting 2-deoxy-D-[1-14C] glucose (2-[14C]DG) (10 mCi) 45 min before the end of clamps (the catheter was used for the injection). During the final 50 min of basal and clamp infusions, 20 l blood samples were collected at 10 min intervals for measurement of [3H] glucose, [3H] H2O and 2-[14C]DG from the tail vein. Samples were stored in -20 C. Figure 1. Right internal jugular vein catheterization. A catheter is placed in the right jugular vein for the infusion of glucose and insulin. This protocol has been adapted from our previously published paper: Paschos et al. (2012). The work during the development of the protocol was supported by the US National Institutes of Health (NIH) grant RO Continue reading >>

Oral Glucose Tolerance Test

Oral Glucose Tolerance Test

This assay is designed to identify genetically modified mice that exhibit alterations of metabolism associated with diabetes, obesity and cardiovascular disease. An oral glucose tolerance test (OGTT), in which the mice are challenged with a bolus of glucose and blood glucose and insulin levels are measured across a two hour time course is performed one week prior to the initiation of the HFD challenge. The mice are subjected to a seven-week HFD challenge using a Western diet. (Circulating levels of insulin, adiponectin, and cholesterol from samples taken before and after the HFD can also be measured, as well as the distribution of cholesterol in the VLDL, LDL, and HDL sub-fractions both pre and post the HFD challenge are analyzed using a polyacrylamide based system (Lipoprint).) The post HFD OGTT results are compared to those obtained before the start of the diet. The ability of genetically modified or pharmacologically treated mice to handle an oral glucose load, in combination with changes in insulin and adiponectin levels in response to the HFD, are assessed to identify genes or pharmacological agents affecting development of a diabetic or pre-diabetic state. Differences in cholesterol levels and cholesterol distribution are examined to establish if the genetic modification or the compound alters the response to the HFD. Prior to the test, the mice were fasted for 16 hours and transferred to a procedure room midway through the light phase of the Light:Dark cycle. Blood was obtained from a tail cut (by removing the distal 2 mm of the tail) and was assessed for baseline glucose levels using a One-touch Ultra 2 (Lifescan, Johnson & Johnson) glucometer. The remaining blood was processed for plasma that was later used to determine the fasting insulin levels. The mice the Continue reading >>

Validation Of Homa-ir In A Model Of Insulin-resistance Induced By A High-fat Diet In Wistar Rats

Validation Of Homa-ir In A Model Of Insulin-resistance Induced By A High-fat Diet In Wistar Rats

Validation of HOMA-IR in a model of insulin-resistance induced by a high-fat diet in Wistar rats 1Programa de Ps-Graduao em Medicina, Cincias Mdicas, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brasil 2Grupo para o Estudo da Resistncia Insulina (GERI), Porto Alegre, RS, Brasil 3Curso de Nutrio, Departamento de Medicina Interna, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brasil 4Servio de Medicina Interna, Hospital de Clnicas de Porto Alegre (HCPA), Porto Alegre, RS, Brasil The present study aimed to validate homeostasis model assessment of insulin resistance (HOMA-IR) in relation to the insulin tolerance test (ITT) in a model of insulin-resistance in Wistar rats induced by a 19-week high-fat diet. A total of 30 male Wistar rats weighing 200-300 g were allocated into a high-fat diet group (HFD) (55% fat-enriched chow, ad lib, n = 15) and a standard-diet group (CD) standard chow, ad lib, n = 15), for 19 weeks. ITT was determined at baseline and in the 19th week. HOMA-IR was determined between the 18-19th week in three different days and the mean was considered for analysis. Area under the curve (AUC-ITT) of the blood glucose excursion along 120 minutes after intra-peritoneal insulin injection was determined and correlated with the corresponding fasting values for HOMA-IR. AUC-ITT and HOMA-IR were significantly greater after 19th week in HFD compared to CD (p < 0.001 for both). AUC-OGTT was also higher in HFD rats (p = 0.003). HOMA-IR was strongly correlated (Pearsons) with AUC-ITT r = 0.637; p < 0.0001. ROC curves of HOMA-IR and AUC-ITT showed similar sensitivity and specificity. HOMA-IR is a valid measure to determine insulin-resistance in Wistar rats. Arch Endocrinol Metab. Continue reading >>

Melior Discovery: Insulin Tolerance Test

Melior Discovery: Insulin Tolerance Test

The Insulin Tolerance Test (ITT) is designed to determine the sensitivity of insulin receptors in tissue by measuring blood glucose levels before and after insulin administration.This is a standard test to determine the diabetic status in humans and experimental animals. This test is used to assess the efficacy of insulin-like compounds and pharmacological agents that can modify insulin responsiveness. The graph above illustrates the difference in response to an insulin challenge (ITT) in insulin-depleted (streptozotocin-treated) mice administered insulin, insulin+MLR-1023 or MLR-1023 alone. This study shows that MLR-1023 significantly extended the duration and magnitude of the insulin response. Data are mean SEM, *p<0.05, ***p<0.001 compared to vehicle. MLR-1023 is a potential "next-generation" insulin sensitizer that works independently of a PPAR mechanism. This compound improves glycemic control by directly and selectively activating the enzyme Lyn kinase. Lyn kinase has been previously shown to modulate the insulin-signaling pathway. MLR-1023 is the first described specific and direct activator of Lyn kinase that elicits glycemic control activity through potentiation of insulin activity. For more information on MLR-1023, please visitour sister site, Melior Pharmaceuticals . In addition to MLR-1023 studies, Melior routinely performs ITT studies in mice fed a "Western diet" that is designed to approximate the "typical" human diet of North Americaand Europe. The "Western diet" contains greater than five times more fat than the normal diet. In this study, mice were fasted four hours prior to study commencement. Insulin tolerance test. A baseline glucose measurement was evaluated one hour prior to dosing. At time 0 minutes, mice received either insulin or vehicle (no in Continue reading >>

Supplement To Fasting-dependent Glucose And Lipid Metabolic Response Through Hepatic Sirtuin 1 | Pnas

Supplement To Fasting-dependent Glucose And Lipid Metabolic Response Through Hepatic Sirtuin 1 | Pnas

Fig. 5. Adenovirus mediated hepatic SIRT1 knockdown and overexpression controls acetylation of PGC-1a. (A) SIRT1 shRNA infection decreases hepatic SIRT1 protein levels. Western blot analysis of SIRT1, PGC-1a, FOXO1, and HNF-4a from nuclear extracts of livers from control and SIRT1 shRNA infected mice. (B) SIRT1 knockdown increases acetylation of endogenous PGC-1a. PGC-1a immunoprecipitated from liver nuclear extracts. (C) Western blot analysis of liver nuclear extracts from mice infected with GFP control or SIRT1 overexpression adenovirus. (D) Overexpression of SIRT1 decreases endogenous PGC-1a acetylation. (E) SIRT1 knockdown increases acetylation on endogenous FOXO1. Acetylation quantification was performed as described in Methods. Data are presented as the average SD. Significance was determined by Student's t test. *, P < 0.05. Fig. 6. (A) Western blot of liver nuclear extracts from mice double infected with GFP or SIRT1 overexpression and Control or PGC-1a shRNA adenoviruses. (B) Glycemia from mice during feeding and after a 5 and 20 h fast. Data are presented as the average SEM from two independent experiments (each bar, n = 9). Control vs. PGC-1a shRNA: #, P < 0.05. (C) Glucose tolerance test. Mice fasted 18 h before i.p. injection of 2 g/kg glucose. Data are presented as the average SEM. Each curve is n = 4. Significance was determined by two-tailed unpaired Student's t test. GFP vs. SIRT1: *, P < 0.05. Control vs. PGC-1alpha shRNA: #, P < 0.05. Glucose tolerance test was performed in two independent experiments with similar results. Fig. 7. (A) Western blot of liver extracts from mice infected with GFP or PGC-1a overexpression and control or SIRT1 shRNA adenovirus. (B) Quantitative RT-PCR analysis of Pepck gene expression from mice fasted for 20 h (GFP: n = 5 Continue reading >>

Glucose And Insulin Tolerance Tests In The Mouse

Glucose And Insulin Tolerance Tests In The Mouse

Search over 50,000 protocols and methods: Glucose and Insulin Tolerance Tests in the Mouse Series: Methods In Molecular Biology > Book: Methods in Mouse Atherosclerosis In vivo metabolic tests are highly valuable to determine whether atherosclerosis progression in mouse models is accompanied by carbohydrate metabolism alterations such as glucose intolerance and insulin resistance. In this chapter, we describe In vivo metabolic tests are highly valuable to determine whether atherosclerosis progression in mouse models is accompanied by carbohydrate metabolism alterations such as glucose intolerance and insulin resistance. In this chapter, we describe protocols to perform in the mouse glucose and insulin tolerance tests, two metabolic assays which evaluate the glucose tolerance and the insulin sensitivity, respectively. Laura N. Castellani et al., 2017, Psychoneuroendocrinology Benetos A, Thomas F, Pannier B et al (2008) All-cause and cardiovascular mortality using the different definitions of metabolic syndrome. Am J Cardiol 102:188191 Zambon S, Zanoni S, Romanato G et al (2009) Metabolic syndrome and all-cause and cardiovascular mortality in an Italian elderly population: the Progetto Veneto Anziani (Pro.V.A.) Study. Diabetes Care 32:153159 Beckman JA, Creager MA, Libby P (2002) Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. JAMA 287:25702581 Nunn AV, Bell JD, Guy GW (2009) Lifestyle-induced metabolic inflexibility and accelerated ageing syndrome: insulin resistance, friend or foe? Nutr Metab (Lond) 6:16 Burks DJ, de Mora JF, Schubert M et al (2000) IRS-2 pathways integrate female reproduction and energy homeostasis. Nature 407:377382 Gonzalez-Rodriguez A, Mas Gutierrez JA, Sanz-Gonzalez S et al (2010) Inhibition of PTP1B restores IRS1-medi Continue reading >>

Improved Insulin Sensitivity And Resistance To Weight Gain In Mice Null For The Ahsg Gene

Improved Insulin Sensitivity And Resistance To Weight Gain In Mice Null For The Ahsg Gene

Fetuin inhibits insulin-induced insulin receptor (IR) autophosphorylation and tyrosine kinase activity in vitro, in intact cells, and in vivo. The fetuin gene (AHSG) is located on human chromosome 3q27, recently identified as a susceptibility locus for type 2 diabetes and the metabolic syndrome. Here, we explore insulin signaling, glucose homeostasis, and the effect of a high-fat diet on weight gain, body fat composition, and glucose disposal in mice carrying two null alleles for the gene encoding fetuin, Ahsg (B6, 129-Ahsgtm1Mbl). Fetuin knockout (KO) mice demonstrate increased basal and insulin-stimulated phosphorylation of IR and the downstream signaling molecules mitogen-activated protein kinase (MAPK) and Akt in liver and skeletal muscle. Glucose and insulin tolerance tests in fetuin KO mice indicate significantly enhanced glucose clearance and insulin sensitivity. Fetuin KO mice subjected to euglycemic-hyperinsulinemic clamp show augmented sensitivity to insulin, evidenced by increased glucose infusion rate (P = 0.077) and significantly increased skeletal muscle glycogen content (P < 0.05). When fed a high-fat diet, fetuin KO mice are resistant to weight gain, demonstrate significantly decreased body fat, and remain insulin sensitive. These data suggest that fetuin may play a significant role in regulating postprandial glucose disposal, insulin sensitivity, weight gain, and fat accumulation and may be a novel therapeutic target in the treatment of type 2 diabetes, obesity, and other insulin-resistant conditions. Worldwide prevalence data indicate that type 2 diabetes has reached epidemic proportions (1). Parallel to the rise in type 2 diabetes is a rapid expansion of obesity, associated with consumption of a high-fat diet (2). Insulin resistance is central to the Continue reading >>

Metabolic Phenotyping Guidelines: Assessing Glucose Homeostasis In Rodent Models

Metabolic Phenotyping Guidelines: Assessing Glucose Homeostasis In Rodent Models

Introduction The incidence of diabetes mellitus, particularly obesity-related type 2 diabetes, is increasing at an alarming rate in the developed world, and this epidemic is driving numerous research programmes into the causes of, and new treatment regimens for, this metabolic disorder. The complex hormonal control of nutrient homeostasis involves numerous tissues and organs, including liver, skeletal muscle, adipose, endocrine pancreas and CNS. While in vitro studies can provide cellular mechanistic insights, it is inevitable that in vivo models are needed to study the integrated control systems. Many animal models for the study of diabetes already exist, with various mechanisms for inducing either type 1 or type 2 diabetes (King 2012). Furthermore, genetically modified mouse models in which genes are up- or down-regulated either globally or in a tissue-specific manner are increasingly used to assess the physiological role of a potential target in glucose homeostasis and the development of diabetes. Consequently, techniques for accurately assessing glucose homeostasis in vivo in rodents are essential tools in current diabetes research. Mice and rats are by far the two most commonly used species for experimental studies of glucose homeostasis, and both models have specific advantages and disadvantages. The primary advantage of using a rat model is a technical consideration in that the larger size of the rat facilitates complex surgical procedures such as catheterisation, and the larger blood volume allows the sampling of more frequent and/or larger blood samples to enable detailed and simultaneous monitoring of multiple plasma hormone levels. Surgical techniques developed in the rat have been successfully miniaturised for use in mouse models, although they are technical Continue reading >>

Standard Operating Procedures For Describing And Performing Metabolic Tests Of Glucose Homeostasis In Mice

Standard Operating Procedures For Describing And Performing Metabolic Tests Of Glucose Homeostasis In Mice

Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice 1Vanderbilt-NIH Mouse Metabolic Phenotyping Center, Nashville, TN 37232, USA 2Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL 32827, USA 1Vanderbilt-NIH Mouse Metabolic Phenotyping Center, Nashville, TN 37232, USA 2Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, FL 32827, USA 3Yale-NIH Mouse Metabolic Phenotyping Center, New Haven, CT 06520, USA 4University of Washington-NIH Mouse Metabolic Phenotyping Center, Seattle, WA 98109, USA 5University of Cincinnati-NIH Mouse Metabolic Phenotyping Center, Cincinnati, OH 45267, USA 6Case Western Reserve University-NIH Mouse Metabolic Phenotyping Center, Cleveland, OH 44106, USA *Author for correspondence ( [email protected] ) This article has been cited by other articles in PMC. The Mouse Metabolic Phenotyping Center (MMPC) Consortium was established to address the need to characterize the growing number of mouse models of metabolic diseases, particularly diabetes and obesity. A goal of the MMPC Consortium is to propose standard methods for assessing metabolic phenotypes in mice. In this article, we discuss issues pertaining to the design and performance of various tests of glucose metabolism. We also propose guidelines for the description of methods, presentation of data and interpretation of results. The recommendations presented in this article are based on the experience of the MMPC Consortium and other investigators. The miniaturization of metabolic techniques for use in the mouse has resulted in important advances in our understanding of the pathophysiology of diabetes and its associated complications. An important goal of the Mouse Metabolic Phenotyping Center (MMPC) Co Continue reading >>

Insulin Tolerance Test And Random Fed Insulin Test Protocol

Insulin Tolerance Test And Random Fed Insulin Test Protocol

Insulintolerancetest and random fed insulin test protocol Mice are maintainedin a normal light/dark cycle according to the standard protocols of the JoslinDiabetes Center Animal Care and Use Committee. Mice aretested with age matched or litter mate controlls and typically at least 12 micepergroup are required. The day of the testmice are prepared for the insulin tolerance test: animals are weighed, the tailis nicked with a fresh razor blade by a horizontal cut of the very end, ~35 to50microliters of blood is very gently massaged from the tail to aneppendorph tube which is immediately placed on ice, baseline bloodglucoseis measured by the glucose oxidase method using aGlucometerElite glucometer, and 0.75 units per kg body weight of dilutedRegular Human Insulin (Lily) is drawn up in a Beckton Dickenson D 29 gage1/2" insulin syringe (insulin is diluted to 1:1000 (0.1 inits/ml) withregular insulin diluent) (3/4 of a unit of 1:1000 insulin for every gram ofbody weight). Animals are transfered to individually labeled 1000cccardboard soup cups with the lid liners removed. When all mice havebeen prepared the test is begun. Inulin is injected into theintraperitoneal cavity. At 15, 30, and 60 minutes blood glucose issampled from the tail of each mouse by gently massaging a small dropofblood onto the glucometer strip. Insulin injections and blood glucosesampling is timed to take approximately the same amount of time per animal(i.e. 25 animals are injected in 12 minutes and blood glucose sampling of thosesame 25 animals should also take about 12 minutes) so that the sample times areaccurate for each animal. Random fedimmunoreactive insulin levels: whole blood samples are spun in a refrigeratedmicrofuge at 14,000 rpm for 10 minutes and transfered toa clean tube. 6microliters of ser Continue reading >>

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