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Glucose Tolerance Test Mice Protocol

Pyruvate Tolerance Test

Pyruvate Tolerance Test

Glucose Test Strips - AccuChek Comfort Curve. Order these through Materiels Service by calling 936-6077 and ordering product 2535. For information see [here] 2.5g pyruvate in 10 mL PBS- This will correspond to 2.5 g/kg injections. Typically starve the mice at 9AM and aim to start injections at 3 PM Weigh mice, mark tails if necessary and take fasting glucose measurement via a tail clip. Prepare pyruvate syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL). At approximately 1 min intervals, inject appropriate amount of pyruvate 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) into cavity At desired intervals (normally 15, 30, 45, 60, 75, 90, 105 and 120 min), take blood glucose measurements from tail vein. If needed re-snip the tail vein. When measuring glucose just lift the tail of the mouse, while leaving it in the cage, rather than removing and restraining the mouse. Analyse data by both% change from fasting glucose and absolute values. Continue reading >>

How Much Time Do I Need To Wait Between A Glucose Tolerance Test And A Insulin Tolerance Test?

How Much Time Do I Need To Wait Between A Glucose Tolerance Test And A Insulin Tolerance Test?

GTT and ITT are stress-inducing experiences for mice, which can elevate blood glucose levels. Also repeated fasts can change physiology. I would wait as long as possible in between these tests/sacrifice especially if you are collecting tissue for further analysis. I am hesitant to say that even 48h is enough. Although you could test this by doing so and seeing if there is a significant difference in ITT between 24 and 48h. Another option is the acclimatize them to the handling to reduce (but not eliminate) the stress response. 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 >>

Lipid Tolerance Test

Lipid Tolerance Test

Unparalleled vision in metabolic disorders and risk assessment expertise for post-marketing studies and drug repositioning. HeartArteryMacrophageLiverMusclePancreasAdipose Tissue In-house predictive models and tracer technologies enable the generation of Proof of Concept efficacy data for your drugs. Screening and lead validation utilize a dedicated research platform which combine with our focused scientific vision. First in vivo proof of concept of the impact of your compound designed to treat type 2 diabetes on cardiovascular complications and/or dyslipidemia, in terms of whole-body lipid homeostasis. Suitable for large-scale screening in vivo (20-30 compounds/week). Yielding in vivo insights into your compounds efficacy in treating cardiovascular complications and/or dyslipidemia associated with type 2 diabetes Sensitivity (15%) to detect a statistically significant impact of your compound on postprandial blood lipid regulation The Lipid tolerance test investigates how dyslipidemic animals treated with your compound regulate triglycerides and free fatty acids after lipid intake. Continue reading >>

Alteration Of De Novo Glucose Production Contributes To Fasting Hypoglycaemia In Fyn Deficient Mice

Alteration Of De Novo Glucose Production Contributes To Fasting Hypoglycaemia In Fyn Deficient Mice

Alteration of De Novo Glucose Production Contributes to Fasting Hypoglycaemia in Fyn Deficient Mice Affiliations Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, Peoples Republic of China, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America Affiliation Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America * E-mail: [email protected] (CCB); [email protected] (GSY) Affiliation Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, Peoples Republic of China Affiliation Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America Affiliations Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America, Department of Chemical Biology and Therapeutics, St. Jude Childrens Research Hospital, Memphis, Tennessee, United States of America Affiliation Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America Affiliations Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America, Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York, United States of America * E-mail: [email protected] (CCB); [email protected] (GSY) Affiliations Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America, Division of Metabolic and Vascular Health, Warwick Medical School, University of Warwick, Coventry, United Kingdom Continue reading >>

Glucose Tolerance Test And Fasting Insulin Test Protocol

Glucose Tolerance Test And Fasting Insulin Test Protocol

Glucosetolerance test and fasting 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 8 micepergroup are required. Mice are fasted for15 hours: beginning around 6 pm the evening prior to the GTT mice aretransfered to a new cage with water but no food and a DO NOT FEED card isplaced in the cardholder. The followingmorning the mice are prepared for the glucose tolerance test: animals areweighed, the tail is nicked with a fresh razor blade by a horizontal cut of thevery end, ~35 to 50microliters of blood is very gently massaged from thetail to an eppendorph tube which is immediately placed on ice, baseline bloodglucoseis measured by the glucose oxidase method using aGlucometerElite glucometer, and 2 grams/kg body weight of 20% D-glucoseis drawn up in a Beckton Dickenson D 29 gage 1/2" insulin syringe (oneunit of D-glucose for every gram of body weight). Animals are transferedto individually labeled 1000cc cardboard soup cups with the lid liners removed. When all mice havebeen prepared the test is begun. Glucose is injected into the intraperitonealcavity. At 15, 30, 60, and 120 minutes blood glucose is sampled from thetail of each mouse by gently massaging a small drop ofblood onto theglucometer strip. Glucose injections and blood glucose sampling is timed totake approximately the same amount of time per animal (i.e. 25 animals areinjected in 12 minutes and blood glucose sampling of those same 25 animalsshould also take about 12 minutes) so that the sample times are accurate foreach animal. Fastingimmunoreactive insulin levels: whole blood samples are spun in a refrigeratedmicrofuge 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 >>

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 >>

Ipgtt

Ipgtt

An oral glucose tolerance test (OGTT) or intra-peritoneal glucose tolerance test (IPGTT) is used to assess the bodys ability to metabolize glucose. In humans an OGTT is commonly used to diagnose type 2 diabetes. In animal research the GTT is used to assess the degree of diabetes and also to test the desired effects of insulin or other drugs on the bodys ability to process glucose. It can also be used to detect the unintended side-effects of drugs intended to treat other unrelated diseases. Figure 1: Oral glucose tolerance test in two groups of n=3 rats. The blue line represents the diabetic group and the red line represents the normal group. The periodic points represent test strip samples. Figure 2: Intraperitoneal glucose tolerance test in two groups of n=3 rats. The blue line represents the diabetic group and the red line represents the normal group. The periodic points represent test strip samples. Traditional methods of glucose tolerance tests consist of the following basic steps: Fast the subject for 6-16 hours, depending on the protocol and IACUC considerations. Calculate the desired glucose dose based on the animals weight. Typical oral dose of 2-5 mg/kg or IP dose of 2 mg/kg. Collect a baseline (predose) blood glucose reading. Collect blood glucose data at multiple points along the curve. For an OGTT this might include points at 5, 15, 30, 60, 120, 180 minutes post-dose. An IPGTT might include an additional point at 10 minutes and omission of the 180 minute point. Relevant Parameters from this test include Peak Glucose, Time to Peak, Area Under Curve, Return to Baseline, Glucose at time X Implantable telemetry will automate this data collection with no need to collect manual measurements other than for (optional) reference purposes. Continuous data will provid 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 >>

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 >>

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 >>

Glucose And Insulin Tolerance Tests In The Mouse

Glucose And Insulin Tolerance Tests In The Mouse

Glucose and Insulin Tolerance Tests in the Mouse Part of the Methods in Molecular Biology book series (MIMB, volume 1339) 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. InsulinGlucoseMetabolismIntraperitoneal injectionMetabolic test This is a preview of subscription content, log in to check access Springer Nature is developing a new tool to find and evaluate Protocols. Learn more This work was supported by grants from the Carlos III Health Institute (CP10/00555, PI13/00834) and from the European Regional Development Fund (FEDER). H. Gonzlez-Navarro is a Miguel Servet Program researcher, and A. Vinu received salary support from Proyecto Paula. 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 CrossRef PubMed Google Scholar 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 PubMedCentral CrossRef PubMed Google Scholar Beckman JA, Creager MA, Libby P (2002) Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. JAMA 287:25702581 CrossRef PubMed Google Scholar 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 C Continue reading >>

Pyruvate Tolerance Test

Pyruvate Tolerance Test

Glucose homeostasis is tightly controlled by the interplay of various hormones and organs. In the fasted state, glucose levels are prevented from dropping too low (hypoglycemia) through two hepatic processes: glycogenolysis, the degradation of glycogen and gluconeogenesis, the generation of glucose from non-carbohydrat e carbon substrates including pyruvate and lactate (produced by anaerobic metabolism of glucose in muscles). The intraperitoneal Pyruvate Tolerance Test (iPTT, 1-2g/kg body weight) in 15h-fasted, awake mice is a variant of the intraqperitoneal glucose tolerance test (GTT) in which pyruvate is injected instead of glucose. The pyruvate bolus elicits a glycemic excursion that reflects hepatic gluconeogenesis. Although the method can be useful in cases of severe alterations in hepatic gluconeogenesis, it is highly dependent on the variables that influence the outcome of a glucose tolerance test (GTT), including glucose-stimulated insulin secretion (GSIS) and insulin sensitivity. It is thus always necessary to analyze results from a pyruvate tolerance test in light of data obtained from a GTT , GSIS and ITT . The gold standard method to assess gluconeogenesis is the measurement of endogenous glucose fluxes with use of tracers (e.g. during the basal period of a euglycemic hyperinsulinemic clamp ). Continue reading >>

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