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Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Determinations of triglycerides in plasma/serum/lymph will be made using a Randox Triglycerides colorimetric kit. The triglycerides are determined after enzymatic hydrolysis with lipases.

Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - Triglyceride Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.xm4fk8w Copy   

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Authors: Vance L. Albaugh
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the protocol for the biliopancreatic diversion procedure in the mouse. This procedure is historically thought to be a malabsorptive procedure and has several variations in the human. In brief, the biliary and pancreatic secretions are physically separated from gastrointestinal chyme flow until a point near the terminal small bowel. In theory this leads to significant malabsorption, though when this procedure is done clinically many times a gastric restriction component is also added.

Proper citation: Vance L. Albaugh 2019. Vandy - Biliopancreatic Diversion in Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.zb4f2qw Copy   

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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Behaviorally, meals are defined as periods of intense feeding and drinking separated by periods of activity, grooming, and rest. A “meal” is the primary data unit for characterizing food intake behavior. Changes in the patterning of meals (number, size or duration) may affect food intake and energy balance indirectly. "Meal", in our SOP, is defined by a minimum food intake of 0.02g and at least 10 min between food bout events. Detailed analysis of food intake behavior includes an assessment of average meal duration, average meal size, number of meals, average inter-meal interval (IMI), as well as calculation of satiety ratio. In addition, the diurnal (light cycle/dark cycle) patterns of food intake behavior are also calculated to examine temporal shifts in feeding behavior. Meal pattern and food intake behavior is measured in the Columbus Instruments Oxymax system within a temperature and light controlled cabinet. Animals are acclimated to the facility for at least 1 week. Animals are then acclimated to the CLAMS cages and powdered diet for 48 hours and to the light and temperature-controlled chamber for 24 hours prior to testing. Analyzed data constitutes data collected from 48 hours of continuous measurement (2 light/2 dark cycles).

Proper citation: Trina Knotts 2019. UC Davis - Meal Pattern Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.yrwfv7e Copy   

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Authors: Anil Singapuri
Group: Mouse Metabolic Phenotyping Centers
Summary: Cardiac hypertrophy is one of the most common causes of heart failure. The development of cardiac hypertrophy and failure can be monitored using electrocardiography (ECG) in conscious animals. ECG is used to measure the rate and regularity of heartbeats as well as the size and position of the chambers, the presence of any damage to the heart, and the effects of drugs or devices used to regulate the heart.

Proper citation: Anil Singapuri 2019. UC Davis - Electrocardiography. protocols.io https://dx.doi.org/10.17504/protocols.io.yidfua6 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum Urea/BUN levels are affected by alterations in systemic protein and nitrogen metabolism. Serum Urea/BUN levels are altered in kidney failure and renal complications of diabetes.

Proper citation: Jason Kim 2019. U Mass - Urea/BUN. protocols.io https://dx.doi.org/10.17504/protocols.io.x5qfq5w Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Glucagon is a 29 amino acid polypeptide processed from proglucagon in pancreatic alpha cells. In intestinal L-cells proglucagon is cleaved into glicentin, corresponding to proglucagon residues no 1-69. Glicentin can further be processed into oxyntomodulin, corresponding to proglucagon residues no 33-69. These peptides are released simultaneously upon stimulation. Moreover, a fragment of glucagon corresponding to its Cterminal part (residues no 19-29), also designated mini-glucagon, is reported to be present in the pancreas in low amounts compared to the total glucagon content. In general, glucagon has an effect opposite that of insulin, i.e. it raises blood glucose levels. It causes the liver to convert glycogen into glucose, which is then released into the blood stream. With longer stimulation, glucagon action in the liver results in a glucose-sparing activation of free fatty acid oxidation and production of ketones. During hypoglycaemia, glucagon secretion offers a protective feedback mechanism, defending the organism against damaging effects of glucose deficiency in the brain and nerves.

Proper citation: Peter Havel 2019. UC Davis - Glucagon. protocols.io https://dx.doi.org/10.17504/protocols.io.63ihgke Copy   

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Authors: Jeff Hodgin
Group: Mouse Metabolic Phenotyping Centers
Summary: Picrosirius red staining (also called Sirius red staining) highlights fibrosis by staining collagen (collagen 1 and III fibers) fibers in paraffin-embedded kidney tissue sections. Sirius red stained collagen appears red by light microscopy. When polarized, the birefringence is highly specific for collagen. The larger collagen fibers are bright yellow or orange, and the thinner ones, including reticular fibers, are green.

Proper citation: Jeff Hodgin 2019. U Michigan - Sirius Red staining. protocols.io https://dx.doi.org/10.17504/protocols.io.56tg9en Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Hyperinsulinemic-euglycemic clamp is the gold-standard method to assess insulin sensitivity. The hyperinsulinemic-euglycemic clamp is widely used in clinics and laboratories to measure insulin action on glucose utilization in humans and animals for clinical and basic science research. Incorporation of radioactive-labeled glucose during hyperinsulinemic-euglycemic clamps makes it possible to measure glucose metabolism in individual organs in awake mice. Impaired insulin sensitivity (insulin resistance) is a major characteristic of obesity and an early requisite event in the development of type 2 diabetes.

Proper citation: Jason Kim 2019. U Mass - Protein metabolism. protocols.io https://dx.doi.org/10.17504/protocols.io.x4ffqtn Copy   

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Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: An intraperitoneal insulin tolerance test or ipITT is designed to 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.

Proper citation: Fawaz G. Haj 2019. UC Davis - Intraperitoneal Insulin Tolerance Test. protocols.io https://dx.doi.org/10.17504/protocols.io.yp5fvq6 Copy   

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Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: Voluntary wheel running provides detailed information on the running capacity and activity patterns of control, treated or mutant mice. Running activity is evaluated from the number of rotations of a stationary wheel detected by sensors attached to running wheel. Running wheel activity provides information regarding when the animal runs, for how long and far, providing temporal and longitudinal data. This system may also be used as a means of providing voluntary exercise in metabolic or exercise based studies.

Proper citation: Jennifer Rutkowsky 2019. UC Davis - Running Wheel. protocols.io https://dx.doi.org/10.17504/protocols.io.yv9fw96 Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: The Wako enzymatic method relies upon the acylation of coenzyme A (CoA) by the fatty acids in the presence of added acyl-CoA synthetase (ACS). The acyl-CoA thus produced is oxidized by added acyl- CoA oxidase (ACOD) with generation of hydrogen peroxide, in the presence of peroxidase (POD) permits the oxidative condensation of 3-methy-N-ethyl-N(β-hydroxyethyl)-aniline (MEFA) with 4-aminoantipyrine to form a purple colored adduct which can be measured colorimetrically at 550 nm.

Proper citation: Peter Havel 2019. UC Davis - Non-Esterified Fatty Acids Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yutfwwn Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of bilirubin reflect bile acid metabolism.

Proper citation: Jason Kim 2019. U Mass - Bilirubin. protocols.io https://dx.doi.org/10.17504/protocols.io.xscfnaw Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: LDL and VLDL are separated from HDL using a precipitation reagent. Then the HDL fraction is measured for either TC or TG using the same reagents for total cholesterol or triglyceride.

Proper citation: Peter Havel 2019. UC Davis - HDL Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ynsfvee Copy   

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Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Summary:Lipidomic analysis by UPLC-QTOF mass spectrometry

Proper citation: Oliver Fiehn 2019. UC Davis - Metabolomics: Lipidomics analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ys7fwhn Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: The EchoMRI 3-in-1 uses ¹H- magnetic resonance spectroscopy to noninvasively measure whole body fat mass, lean mass, and water mass in awake mice. Whole body fat mass is a measure of adiposity that is altered in obesity. Whole body lean mass may be altered by exercise and in mouse models of altered metabolism.

Proper citation: Jason Kim 2019. U Mass - Body composition (whole body). protocols.io https://dx.doi.org/10.17504/protocols.io.xsqfndw Copy   

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Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Total body composition in live, un-anaesthetized small animals and carcasses will reveal absolute amounts of body fat, lean tissue and body water via a quantitative magnetic resonance (QMR) instrument, EchoMRI, (Echo Medical Systems, LLC, Houston, TX). This instrument uses the differences in the nuclear magnetic resonance properties of hydrogen atoms in organic and non-organic environments to fractionate signals originating from fat, lean tissue and free water.

Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - Body Composition & Carcass Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.2qigdue Copy   

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Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: This test is designated to determine if rodents exhibit signs of endoplasmic reticulum stress, through evaluation of the activation state of the 3 sub-arms: PERK/EiF2α, Ire1α/sXBP1, and ATF6α pathways such. We will examine induction of ER stress in adipose and liver tissues.

Proper citation: Fawaz G. Haj 2019. UC Davis - Endoplasmic reticulum stress. protocols.io https://dx.doi.org/10.17504/protocols.io.yihfub6 Copy   

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Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Total glycerol is determined by first hydrolyzing glycerides (in plasma or tissues) in 1 N KOHethanol + heat and then the hydrolysate extracted following acidification ~pH 1.0. A two phase extract is created using chloroform. The aqueous phase contains the total glycerol and the organic phase contains total fatty acids. An aliquot of aqueous phase in dried down by nitrogen gas and the concentration and/or 2H-labelled glycerol is then determined by GC-MS following conversion of glycerol to its triacetate derivative. Glucose assay involves extraction of glucose from blood, plasma or tissues and the concentration and/or 2H/13C-labelled glucose is also determined by GC-MS following conversion to its pentaacetate derivative. Since the derivatizing methods are the same for glucose as glycerol, the can be assayed from a single sample preparation under the same GC-MS conditions and single run-time.References: 1. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources. Bederman IR, Foy S, Chandramouli V, Alexander JC, Previs SF. J Biol Chem.;284(10):6101-8 (2009)

Proper citation: Henri Brunengraber 2019. Case - Glycerol and Glucose Assays by GC-mass spectrometry. protocols.io https://dx.doi.org/10.17504/protocols.io.ydkfs4w Copy   

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Authors: Richard Mcindoe, Mark Kelly, Timothy P. Fitzgibbons
Group: Mouse Metabolic Phenotyping Centers
Summary: This is a mouse model of acute myocardial infarction. This protocol is suitable for studying mouse models thought to be relevant to coronary ischemia or the development of congestive heart failure.

Proper citation: Richard Mcindoe, Mark Kelly, Timothy P. Fitzgibbons 2019. UMass - Coronary Artery Ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.wc4fayw Copy   

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Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: The glucose tolerance test measures the clearance of an intraperitoneally injected glucose load from the body. It is used to detect disturbances in glucose metabolism that can be linked to diabetes or metabolic syndrome. Animals are fasted for 5 hours, fasted blood glucose levels are determined before a solution of glucose is administered by intra-peritoneal (IP) injection. Subsequently, the blood glucose level is measured at different time points during the following 90 minutes.

Proper citation: Louise Lantier 2019. Vandy – IP glucose tolerance test. protocols.io https://dx.doi.org/10.17504/protocols.io.yzzfx76 Copy   

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