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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 dx.doi.org/10.17504/protocols.io.ydkfs4w Copy
Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: The Promethion from Sable Systems (Las Vegas, NV) assesses several key metabolic characteristics of mice such as energy expenditure, preferred metabolic substrate, meal patterns, activity, live body mass. These are measured by individually housing the mice in the Promethion System cages for several days during which numerous parameters are continuously measured (food and water intake, weight, activity, O² and CO²).
Proper citation: Louise Lantier 2019. Vandy – Energy Balance with Promethion. protocols.io dx.doi.org/10.17504/protocols.io.yzxfx7n Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
This experiment provides the quantification of multiple cytokines and chemokines using multiplexed-Luminex technology based on beads containing specific antibodies. Serum cytokine levels reflect chronic or acute inflammation, and circulating cytokines and chemokines are altered in obesity.
Cytokines Panel I include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, KC (IL-8 homologue), IL-9, IL-10 (interleukin-10), IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, TNFα (tumor necrosis factor alpha), IFNγ (interferon gamma), IP-10 (interferon gamma-induced protein 10; CXCL-10), Eotaxin (CCL-11), G-CSF (granulocyte colony stimulating factor), GM-CSF (granulocyte macrophage colony stimulating factor), LIF (leukemia inhibitory factor), LIX (LPS- induced CXC chemokine), MCP-1 (monocyte chemotactic protein-1; CCL-2), M-CSF (macrophage colony stimulating factor), MIG (monokine induced by gamma interferon; CXCL- 9), MIP-1α (macrophage inflammatory protein alpha; CCL-3), MIP-1β (macrophage inflammatory protein beta; CCL-4), and MIP-2/RANTES (macrophage inflammatory protein 2- alpha; CXCL-2).
A service can be requested for all or any combination of listed cytokines/chemokines for customized multiplexed Luminex assay.
Proper citation: Jason Kim 2019. U Mass - Electrolytes. protocols.io dx.doi.org/10.17504/protocols.io.xwafpae Copy
Authors: Lin Zhong, Jeffrey Rottman, Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The most common cause of cardiovascular mortality in man is the outcome from myocardial ischemic injury. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocol describes the surgical induction of myocardial injury via transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).
Proper citation: Lin Zhong, Jeffrey Rottman, Chee Lim 2019. Vandy - Myocardial Ischemia Reperfusion. protocols.io dx.doi.org/10.17504/protocols.io.6xfhfjn Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Glucose tolerance test measures systemic clearance of glucose following an intraperitoneal bolus injection of 20% dextrose. This experiment measures insulin sensitivity in awake mice assuming that there are no alterations in the animal’s pancreatic -cell function and insulin secretion. Insulin sensitivity is altered in obese mice.
Proper citation: Jason Kim 2019. U Mass - Glucose Tolerance Test. protocols.io dx.doi.org/10.17504/protocols.io.xxafpie Copy
Authors: Eva L. Feldman
Group: Mouse Metabolic Phenotyping Centers
Summary: Intra-epidermal nerve fiber density (IENFD) is used as a tool to assess small fiber neuropathy.
Proper citation: Eva L. Feldman 2019. U Michigan - Intra-Epidermal Fiber Density. protocols.io dx.doi.org/10.17504/protocols.io.563g9gn Copy
Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: Mouse Insulin ELISA kit is used for the non radioactive quantification of insulin in mouse plasma.
Proper citation: Henri Brunengraber 2019. Case - Plasma Insulin Measurement by ELISA. protocols.io dx.doi.org/10.17504/protocols.io.yesftee Copy
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 dx.doi.org/10.17504/protocols.io.yrwfv7e Copy
Authors: Jeff Hodgin
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the protocol for measuring Glomerular Filtration Rate (GFR) with minipump FITC-inulin clearance in mice. In brief, Alzet micro-osmotic pumps (minipumps, Model 1007D) are filled with 3% FITC-inulin solution. These pumps release FITC-inulin at a rate of 0.5 μl/hr at least seven days. Mouse is temporarily anesthetized and then two mini-pumps are implanted in mouse peritoneal cavity through a tiny midline incision. After surgery, 24-hour urine is collected with metabolic cage and 100 μl of blood from saphenous vein is obtained at the same time. The concentrations of FITC-inulin in urine and blood samples are measured by flurometer and GFR is calculated by the concentration of FITC-inulin in 24-hour-urine and the concentration of FITC-inulin in plasma.
Proper citation: Jeff Hodgin 2019. U Michigan - Glomerular Filtration Rate Determination with Minipump Inulin Clearance. protocols.io dx.doi.org/10.17504/protocols.io.x63frgn Copy
Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to measure the activity of Aspartate Amino Transferase (AST). AST activity is measured by the enzymatically coupled reactions of AST (to form oxaloacetate from aspartate and α-ketoglutarate) and malate dehydrogenase (conversion of oxaloacetate to malate with oxdiation of NADH to NAD). The rate of NAD formation is monitored by the change in absorbance at 340 nm.
Proper citation: Gary Cline, John Stack 2019. Yale - Aspartate Amino Transferase. protocols.io dx.doi.org/10.17504/protocols.io.yz9fx96 Copy
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 dx.doi.org/10.17504/protocols.io.x5qfq5w Copy
Authors: John Rutledge
Group: Mouse Metabolic Phenotyping Centers
Summary: Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) is used to assess blood-brain barrier permeability in vivo in rodent models. Post-processing image analysis is done using our in-house matlab script with patlak linearized regression mathematical modeling.
Proper citation: John Rutledge 2019. UC Davis - Dynamic contrast enhanced magnetic resonance imaging. protocols.io dx.doi.org/10.17504/protocols.io.yh9ft96 Copy
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 dx.doi.org/10.17504/protocols.io.63ihgke Copy
Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Description of standard setting for analysis of derivatized samples ready for injection into a Leco Pegasus III or Pegasus IV gas chromatograph-time of flight mass spectrometer.References: Fiehn O, Wohlgemuth G, Scholz M, Kind T, Lee DY, Lu Y, Moon S, Nikolau BJ (2008) Quality control for plant metabolomics: Reporting MSI-compliant studies. Plant J. 53, 691-704
Proper citation: Oliver Fiehn 2019. UC Davis - Metabolomics: Primary metabolites by GC-TOF MS. protocols.io dx.doi.org/10.17504/protocols.io.64nhgve Copy
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 dx.doi.org/10.17504/protocols.io.56tg9en Copy
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 dx.doi.org/10.17504/protocols.io.yv9fw96 Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Lipoprotein profiling and Lipoprotein size will be provided for CM, VLDL, LDL and HDL as well as 20 additional subclasses of lipoprotein. Testing requires as little as10µL for full analytical testing.
Proper citation: Peter Havel 2019. UC Davis - Lipoprotein profiling and partical size. protocols.io dx.doi.org/10.17504/protocols.io.yrifv4e Copy
Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to measure glucose concentrations. Glucose is measured by the enzymatically coupled reactions of hexokinase and glucose-6-P dehydrogenase. The rate of NADH formation is monitored by the change in absorbance at 340 nm.
Proper citation: John Stack, Gary Cline 2019. Yale - Blood Glucose. protocols.io dx.doi.org/10.17504/protocols.io.y2kfycw Copy
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 dx.doi.org/10.17504/protocols.io.ynsfvee Copy
Authors: Vance L. Albaugh
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the protocol for conducting the bile diversion procedure in the mouse. In this procedure the common bile duct is ligated proximal to the confluence of the common bile and the pancreatic ducts and an anastomosis is created at a prespecified point along the gastrointestinal tract to re-establish drainage of the biliary tree.
Proper citation: Vance L. Albaugh 2019. Vandy – Bile Diversion in Mice. protocols.io dx.doi.org/10.17504/protocols.io.zdcf22w Copy
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