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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment measures blood or plasma glucose concentration using Analox GM9 Glucose Analyzer or GM7 Micro-Stat Rapid Multi-assay Analyzer. The measurement is based on the rate of oxygen uptake in the reaction between sample glucose and glucose oxidase. Plasma glucose levels are elevated (i.e., hyperglycemia) in obesity, insulin resistance, and type 2 diabetes.
Proper citation: Jason Kim 2019. U Mass - Glucose. protocols.io dx.doi.org/10.17504/protocols.io.xw5fpg6 Copy
Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
Food Intake Monitoring - Oxymax/CLAMS monitors the cumulative amount of food eaten as well as the amount eaten in each bout with the use of a Mettler Toledo balance with a resolution of 0.01g. Feeders are designed to account for spillage of food and to prevent foraging, a combination which provides the most accurate food intake monitor on the market.
Food Access Control - Access to food can be placed under automatic computerized control. A pneumatic driven shield prevents access to the food, and can be controlled by times intervals and/or by mass of food. Additionally, this option also includes paradigms for Yolked or Paired feeding.
Center Feeder Cages: The Center Feeder Cage maintains the same sealed environment but adds a feeder to the center of the cage to allow food intake measurements. The Center Feeder is the most popular and universal in application, supporting all subject body types from lean to obese. An adjustable food guard allows mechanical fine tuning of food access to minimize foraging.
Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - Meal Pattern Analysis Food Intake Procedure. protocols.io dx.doi.org/10.17504/protocols.io.xjqfkmw Copy
Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
This test is designated to determine defects in the insulin signaling pathway, through evaluation of the activation state of the insulin receptor (IR) and its substrate (IRS1/2), as well as downstream target, mainly Akt and MAP kinases.
Proper citation: Fawaz G. Haj 2019. UC Davis - Insulin signaling pathway. protocols.io dx.doi.org/10.17504/protocols.io.yp2fvqe Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Hepatic gluconeogensis is estimated using pyruvate tolerance test that measures systemic elevation of glucose partly derived from pyruvate and hepatic gluconeogensis following an intraperitoneal bolus injection of pyruvate in awake mice. Hepatic gluconeogenesis is affected by obesity and regulates glucose homeostasis.
Proper citation: Jason Kim 2019. U Mass - Hepatic gluconeogenesis. protocols.io dx.doi.org/10.17504/protocols.io.xy7fpzn Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
The lipid extraction method is based on work by Folch 1957. Tissue is homogenized and put into a 2:1 chloroform and methanol mix. The mix is separated into two phases by adding a salt solution. The upper phase contains any non-lipid substances and the lower phase contains the chloroform with the lipids. The upper phase is removed and a sample is taken from the bottom phase and evaporated. The sample is reconstituted with 2-propanol with a volume to bring it to the desired concentration to assay.
Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3- phosphate and adenosine diphosphate. Glycerol-3-phosphate is oxidized by dihydroxyacetone phosphate (DAP) by glycerolphosphate oxidase producing hydrogen peroxide (H²O²). In a Trinder5 type color reaction catalyzed by peroxidase, the H²O² reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2- hydroxybenzene sulfonate (DHBS) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglycerides present in the sample.
Cholesterol esters are enzymatically hydrolysed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-en-3- one and hydrogen peroxide. The hydrogen peroxide combines with HBA and 4-aminoantipyrine to form
a chromophore (quinoneimine dye) which may be quantitated at 500-550nm.
Proper citation: Peter Havel 2019. UC Davis - Tissue TG & TC Protocol. protocols.io dx.doi.org/10.17504/protocols.io.62thgen Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
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 (LPSinduced CXC chemokine), MCP-1 (monocyte chemotactic protein-1; CCL-2), M-CSF (macrophage colony stimulating factor), MIG (monokine induced by gamma interferon; CXCL9), MIP-1α (macrophage inflammatory protein alpha; CCL-3), MIP-1β (macrophage inflammatory protein beta; CCL-4), and MIP-2/RANTES (macrophage inflammatory protein 2alpha; 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 - Gamma-glutamyl Transferase. protocols.io dx.doi.org/10.17504/protocols.io.xwzfpf6 Copy
Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of magnesium in blood, serum, and plasma. Magnesium is measured as the chlorophosphonazo III complex (addition of EGTA prevents calcium interference) and monitored at 600 nm.
Proper citation: John Stack, Gary Cline 2019. Yale - Magnesium. protocols.io dx.doi.org/10.17504/protocols.io.y3zfyp6 Copy
Authors: Oliver Fiehn
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Summary:This SOP describes sample extraction and sample preparation for lipid profiling by liquid chromatography / quadrupole time of flight mass spectrometry (LC-QTOF) or nanoelectrospray ion trap-FTICR MS.
Proper citation: Oliver Fiehn 2019. UC Davis - Metabolomics: Sample preparation for Lipidomics. protocols.io dx.doi.org/10.17504/protocols.io.ytpfwmn Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Hyperglycemic clamp measures insulin secretion and pancreatic -cell function in awake mice. Islet function is affected by obesity and insulin resistance, and abnormal islet function predisposes to the development of type 2 diabetes.
Proper citation: Jason Kim 2019. U Mass - Hyperglycemic clamp. protocols.io dx.doi.org/10.17504/protocols.io.xzafp2e Copy
Authors: Mark Kelly, Timothy P. Fitzgibbons
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:This is a mouse model of hind limb ischemia, a technique involving an interruption in the arterial blood supply to tissue in the hind limb. This model is used to study peripheral artery disease and vascular remodeling.
Proper citation: Mark Kelly, Timothy P. Fitzgibbons 2019. U Mass - Hind Limb Ischemia. protocols.io dx.doi.org/10.17504/protocols.io.56jg9cn Copy
Authors: David A. Antonetti
Group: Mouse Metabolic Phenotyping Centers
Summary: The vascular permeability is quantified by measuring albumin leakage form blood vessels into the retina. Fluorescent dye (FITC-BSA) is used to measure the breakdown in blood-retinal barrier to be detected when increased vessel leakage is extravasated into the interstitial space.
Proper citation: David A. Antonetti 2019. U Michigan - Retinal Vascular Permeability. protocols.io dx.doi.org/10.17504/protocols.io.yagfsbw Copy
Authors: Mari Golub
Group: Mouse Metabolic Phenotyping Centers
Summary: The y-maze is a modification of the T- maze which evaluates memory and special learning in rodents though quantification of spontaneous alternation (a measure of spatial cognition). This measure has previously been shown to be affected in mouse models of Alzheimer’s.
References: Ohno et al. Neuron 41:27-33, 2004. Oakley et al. J Neuroscience 26(40):10129-10140, 2006. Ohno et al. Neurobiol Dis 26:134-145, 2007. Holcomb et al. Nat Med 4:97-100, 1998.
Proper citation: Mari Golub 2019. UC Davis - Y-Maze. protocols.io dx.doi.org/10.17504/protocols.io.yw7fxhn Copy
Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: The glucose tolerance test measures the clearance of an oral 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 oral gavage. Subsequently, the blood glucose and plasma insulin levels are measured at different time points during the following 120 minutes.
Proper citation: Louise Lantier 2019. Vandy – Oral glucose tolerance test. protocols.io dx.doi.org/10.17504/protocols.io.yz2fx8e Copy
Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure followed to detect the concentration of creatinine in serum, plasma, and urine.
Proper citation: Gary Cline, John Stack 2019. Yale - Blood and Urine Creatinine. protocols.io dx.doi.org/10.17504/protocols.io.y2dfya6 Copy
Authors: Li-Ming Jin
Group: Mouse Metabolic Phenotyping Centers
Summary: Erectile function is well regulated by vasorelaxant and vasoconstrictive mechanisms, which can be perturbed by various disease states including cardiovascular disease, diabetes, and hormonal insufficiencies. This protocol enables us to asses erectile function by measuring the mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) upon electrical stimulation of the cavernous nerve.Modified from: Burnett et al Science. 1992 Jul 17;257(5068):401-3.; Quinlan et al J Urol. 1989 Mar;141(3):656-61.; and Jin et al J Sex Med. 2008 Mar;5(3):544-51.
Proper citation: Li-Ming Jin 2019. UC Davis - Erectile Dysfunction. protocols.io dx.doi.org/10.17504/protocols.io.yikfucw Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary:
Insulin tolerance test measures systemic clearance of glucose following an intraperitoneal bolus injection of a physiological dose of insulin. This experiment measures insulin sensitivity in awake mice assuming that there are no alterations in the animal’s counterregulatory response. Insulin sensitivity is altered in obese mice.
Proper citation: Jason Kim 2019. U Mass - Insulin tolerance test. protocols.io dx.doi.org/10.17504/protocols.io.x2xfqfn Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The insulin enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay for the detection of insulin levels in serum samples. This experiment uses a 96-well plate coated with anti-insulin antibodies and developed with a secondary anti-insulin antibody linked to horseradish peroxidase. The ELISA is developed with 3,3',5,5'-Tetramethylbenzidine (TMB), a chromogenic substrate and read on an automated plate spectrophotometer at 450 nm. Insulin ELISA may be processed with normal or ultra-sensitive assay kit depending on the expected range of sample concentrations. Serum insulin levels are affected by pancreatic β-cell function and insulin sensitivity. Serum insulin levels are elevated in obesity and insulin resistance.
Proper citation: Jason Kim 2019. U Mass - Insulin. protocols.io dx.doi.org/10.17504/protocols.io.xznfp5e Copy
Authors: Mari Golub
Group: Mouse Metabolic Phenotyping Centers
Summary: The Morris water maze (MWM) is a widely used tool to study spatial learning and memory. It can assess learning, memory, and spatial working memory and can also indicative of damage to cortical regions of the brain. It can measure the effect of neurocognitive disorders on spatial learning and possible neural treatments, to test the effect of lesions to the brain in areas focused on memory, and to study how age influences cognitive function and spatial learning.
Proper citation: Mari Golub 2019. UC Davis - Morris Water Maze. protocols.io dx.doi.org/10.17504/protocols.io.yuqfwvw Copy
Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Changes in fecal energy loss can result in differences in net energy balance and feed efficiency, and may result from, e.g., alterations in bile acid production, secretion or re-uptake, and changes in activities of enzymes involved in macronutrient digestion or uptake (e.g., pancreatic lipase). This assay will be coupled to food intake determinations and will involve fecal collection and feces weight measures for 48h, which can be coordinated with the meal pattern studies (catalog item D4003) or indirect calorimetry (catalog item D4007). Specific components (crude protein, dry matter, ash, and fat), will be measured by proximate analysis in fecal samples to in order to assess fat or protein absorption.
Proper citation: Trina Knotts 2019. UC Davis - Digestible Energy by Proximate Analysis. protocols.io dx.doi.org/10.17504/protocols.io.2vbge2n Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Catalase (EC 1.11.1.6; 2H2O2 oxidoreductase) is an ubiquitous antioxidant enzyme that is present in most aerobic cells. Catalase (CAT) is involved in the detoxification of hydrogen peroxide (H2O2), a reactive oxygen species (ROS), which is a toxic product of both normal aerobic metabolism and pathogenic ROS production. This enzyme catalyzes the conversion of two molecules of H2O2 to molecular oxygen and two molecules of water (catalytic activity). CAT also demonstrates peroxidatic activity, in which low molecular weight alcohols can serve as electron donors. While the aliphatic alcohols serve as specific substrates for CAT, other enzymes with peroxidatic activity do not utilize these substrates. In humans, the highest levels of catalase are found in liver, kidney, and erythrocytes, where it is believed to account for the majority of hydrogen peroxide decomposition. The Cayman Chemical Catalase Assay Kit utilizes the peroxidatic function of CAT for determination of enzyme activity. The method is based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced is measured spectrophotometrically with 4-amino3-hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the chromogen. Purpald specifically forms a bicyclic heterocycle with aldehydes, which upon oxidation changes from colorless to a purple color. The assay can be used to measure CAT activity in plasma, serum, erythrocyte lysates, tissue homogenates, and cell lysates.
Proper citation: Peter Havel 2019. UC Davis - Catalase. protocols.io dx.doi.org/10.17504/protocols.io.ygbftsn Copy
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